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Sample records for polyamine oxidase activity

  1. Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

    PubMed

    Schenkel, E; Dubois, J G; Helson-Cambier, M; Hanocq, M

    1996-02-01

    It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.

  2. Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

    PubMed

    Schenkel, E; Dubois, J G; Helson-Cambier, M; Hanocq, M

    1996-02-01

    It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity. PMID:8882384

  3. 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

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

    PubMed Central

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

    2016-01-01

    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. Newborn 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

  5. 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.

  6. 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

  7. Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

    PubMed

    Agostinelli, Enzo; Vianello, Fabio; Magliulo, Giuseppe; Thomas, Thresia; Thomas, T J

    2015-01-01

    Nanotechnology for cancer gene therapy is an emerging field. Nucleic acids, polyamine analogues and cytotoxic products of polyamine oxidation, generated in situ by an enzyme-catalyzed reaction, can be developed for nanotechnology-based cancer therapeutics with reduced systemic toxicity and improved therapeutic efficacy. Nucleic acid-based gene therapy approaches depend on the compaction of DNA/RNA to nanoparticles and polyamine analogues are excellent agents for the condensation of nucleic acids to nanoparticles. Polyamines and amine oxidases are found in higher levels in tumours compared to that of normal tissues. Therefore, the metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, can be targets for antineoplastic therapy since these naturally occurring alkylamines are 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. In particular, polyamine catabolism involves copper-containing amine oxidases. Several studies showed an important role of these enzymes in developmental and disease-related processes in animals through the control of polyamine homeostasis in response to normal cellular signals, drug treatment, and environmental and/or cellular stress. The production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these oxidases suggests a mechanism by which amine oxidases can be exploited as antineoplastic drug targets. The combination of bovine serum amine oxidase (BSAO) and polyamines prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. The findings described herein suggest that enzymatically formed cytotoxic agents activate stress signal transduction pathways, leading to apoptotic cell death. Consequently, superparamagnetic nanoparticles or other

  8. Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion.

    PubMed Central

    Vujcic, Slavoljub; Liang, Ping; Diegelman, Paula; Kramer, Debora L; Porter, Carl W

    2003-01-01

    In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic

  9. 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

  10. 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.

  11. 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

  12. Metabolism of an alkyl polyamine analog by a polyamine oxidase from the microsporidian Encephalitozoon cuniculi.

    PubMed

    Bacchi, Cyrus J; Yarlett, Nigel; Faciane, Evangeline; Bi, Xiangdong; Rattendi, Donna; Weiss, Louis M; Woster, Patrick M

    2009-06-01

    Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{N-[o-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N'-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K(m) of 2 microM. In the presence of N'-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of (13)C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of (13)C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.

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

    PubMed

    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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae.

    PubMed

    Mo, Huijuan; Wang, Xingfen; Zhang, Yan; Zhang, Guiyin; Zhang, Jinfa; Ma, Zhiying

    2015-09-01

    Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2 O2 ), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2 O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling.

  1. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants.

    PubMed

    Valdés-Santiago, Laura; Guzmán-de-Peña, Doralinda; Ruiz-Herrera, José

    2010-11-01

    In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

  2. 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

  3. 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

  4. 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.

  5. Polyamine homoeostasis.

    PubMed

    Persson, Lo

    2009-11-04

    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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. Glucose oxidase activity of actinomycetes.

    PubMed

    St Vlahov, S

    1978-01-01

    The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established. PMID:76424

  11. Dependence of Trichomonas vaginalis upon polyamine backconversion.

    PubMed

    Yarlett, N; Martinez, M P; Goldberg, B; Kramer, D L; Porter, C W

    2000-10-01

    Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellular pool of [(14)C]spermidine and a small but detectable pool of [(14)C]putrescine. When [(3)H]putrescine was added to the growth medium, a large intracellular pool of [(3)H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0. 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM. The enzyme was competitively inhibited by di(ethyl)norspermine with a K:(i) of 28 microM. Growth studies indicated that 50 microM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively. The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K:(m) of 6.0 microM for N(1)-acetylspermine. The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed. PMID:11021947

  12. Activation of Dbl restores migration in polyamine-depleted intestinal epithelial cells via Rho-GTPases.

    PubMed

    Ray, Ramesh M; Bavaria, Mitulkumar N; Bhattacharya, Sujoy; Johnson, Leonard R

    2011-06-01

    Integrin binding to the extracellular matrix (ECM) activated Rho GTPases, Src, and focal adhesion kinase in intestinal epithelial cells (IEC)-6. Polyamine depletion inhibited activities of Rac1, RhoA, and Cdc42 and thereby migration. However, constitutively active (CA) Rac1 expression abolished the inhibitory effect of polyamine depletion, indicating that polyamines are involved in a process upstream of Rac1. In the present study, we examined the role of polyamines in the regulation of the guanine nucleotide exchange factor, diffuse B-cell lymphoma (Dbl), for Rho GTPases. Polyamine depletion decreased the level as well as the activation of Dbl protein. Dbl knockdown by siRNA altered cytoskeletal structure and decreased Rac1 activity and migration. Cells expressing CA-Dbl increased migration, Rac1 activity, and proliferation. CA-Dbl restored migration in polyamine-depleted cells by activating RhoA, Rac1, and Cdc42. CA-Dbl caused extensive reorganization of the F-actin cortex into stress fibers. Inhibition of Rac1 by NSC23766 significantly decreased migration of vector-transfected cells and CA-Dbl-transfected cells. However, the inhibition of migration was significantly higher in the vector-transfected cells compared with that seen in the CA-Dbl-transfected cells. Dbl localized in the perinuclear region in polyamine-depleted cells, whereas it localized with the stress fibers in control cells. CA-Dbl localized with stress fibers in both the control and polyamine-depleted cells. These results suggest that polyamines regulate the activation of Dbl, a membrane-proximal process upstream of Rac1.

  13. The polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike that of angiosperms, back-converts thermospermine to norspermidine.

    PubMed

    Sagor, G H M; Inoue, Masataka; Kim, Dong Wook; Kojima, Seiji; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2015-10-01

    In the phylogeny of plant polyamine oxidases (PAOs), clade III members from angiosperms, such as Arabidopsis thaliana PAO5 and Oryza sativa PAO1, prefer spermine and thermospermine as substrates and back-convert both of these substrates to spermidine in vitro. A clade III representative of lycophytes, SelPAO5 from Selaginella lepidophylla, also prefers spermine and thermospermine but instead back-converts these substrates to spermidine and norspermidine, respectively. This finding indicates that the clade III PAOs of lycophytes and angiosperms oxidize thermospermine at different carbon positions. We discuss the physiological significance of this difference.

  14. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    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 x mg(-1) chlorophyll x 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

  15. 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

  16. Polyamines reverse non-steroidal anti-inflammatory drug-induced toxicity in human colorectal cancer cells.

    PubMed Central

    Hughes, Alun; Smith, Nicholas I; Wallace, Heather M

    2003-01-01

    Naproxen, sulindac and salicylate, three NSAIDs (non-steroidal anti-inflammatory drugs), were cytotoxic to human colorectal cancer cells in culture. Toxicity was accompanied by significant depletion of intracellular polyamine content. Inhibition of ornithine decarboxylase (the first enzyme of the polyamine biosynthetic pathway), induction of polyamine oxidase and spermidine/spermine N(1)-acetyltransferase (the enzymes responsible for polyamine catabolism) and induction of polyamine export all contributed to the decreased intracellular polyamine content. Morphological examination of the cells showed typical signs of apoptosis, and this was confirmed by DNA fragmentation and measurement of caspase-3-like activity. Re-addition of spermidine to the cells partially prevented apoptosis and recovered the cell number. Thus polyamines appear to be an integral part of the signalling pathway mediating NSAID toxicity in human colorectal cancer cells, and may therefore also be important in cancer chemoprevention in humans. PMID:12793857

  17. Xanthine oxidase inhibitory activity of alkyl gallates.

    PubMed

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Kubo, Isao

    2006-08-01

    A series (C1-C12) of alkyl gallates was examined for their effects on the activity of xanthine oxidase. Octyl (C8), decyl (C10), and dodecyl (C12) gallates competitively inhibited uric acid formation generated by xanthine oxidase, and the inhibition increased upon increasing the alkyl chain length. Interestingly, neither menthyl nor bornyl gallates inhibited uric acid formation. These data indicate that the hydrophobic alkyl portion is associated with the xanthine-binding site in the Mo-binding domain. It is likely that the linear alkyl portion interacts with the hydrophobic domain close to the binding site, and the hydrophobic interaction is crucial to inhibit the xanthine oxidase reaction. On the other hand, all of gallic acid and its esters equally suppress superoxide anion generation catalyzed by xanthine oxidase at low concentration. The suppression is not due to scavenging activity of these gallates but due to reduction of xanthine oxidase by these gallates. The reduced enzyme catalyzes the reaction to generate hydrogen peroxide and uric acid.

  18. Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism.

    PubMed

    Cerrada-Gimenez, Marc; Häyrinen, Jukka; Juutinen, Sisko; Reponen, Tuula; Jänne, Juhani; Alhonen, Leena

    2010-02-01

    We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor gamma co-activator 1alpha in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.

  19. Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues

    PubMed Central

    Bacchi, Cyrus J.; Rattendi, Donna; Faciane, Evangeline; Yarlett, Nigel; Weiss, Louis M.; Frydman, Benjamin; Woster, Patrick; Wei, Benjamin; Marton, Laurence J.; Wittner, Murray

    2011-01-01

    The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N1-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials. PMID:15133083

  20. 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...

  1. Inhibition of mast cell secretion by oxidation products of natural polyamines.

    PubMed

    Vliagoftis, H; Boucher, W S; Mak, L L; Theoharides, T C

    1992-05-28

    Mast cells secrete many biologically active compounds upon stimulation by immunoglobulin E (IgE) and specific antigen (Ag), anaphylatoxins, as well as a number of cationic compounds which include drugs, kinins and neuropeptides. The effects of the two naturally occurring polyamines, spermine (SP) and spermidine (SPD), on mast cell secretion were studied because they have been implicated in the modulation of cellular processes, possibly through their cationic charge or the regulation of calcium ions. SP and SPD over the range of 10(-7) to 10(-4) M inhibited the release of 5-hydroxytryptamine (5-HT, serotonin) triggered by compound 48/80 (C48/80) in a time- and concentration-dependent manner, as long as at least 2% calf serum (CS) was present. SP also inhibited secretion of both histamine and serotonin stimulated immunologically by using IgE and anti-rat IgE. This inhibition was not accompanied by cytotoxicity. The major available polyamine metabolites tested, N1-acetyl spermine (N1-acSP) and N8-acetyl spermidine (N8-acSPD), also showed inhibition in the presence of CS, whereas putrescine, N8,N1-hexamethylene-bis-acetamide (HMBA) and benzylamine did not. Fetal bovine serum (FBS), as well as human and rat serum, which do not contain polyamine oxidase, did not result in any inhibition with the polyamines tested. Inhibitors of the polyamine oxidase blocked the polyamine effect, indicating that the inhibition of mast cell secretion must derive from aldehydes produced from these polyamines. Addition of the aldehyde inhibitor phenylhydrazine (phi-HDZ), simultaneously with, but not following the polyamines, blocked their inhibitory effect, further strengthening the involvement of aldehydes. These results indicate that naturally occurring polyamines may regulate mast cell secretion through metabolic products of polyamine oxidase, a similar enzyme of which is also present in human liver, placenta and pregnant serum.

  2. 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.

  3. 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

  4. 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

  5. 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.

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

    PubMed Central

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

    2009-01-01

    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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Tamoxifen metabolite endoxifen interferes with the polyamine pathway in breast cancer.

    PubMed

    Thomas, T J; Thomas, Thresia; John, Shali; Hsu, Hui-Chen; Yang, PingAr; Keinänen, Tuomo A; Hyvönen, Mervi T

    2016-10-01

    Tamoxifen is the most widely used drug to treat women with estrogen receptor α (ERα)-positive breast cancer. Endoxifen is recognized as the active metabolite of tamoxifen in humans. We studied endoxifen effects on ERα-positive MCF-7 breast cancer cells. Estradiol increased the proliferation of MCF-7 cells by two- to threefold and endoxifen suppressed its effects. Endoxifen suppressed c-myc, c-fos and Tff1 oncogene expression, as revealed by RT-PCR. Estradiol increased the activity of ornithine decarboxylase (ODC) and adenosyl methioninedecarboxylase (AdoMetDC), whereas endoxifen suppressed these enzyme activities. Endoxifen increased activities of spermine oxidase (SMO) and acetyl polyamine oxidase (APAO) significantly, and reduced the levels of putrescine and spermidine. These data suggest a possible mechanism for the antiestrogenic effects of tamoxifen/endoxifen, involving the stimulation of polyamine oxidase enzymes. Therefore, SMO and APAO stimulation might be useful biomarkers for the efficacy of endoxifen treatment of breast cancer.

  14. Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

    PubMed

    Xiong, Xiao-Feng; Poulsen, Mette H; Hussein, Rama A; Nørager, Niels G; Strømgaard, Kristian

    2014-12-01

    The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.

  15. 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.

  16. 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

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

    PubMed

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

    2015-04-01

    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 surprising 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

  18. 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

  19. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  20. 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

  1. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    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

  2. 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

  3. 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.

  4. Polyamines in plant physiology.

    PubMed Central

    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. PMID:11537482

  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. 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.

  7. Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity.

    PubMed

    Bartl, Jasmin; Müller, Thomas; Grünblatt, Edna; Gerlach, Manfred; Riederer, Peter

    2014-04-01

    Patients with Parkinson's disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.

  8. Effect of salicylic acid treatment on postharvest quality, antioxidant activities, and free polyamines of asparagus.

    PubMed

    Wei, Yunxiao; Liu, Zhenfeng; Su, Yujing; Liu, Donghong; Ye, Xingqian

    2011-03-01

    The effects of salicylic acid (SA) on the quality and antioxidant activity of asparagus stored at 18 ± 2 °C were investigated by analyzing the color, chlorophyll, shear force, and the activity of antioxidant compounds such as ascorbic acid, phenolics, flavonoids, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity ferric reducing antioxidant power (FRAP), and polyamines (PAs). The results showed that SA improved the color and maintained the chlorophyll, phenolic, flavonoid, and ascorbic acid content of asparagus. High concentrations of SA caused a deterioration in asparagus would harm to color and had no effect on shear force within 6 d. SA induced the maximum concentration of phenolics in postharvest asparagus, promoted the increase in total flavonoids before 6 to 9 d, affected the antioxidant activity positively as indicated by the resultant increase in FRAP concentration; however, SA was only active with regard to DPPH scavenging activity within 6 d of treatment. Spermidine (Spd) is the most common form of PA in asparagus, and free putrescine (Put) contents increased over the first 3 d following harvest and then decreased. Spd and Spm concentrations evolved in a similar way and decreased during storage. Higher Spd and Spm contents in the SA pre-treatment Put was inhabited and its peaks appeared later.

  9. 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...

  10. Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae).

    PubMed

    Silva, E C C; Masui, D C; Furriel, R P M; Mantelatto, F L M; McNamara, J C; Barrabin, H; Leone, F A; Scofano, H M; Fontes, C F L

    2008-04-01

    Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

  11. 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

  12. Polyamines alter intestinal glucose transport.

    PubMed

    Johnson, L R; Brockway, P D; Madsen, K; Hardin, J A; Gall, D G

    1995-03-01

    Polyamines are required for the growth of all eukaryotic cells. Enterocytes respond to luminal nutrients with large increases in polyamine synthesis, even though they are mature, nonproliferating cells. The role of polyamines in these cells is unknown. The current experiments examined whether polyamines affected intestinal transport of glucose, since absorption is the primary activity of enterocytes and since polyamines are known to affect membrane function and stability. Glucose transport was examined in rabbit brush-border membrane vesicles (BBMV). BBMV from rabbits given 5% alpha-difluoromethylornithine (DFMO) in their drinking water 24 h before they were killed transported significantly less glucose than control vesicles [38% decrease in maximal transport rate (Jmax)]. Orogastric administration of spermine, spermidine, or putrescine to DFMO-treated animals 24 h before they were killed prevented the decrease. In rabbits receiving only orogastric spermine, glucose transport was significantly increased (64% increase in Jmax), whereas in vivo spermidine and putrescine decreased Jmax. This increase in Jmax caused by in vivo administration of spermine was not dependent on protein synthesis. Addition of polyamines whether in vivo or in vitro decreased Michaelis constant in vesicles from control and DFMO-treated animals. The change in glucose transport induced by DFMO or polyamines was not related to altered membrane lipid composition or fluidity.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Drought stress tolerance in grapevine involves activation of polyamine oxidation contributing to improved immune response and low susceptibility to Botrytis cinerea.

    PubMed

    Hatmi, Saloua; Gruau, Charlotte; Trotel-Aziz, Patricia; Villaume, Sandra; Rabenoelina, Fanja; Baillieul, Fabienne; Eullaffroy, Philippe; Clément, Christophe; Ferchichi, Ali; Aziz, Aziz

    2015-02-01

    Environmental factors including drought stress may modulate plant immune responses and resistance to pathogens. However, the relationship between mechanisms of drought tolerance and resistance to pathogens remained unknown. In this study, the effects of drought stress on polyamine (PA) homeostasis and immune responses were investigated in two grapevine genotypes differing in their drought tolerance; Chardonnay (CHR), as sensitive and Meski (MSK), as tolerant. Under drought conditions, MSK plants showed the lowest leaf water loss and reduction of photosynthetic efficiency, and expressed a lower level of NCED2, a gene involved in abscisic acid biosynthesis, compared with CHR plants. The improved drought tolerance in MSK was also coincident with the highest change in free PAs and up-regulation of the genes encoding arginine decarboxylase (ADC), copper amine-oxidase (CuAO), and PA-oxidases (PAO) and their corresponding enzyme activities. MSK plants also accumulated the highest level of amino acids, including Arg, Glu, Gln, Pro, and GABA, emphasizing the participation of PA-related amino acid homeostasis in drought tolerance. Importantly, drought-tolerant plants also exhibited enhanced phytoalexin accumulation and up-regulation of PR genes, especially PR-2 and Chit4c, compared with the sensitive plants. This is consistent with a lower susceptibility of MSK than CHR to Botrytis cinerea. Data suggest a possible connection between water stress tolerance and immune response in grapevine. Pharmacological experiments revealed that under drought conditions CuAO and PAO pathways were involved in the regulation of photosynthetic efficiency, and also of immune response and resistance of grapevine to a subsequent pathogen attack. These results open new views to improve our understanding of crosstalk between drought tolerance mechanisms and immune response.

  14. 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

  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. 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.

  17. 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.

  18. Quantitative analysis of phenol oxidase activity in insect hemolymph.

    PubMed

    Sorrentino, Richard Paul; Small, Chiyedza N; Govind, Shubha

    2002-04-01

    We describe a simple, inexpensive, and robust protocol for the quantification of phenol oxidase activity in insect hemolymph. Discrete volumes of hemolymph from Drosophila melanogaster larvae are applied to pieces of filter paper soaked in an L-3, 4-dihydroxyphenylalanine (L-DOPA) solution. Phenol oxidase present in the samples catalyzes melanin synthesis from the L-DOPA precursor, resulting in the appearance of a roughly circular melanized spot on the filter paper. The filter paper is then scanned and analyzed with image-processing software. Each pixel in an image is assigned a grayscale value. The mean of the grayscale values for a circular region of pixels at the center of the image of each spot is used to compute a melanization index (MI) value, the computation is based on a comparison to an external standard (India ink). Numerical MI values for control and experimental larvae can then be pooled and subjected to statistical analysis. This protocol was used to evaluate phenol oxidase activity in larvae of different backgrounds: wild-type, lozenge, hopscotch(Tumorous-lethal) (which induces the formation of large melanotic tumors), and body-color mutations ebony and yellow. Our results demonstrate that this assay is sensitive enough for use in genetic screens with D. melanogaster and could conceivably be used for evaluation of MI from hemolymph of other insects.

  19. 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

  20. 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.

  1. Partial characterization of polyphenol oxidase activity in raspberry fruits.

    PubMed

    González, E M; de Ancos, B; Cano, M P

    1999-10-01

    A partial characterization of polyphenol oxidase (PPO) activity in raspberry fruits is described. Two early cultivars harvested in May/June (Heritage and Autumm Bliss) and two late cultivars harvested in October-November (Ceva and Rubi) were analyzed for PPO activity. Stable and highly active PPO extracts were obtained using insoluble poly(vinylpyrrolidone) (PVP) and Triton X-100 in sodium phosphate, pH 7.0 buffer. Polyacrylamide gel electrophoresis of raspberry extracts under nondenaturing conditions resolved in one band (R(f)()(1) = 0.25). Raspberry PPO activity has pH optima of 8.0 and 5.5, both with catechol (0.1 M). Maximum activity was with D-catechin (catecholase activity), followed by p-coumaric acid (cresolase activity). Heritage raspberry also showed PPO activity toward 4-methylcatechol. Ceva and Autumm Bliss raspberries showed the higher PPO activity using catechol as substrate.

  2. Iron regulates xanthine oxidase activity in the lung.

    PubMed

    Ghio, Andrew J; Kennedy, Thomas P; Stonehuerner, Jacqueline; Carter, Jacqueline D; Skinner, Kelly A; Parks, Dale A; Hoidal, John R

    2002-09-01

    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 oxidoreductase activity in cultured V79 cells was increased with exposure to ferric ammonium sulfate and inhibited by deferoxamine. Lung XO and total xanthine oxidoreductase activities were reduced in rats fed an iron-depleted diet and increased in rats supplemented with iron, without change in the ratio of XO to total oxidoreductase. Intratracheal injection of an iron salt or silica-iron, but not aluminum salts or silica-zinc, significantly increased rat lung XO and total xanthine oxidoreductase activities, immunoreactive xanthine oxidoreductase, and the concentration of urate in bronchoalveolar fluid. These results suggest the possibility that the production of uric acid, a major chelator of iron in extracellular fluid, is directly influenced by iron-mediated regulation of the expression and/or activity of its enzymatic source, xanthine oxidase.

  3. Xanthine oxidase inhibitory activity of Hungarian wild-growing mushrooms.

    PubMed

    Ványolós, Attila; Orbán-Gyapai, Orsolya; Hohmann, Judit

    2014-08-01

    Mushrooms represent a remarkable and yet largely unexplored source of new, biologically active natural products. In this work, we report on the xanthine oxidase (XO) inhibitory activity of 47 wild-growing mushrooms native to Hungary. Aqueous and organic (n-hexane, chloroform, and 50% methanol) extracts of selected mushrooms from different families were screened for their XO inhibitory activities. Among the 188 extracts investigated, the chloroform and 50% methanol fractions proved to be the most effective. Some species exhibited high inhibitory activity, e.g., Hypholoma fasciculare (IC50  =67.76 ± 11.05 µg/mL), Suillus grevillei (IC50  =13.28 ± 1.58 µg/mL), and Tricholoma populinum (IC50  =85.08 ± 15.02 µg/mL); others demonstrated moderate or weak activity. Additional studies are warranted to characterize the compounds responsible for the XO inhibitory activity of mushroom extracts.

  4. Changes in free polyamines and related enzymes during stipule and pod wall development in Pisum sativum.

    PubMed

    Chattopadhyay, Soumen; Lahiri, Kajari; Bharati, Ghosh

    2002-08-01

    Level of free polyamines, their key metabolic enzymes, and other features related to ageing were examined during stipule and pod wall development in pea (Pisum sativum). Free polyamine titre (per unit fresh mass) in both the organs, the specific activities of arginine decarboxylase and ornithine decarboxylase in the pod wall, gradually decreased with maturation. In stipule, these enzymes attained peak activity at 15 days after pod emergence and declined thereafter. Ornithine decarboxylase activity was greater in pod wall than in stipule; while, arginine decarboxylase activity was higher in stipule. Activity of degradative enzyme diamine oxidase increased with the onset of senescence in both the organs. Chlorophyll and electrical conductance had a inverse relationship throughout the experimental period, whereas, the chlorophyll content was directly related with polyamine levels in both stipule and pod wall during aging. On the other hand, protein and RNA contents were positively correlated with free polyamines throughout the test period in stipule, but in the pod wall this was true only for the later stages of development.

  5. Xanthine oxidase inhibitory activity of some Indian medical plants.

    PubMed

    Umamaheswari, Muthuswamy; AsokKumar, Kuppusamy; Somasundaram, Arumugam; Sivashanmugam, Thirumalaisamy; Subhadradevi, Varadharajan; Ravi, Thenvungal Kochupapy

    2007-02-12

    Xanthine oxidase inhibitory activity was assayed from six species belonging to different families traditionally used for the treatment of gout and related symptoms by indigenous people of India. The aqueous, methanol-water mixture and methanolic extract of these plants were used for the experiment. Of the 18 extracts assayed, 14 extracts demonstrated xanthine oxidase inhibitory activity at 100 microg/ml, among which 10 extracts showed an inhibition greater than 50% and IC(50) values below 100 microg/ml. The methanolic extracts of Coccinia grandis, Datura metel, Strychnos nux-vomica and Vitex negundo showed more than 50% inhibition, hence, they were screened for their in vivo hypouricaemic activity against potassium oxonate-induced hyperuricaemia in mice. Methanolic extracts of Coccinia grandis and Vitex negundo showed a significant decrease in the serum urate level (3.90+/-0.07 mg/dl, P<0.001) and (6.26+/-0.06 mg/dl, P<0.01), respectively, when compared to hyperuricaemic control (11.42+/-0.14 mg/dl). This effect is almost similar to the serum urate level of allopurinol (3.89+/-0.07 mg/dl).

  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.

  7. 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

  8. 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...

  9. 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.

  10. 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

  11. 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

  12. Xanthine oxidase inhibitory activity of Lychnophora species from Brazil ("Arnica").

    PubMed

    Filha, Z S Ferraz; Vitolo, I F; Fietto, L G; Lombardi, J A; Saúde-Guimarães, D A

    2006-08-11

    Twenty-two extracts from five Lychnophora species and one Lychnophoriopsis species, traditionally used in Brazil as analgesic, anti-inflammatory, and to treat bruise and rheumatism were examined for the inhibition of xanthine oxidase (XO), the enzyme that catalyses the metabolism of hypoxanthine and xanthine into uric acid. Sixteen extracts were tested. All of them were found to have excellent XO inhibitory activity, with inhibitions greater than 38% at 100 microg/mL in the assay mixture. The most active plants examined were Lychnophora trichocarpha, Lychnophora ericoides, Lychnophora staavioides and Lychnophoriopsis candelabrum, with inhibitions of 77%, 78%, 66% and 63% at 100 microg/mL, respectively, and IC(50) values of 6.16, 8.28, 33.97 and 37.70 microg/mL, respectively.

  13. 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

  14. 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

  15. Abscisic acid signals reorientation of polyamine metabolism to orchestrate stress responses via the polyamine exodus pathway in grapevine.

    PubMed

    Toumi, Imene; Moschou, Panagiotis N; Paschalidis, Konstantinos A; Bouamama, Badra; Ben Salem-Fnayou, Asma; Ghorbel, Abdel Wahed; Mliki, Ahmed; Roubelakis-Angelakis, Kalliopi A

    2010-05-01

    Polyamines (PAs) have been suggested to be implicated in plant responses to abiotic and biotic stress. Grapevine is a model perennial plant species whose cultivars respond differently to osmotic stress. In this study, we used two cultivars, one sensitive (S) and one tolerant (T) to drought. In adult vines subjected to drought under greenhouse conditions, total PAs were significantly lower in the control T- and higher in the control S-genotype and significantly increased or decreased, respectively, post-treatment. Soluble Put and Spd exhibited the greatest increase on d 8 post-treatment in the T- but not in the S-genotype, which accumulated soluble Spm. Abscisic acid (ABA) was differentially accumulated in T- and S-genotypes under drought conditions, and activated the PA biosynthetic pathway, which in turn was correlated with the differential increases in PA titers. In parallel, polyamine oxidases (PAOs) increased primarily in the S-genotype. ABA at least partially induced PA accumulation and exodus into the apoplast, where they were oxidized by the apoplastic amine oxidases (AOs), producing H2O2, which signaled secondary stress responses. The results here show that the ABA signaling pathway integrates PAs and AOs to regulate the generation of H2O2, which signals further stress responses or the PCD syndrome.

  16. Nox family NADPH oxidases: Molecular mechanisms of activation.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-11-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS). Numerous homologue-specific mechanisms control the activity of this enzyme family involving calcium, free fatty acids, protein-protein interactions, intracellular trafficking, and posttranslational modifications such as phosphorylation, acetylation, or sumoylation. After a brief review on the classic pathways of Nox activation, this article will focus on novel mechanisms of homologue-specific activity control and on cell-specific aspects which govern Nox activity. From these findings of the recent years it must be concluded that the activity control of Nox enzymes is much more complex than anticipated. Moreover, depending on the cellular activity state, Nox enzymes are selectively activated or inactivated. The complex upstream signaling aspects of these events make the development of "intelligent" Nox inhibitors plausible, which selectively attenuate disease-related Nox-mediated ROS formation without altering physiological signaling ROS. This approach might be of relevance for Nox-mediated tissue injury in ischemia-reperfusion and inflammation and also for chronic Nox overactivation as present in cancer initiation and cardiovascular disease.

  17. Characterization of ent-kaurene oxidase activity from Gibberella fujikuroi.

    PubMed

    Ashman, P J; Mackenzie, A; Bramley, P M

    1990-11-01

    Cell extracts of wild-type and mutant strains of Gibberella fujikuroi were assayed for kaurene oxidase activity, using ent-[3H]kaurene as the substrate. Extracts from strain SG78 exhibited the highest specific activity, and were used in subsequent experiments. The microsomal enzyme activity was solubilized with buffers or salt solutions at a concentration of 400 mM. Both the soluble and microsomal preparations showed characteristic cytochrome P-450 spectra, ligand binding spectra with the substrate and with the plant growth regulator, paclobutrazol, and inhibition of enzymic activity by carbon monoxide. The addition of 20% glycerol to the extraction buffer stabilized the activity to some extent. Loss of enzymic activity on storage was accompanied by conversion of P-450 to P-420. Michaelis-Menten kinetic parameters for the membrane-bound and soluble enzyme have been estimated, as have constants for the binding of ent-kaurene and paclobutrazol to the P-450 and P-420 forms of the protein.

  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. 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.

  20. Salivary Ceruloplasmin Ferroxidase & Oxidase Activities in Celiac Patients

    PubMed Central

    Hasan, Hathama R.; Ghadhban, Jasim M.; Abudal Kadhum, Zahraa I.

    2012-01-01

    The aim of the current study was to evaluate salivary ferroxidase ceruloplasmin activities in celiac patients with different histopathological severity. This study included 75 celiac patients with different mean age (18.68 ± 11.13) year, who had positive screen for celiac antibodies, and who had gastrointestinal symptoms. In order to simplify the comparison with the healthy control group, celiac patients were divided into two groups according to their histopathological severity: severe (marsh IIIa, b, c) & less severe (marsh 0, I). All these patients have been evaluating for salivary ceruloplasmin (Cp) concentration and Cp ferroxidase activities. To confirm the presence of the enzymatic activity of this protein, polyacrylamide gel electrophoresis was carried out and then stained for Cp ferroxidase, as well as for Cp oxidase activity. Furthermore, the concentrations of salivary total protein, albumin, and globulin were measured in the studied groups. A significant increase (p<0.05) in salivary concentration of ceruloplasmin was found in all above mentioned patients groups in comparison to that of the control group, except for total villous atrophy (marsh IIIc) patients subgroup. Salivary Cp ferroxidase activity revealed statistically significant decrease among the patient groups as well as between them and the control group. The result of salivary total protein and globulin showed presence a significant increase (p<0.05) in comparison to that of the control group. Meanwhile albumin levels was found to increase non-significantly (p=0.186). PMID:23675269

  1. 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.

  2. 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

  3. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

    PubMed

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

  4. 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.

  5. [Increasing activity of a monoamine oxidase by random mutation].

    PubMed

    Chen, Xuejun; Ma, Yuanhui; Shao, Jianhua; Lai, Dunyue; Wang, Zhiguo; Chen, Zhenming

    2014-01-01

    The monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic k(cat)/K(m) value increased from 0.001 51 mmol/(L x s) to 0.002 89 mmol/(L x s), suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that T162A may affect the secondary structure of the substrate channel and expand the binding pocket. PMID:24818485

  6. 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

  7. Xanthine oxidase inhibitory activity of extracts prepared from Polygonaceae species.

    PubMed

    Orbán-Gyapai, Orsolya; Lajter, Ildikó; Hohmann, Judit; Jakab, Gusztáv; Vasas, Andrea

    2015-03-01

    The xanthine oxidase (XO) inhibitory activity of aqueous and organic extracts of 27 selected species belonging in five genera (Fallopia, Oxyria, Persicaria, Polygonum and Rumex) of the family Polygonaceae occurring in the Carpathian Basin were tested in vitro. From different plant parts (aerial parts, leaves, flowers, fruits and roots), a total of 196 extracts were prepared by subsequent extraction with methanol and hot H2O and solvent-solvent partition of the MeOH extract yielding n-hexane, chloroform and 50% MeOH subextracts. It was found that the chloroform subextracts and/or the remaining 50% MeOH extracts of Fallopia species (F. bohemica, F. japonica and F. sachalinensis), Rumex species (R. acetosa, R. acetosella, R. alpinus, R. conglomeratus, R. crispus, R. hydrolapathus, R. pulcher, R. stenophyllus, R. thyrsiflorus, R. obtusifolius subsp. subalpinus, R. patientia) and Polygonum bistorta, Polygonum hydropiper, Polygonum lapathifolium and Polygonum viviparum demonstrated the highest XO inhibitory activity (>85% inhibition) at 400 µg/mL. The IC50 values of the active extracts were also determined. On the basis of the results, these plants, and especially P. hydropiper and R. acetosella, are considered worthy of activity-guided phytochemical investigations.

  8. The Escherichia coli CydX protein is a member of the CydAB cytochrome bd oxidase complex and is required for cytochrome bd oxidase activity.

    PubMed

    VanOrsdel, Caitlin E; Bhatt, Shantanu; Allen, Rondine J; Brenner, Evan P; Hobson, Jessica J; Jamil, Aqsa; Haynes, Brittany M; Genson, Allyson M; Hemm, Matthew R

    2013-08-01

    Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

  9. 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

  10. The mechanisms by which polyamines accelerate tumor spread.

    PubMed

    Soda, Kuniyasu

    2011-10-11

    Increased polyamine concentrations in the blood and urine of cancer patients reflect the enhanced levels of polyamine synthesis in cancer tissues arising from increased activity of enzymes responsible for polyamine synthesis. In addition to their de novo polyamine synthesis, cells can take up polyamines from extracellular sources, such as cancer tissues, food, and intestinal microbiota. Because polyamines are indispensable for cell growth, increased polyamine availability enhances cell growth. However, the malignant potential of cancer is determined by its capability to invade to surrounding tissues and metastasize to distant organs. The mechanisms by which increased polyamine levels enhance the malignant potential of cancer cells and decrease anti-tumor immunity are reviewed. Cancer cells with a greater capability to synthesize polyamines are associated with increased production of proteinases, such as serine proteinase, matrix metalloproteinases, cathepsins, and plasminogen activator, which can degrade surrounding tissues. Although cancer tissues produce vascular growth factors, their deregulated growth induces hypoxia, which in turn enhances polyamine uptake by cancer cells to further augment cell migration and suppress CD44 expression. Increased polyamine uptake by immune cells also results in reduced cytokine production needed for anti-tumor activities and decreases expression of adhesion molecules involved in anti-tumor immunity, such as CD11a and CD56. Immune cells in an environment with increased polyamine levels lose anti-tumor immune functions, such as lymphokine activated killer activities. Recent investigations revealed that increased polyamine availability enhances the capability of cancer cells to invade and metastasize to new tissues while diminishing immune cells' anti-tumor immune functions.

  11. Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation.

    PubMed

    Moffatt, J; Hashimoto, M; Kojima, A; Kennedy, D O; Murakami, A; Koshimizu, K; Ohigashi, H; Matsui-Yuasa, I

    2000-12-01

    The efficacy of the antitumor activity of 1'-acetoxychavicol acetate (ACA), reported to be a suppressor of chemically induced carcinogenesis, was evaluated in Ehrlich ascites tumor cells. ACA treatment resulted in changes in morphology and a dose-dependent suppression of cell viability. Apoptosis, characterized by nuclear condensation, membrane blebbing, cell shrinkage and a significant induction of caspase-3-like protease activity at 8 h in a time-course study were observed. Formation of apoptotic bodies was preceded by lowering of intracellular polyamines, particularly putrescine, and both dose- and time-dependent inhibitory and activation effect by ACA on ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT), respectively. Administration of exogenous polyamines prevented ACA-induced apoptosis represented by a reduction in the number of apoptotic bodies and also caused reduction in the induced caspase-3-like protease activity at 8 h. These findings suggest that the anticarcinogenic effects of ACA might be partly due to perturbation of the polyamine metabolic pathway and triggering of caspase-3-like activity, which result in apoptosis.

  12. 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

  13. 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%.

  14. 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

  15. 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

  16. Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens).

    PubMed

    Serapiglia, Michelle J; Minocha, Rakesh; Minocha, Subhash C

    2008-12-01

    We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount of N in the culture medium was reduced. When total N in the medium was increased, cell mass increased without significant changes in glutamine synthetase activity or polyamine concentration. Reductions in the amount of nitrate or total N in the culture medium resulted in increased accumulations of Ca, Mn and Zn in the cells, and K accumulation decreased in response to decreasing nitrate:ammonium ratios. The data indicate that changes in total N availability as well as the forms of N play important roles in the physiological responses of in-vitro-grown red spruce cells that mimic the observed responses of forest trees to soil N deficiency and N fertilization.

  17. 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

  18. Clinical role of polyamine analysis: problem and promise.

    PubMed

    Jeevanandam, M; Petersen, S R

    2001-09-01

    Polyamines are ubiquitous cell components for growth. They play an important role in cell proliferation, cell growth and synthesis of protein and nucleic acids. Cells that are stimulated to reproduce demonstrated early increases in biosynthetic enzymes involved in polyamine synthesis and subsequent elevations in polyamine levels. Extracellular fluid polyamine concentrations that reflect the intracellular events may be useful as rapid indicators of therapy in disorders which involve altered cell growth. More complex analytical approaches are required to isolate, identify and quantitate these polyamines. Most of the methods require an extraction procedure to remove interfering amino acid derivatives. Daily monitoring of plasma and urine polyamine levels in many pathological states may provide a non-invasive biochemical marker of the existing disease activity or response to therapy or to screen for drug efficacy. Automated high-performance liquid chromatography, with post or pre-column derivatization and fluorescence or electrochemical detection is frequently used for the simultaneous quantitation of picomolar quantities of polyamines. Recently, a new immuno-cytochemical model system incorporating an enzyme-linked immunosorbent assay for specific polyamines has been developed. The increasing momentum of basic science information in the polyamine discipline may lead clinicians to regard polyamines, their metabolites and antimetabolites as sources of effective treatment.

  19. 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.

  20. The active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation

    SciTech Connect

    Thompson, D.A.; Suarez-Villafane, M.; Ferguson-Miller, S.

    1982-01-01

    Cytochrome oxidase is a multisubunit, intrinsic membrane protein with a complex function that includes oxidation of cytochrome c, reduction of oxygen and generation of a membrane potential. To clarify the relationship of its normal function to protein and membrane structure, we have examined the kinetic behavior of rat liver cytochrome oxidase in the intace inner mitochondrial membrane and in detergent solubilized states. Dissolution of rat liver mitochondrial membranes alters the kinetic parameters of the oxidase in a manner dependent in part on the dispersing agent, and characterized by a large increase in maximal activity which is not attributable to exposure of more oxidase or diminished affinity for cytochrome c. The most profound effect of solubilization of the membrane is seen on the low affinity reaction of cytochrome c, suggesting that the electron transfer pathway from this site to oxygen is sensitive to alterations in hydrophobic interactions within the oxidase. Purified rat liver and beef heart oxidase exists predominantly in a monodisperse, 300 kilodalton form in laurylmaltoside (Rosevear et al., 1980). However, a smaller, 130 kd species that exhibits high turnover rates equal to the 300 kd form is detected in some beef heart preparations, implying that the dimer may not be essential for high activity. Radiation inactivation studies on purified oxidase reveal a molecular weight for the functional unit of approx.70 kd. It is concluded that less than a complete set of subunits may be sufficient for both normal bindings of cytochrome c and rapid electron transfer to oxygen.

  1. Active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation

    SciTech Connect

    Thompson, D.A.; Suarez-Villafane, M.; Ferguson-Miller, S.

    1982-01-01

    Cytochrome oxidase is a multisubunit, intrinsic membrane protein with a complex function that includes oxidation of cytochrome c, reduction of oxygen and generation of a membrane potential. To clarify the relationship of its normal function to protein and membrane structure, we have examined the kinetic behavior of rat liver cytochrome oxidase in the intact inner mitochondrial membrane and in detergent solubilized states. Dissolution of rat liver mitochondrial membranes alters the kinetic parameters of the oxidase in a manner dependent in part on the dispersing agent, and characterized by a large increase in maximal activity which is not attributable to exposure of more oxidase or diminished affinity for cytochrome c. The most profound effect of solubilization of the membrane is seen on the low affinity reaction of cytochrome c, suggesting that the electron transfer pathway from this site to oxygen is sensitive to alterations in hydrophobic interactions within the oxidase. Purified rat liver and beef heart oxidase exists predominantly in a monodisperse, 300 kilodalton form in laurylmaltoside (Rosevear et al., 1980). However, a smaller, 130 kd species that exhibits high turnover rates equal to the 300 kd form is detected in some beef heart preparations, implying that the dimer may not be essential for high activity. Radiation inactivation studies on purified oxidase reveal a molecular weight for the functional unit of approx.70 kd. It is concluded that less than a complete set of subunits may be sufficient for both normal binding of cytochrome c and rapid electron transfer to oxygen.

  2. Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress.

    PubMed

    Jakubowska, Dagmara; Janicka-Russak, Małgorzata; Kabała, Katarzyna; Migocka, Magdalena; Reda, Małgorzata

    2015-05-01

    The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxidase activity in cucumber roots. Plants were treated with cadmium for 1, 3 or 6 days. Some of the plants after 3-day exposure to cadmium were transferred to a medium without the heavy metal for the next 3 days. Treatment of plants with cadmium for 6 days stimulated the activity of NADPH oxidase. The highest stimulation of O2(•-) production by NADPH oxidase was observed in post-stressed plants, which was correlated with the stimulation of activity of PM H(+)-ATPase in the same conditions. In order to examine the effects of cadmium stresses on the expression level of genes encoding NADPH oxidase, putative cucumber homologs encoding RBOH proteins were selected and a real-time PCR assay was performed. NADPH is a substrate for oxidase; thus alterations in the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and NADP-malic enzyme under cadmium stress were studied. The activity of NADPH dehydrogenases was increased under cadmium stress. The results indicate that PM NADPH oxidase could be involved in plants' response to cadmium stress by affecting the activity of PM H(+)-ATPase, and NADPH-generating enzymes could play important roles in this process.

  3. F14512, a polyamine-vectorized inhibitor of topoisomerase II, exhibits a marked anti-tumor activity in ovarian cancer.

    PubMed

    Thibault, Benoît; Clement, Emily; Zorza, Grégoire; Meignan, Samuel; Delord, Jean-Pierre; Couderc, Bettina; Bailly, Christian; Narducci, Fabrice; Vandenberghe, Isabelle; Kruczynski, Anna; Guilbaud, Nicolas; Ferré, Pierre; Annereau, Jean-Philippe

    2016-01-01

    Epithelial ovarian cancer is the fourth cause of death among cancer-bearing women and frequently associated with carboplatin resistance, underlining the need for more efficient and targeted therapies. F14512 is an epipodophylotoxin-core linked to a spermine chain which enters cells via the polyamine transport system (PTS). Here, we investigate this novel concept of vectorization in ovarian cancer. We compared the effects of etoposide and F14512 on a panel of five carboplatin-sensitive or resistant ovarian cancer models. We assessed the incorporation of F17073, a spermine-linked fluorescent probe, in these cells and in 18 clinical samples. We then showed that F14512 exhibits a high anti-proliferative and pro-apoptotic activity, particularly in cells with high levels of F17073 incorporation. Consistently, F14512 significantly inhibited tumor growth compared to etoposide, in a cisplatin-resistant A2780R subcutaneous model, at a dose of 1.25 mg/kg. In addition, ex vivo analysis indicated that 15 out of 18 patients presented a higher F17073 incorporation into tumor cells compared to normal cells. Overall, our data suggest that F14512, a targeted drug with a potent anti-tumor efficacy, constitutes a potential new therapy for highly PTS-positive and platinum-resistant ovarian cancer-bearing patients.

  4. Bifunctionalized mesoporous silica-supported gold nanoparticles: intrinsic oxidase and peroxidase catalytic activities for antibacterial applications.

    PubMed

    Tao, Yu; Ju, Enguo; Ren, Jinsong; Qu, Xiaogang

    2015-02-11

    Bifunctionalized mesoporous silica-supported gold nanoparticles as oxidase and peroxidase mimics for antibacterial applications are demonstrated. For the first time, these mesoporous silica-supported gold nanoparticles are applied as oxidase and peroxidase mimics. Taking advantage of their prominent enzyme activities, the MSN-AuNPs show excellent antibacterial properties against both Gram-negative and Gram-positive bacteria. Furthermore, MSN-AuNPs also exhibit outstanding performance in biofilm elimination . PMID:25655182

  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.

  7. 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

  8. 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

  9. Inhibitory action of NoxA1 on dual oxidase activity in airway cells.

    PubMed

    Pacquelet, Sandrine; Lehmann, Mandy; Luxen, Sylvia; Regazzoni, Karine; Frausto, Monika; Noack, Deborah; Knaus, Ulla G

    2008-09-01

    Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.

  10. 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.

  11. 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

  12. Polyamines and polyamine biosynthesis in cells exposed to hyperthermia

    SciTech Connect

    Gerner, E.W.; Stickney, D.G.; Herman, T.S.; Fuller, D.J.

    1983-02-01

    The issue of how polyamines act to sensitize cultured cells to the lethal effects of hyperthermia was investigated using Chinese hamster cells which were induced to express thermotolerance. Intracellular levels of these naturally occurring polycations were manipulated in certain situations by treating whole cells with methylglyoxal bis-(guanylhydrazone), an inhibitor of the S-adenosyl-L-methionine decarboxylases. Exogenous spermine as low as 100 ..mu..M in the culture media dramatically sensitized cells expressing thermotolerance to the lethal effects of subsequent 42/sup 0/C exposures. When thermotolerance was differentially induced in cultures exposed to 42.4/sup 0/C by varying the rate of heating from 37 to 42.4/sup 0/C, the most resistant cells and the highest levels of intracellular spermidine and spermine. This finding was explainable in part by the observation that the putrescine-dependent S-adenosyl-L-methionine decarboxylase activity was minimally affected in cells expressng the greatest degree of thermotolerance. When this enzyme activity was inhibited by drug, lowered intracellular polyamine levels did not correspond with subsequent survival responses to heat. Interestingly, cultures treated with methylglyoxal bis-(guanylhydrazone) 24 hr previous to heat exposure showed a reduced capacity to express rate of heating-induced thermotolerance. Together, these results demonstrate that the polyamines, especially spermidine and spermine, enhance hyperthermia-induced cell killing by some mechanism involving the plasma membrane. Further, our data suggest that methylglyoxal bis-(guanylhydrazone) can act to affect thermal responses by a mechanism(s) other than modification of intracellular polyamine levels.

  13. 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

  14. 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.

  15. Complement-dependent NADPH oxidase enzyme activation in renal ischemia/reperfusion injury.

    PubMed

    Simone, S; Rascio, F; Castellano, G; Divella, C; Chieti, A; Ditonno, P; Battaglia, M; Crovace, A; Staffieri, F; Oortwijn, B; Stallone, G; Gesualdo, L; Pertosa, G; Grandaliano, G

    2014-09-01

    NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as α-SMA expression and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and α-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting.

  16. Complement-dependent NADPH oxidase enzyme activation in renal ischemia/reperfusion injury.

    PubMed

    Simone, S; Rascio, F; Castellano, G; Divella, C; Chieti, A; Ditonno, P; Battaglia, M; Crovace, A; Staffieri, F; Oortwijn, B; Stallone, G; Gesualdo, L; Pertosa, G; Grandaliano, G

    2014-09-01

    NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as α-SMA expression and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and α-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting. PMID:25017967

  17. Perturbation of polyamine catabolism affects grape ripening of Vitis vinifera cv. Trincadeira.

    PubMed

    Agudelo-Romero, Patricia; Ali, Kashif; Choi, Young H; Sousa, Lisete; Verpoorte, Rob; Tiburcio, Antonio F; Fortes, Ana M

    2014-01-01

    Grapes are economically the most important fruit worldwide. However, the complexity of biological events that lead to ripening of nonclimacteric fruits is not fully understood, particularly the role of polyamines' catabolism. The transcriptional and metabolic profilings complemented with biochemical data were studied during ripening of Trincadeira grapes submitted to guazatine treatment, a potent inhibitor of polyamine oxidase activity. The mRNA expression profiles of one time point (EL 38) corresponding to harvest stage was compared between mock and guazatine treatments using Affymetrix GrapeGen(®) genome array. A total of 2113 probesets (1880 unigenes) were differentially expressed between these samples. Quantitative RT-PCR validated microarrays results being carried out for EL 35 (véraison berries), EL 36 (ripe berries) and EL 38 (harvest stage berries). Metabolic profiling using HPLC and (1)H NMR spectroscopy showed increase of putrescine, proline, threonine and 1-O-ethyl-β-glucoside in guazatine treated samples. Genes involved in amino acid, carbohydrate and water transport were down-regulated in guazatine treated samples suggesting that the strong dehydrated phenotype obtained in guazatine treated samples may be due to impaired transport mechanisms. Genes involved in terpenes' metabolism were differentially expressed between guazatine and mock treated samples. Altogether, results support an important role of polyamine catabolism in grape ripening namely in cell expansion and aroma development.

  18. 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.

  19. 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

  20. Polyamine-Induced Rapid Root Abscission in Azolla pinnata

    PubMed Central

    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

  1. 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

  2. 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

  3. 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.

  4. Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza.

    PubMed

    Liu, Xiaoyu; Chen, Ruohua; Shang, Yanjun; Jiao, Binghua; Huang, Caiguo

    2009-06-01

    In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in beta-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC(50) of 29.8 microg/mL and 4.06 microg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.

  5. 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.

  6. 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...

  7. Human platelet monoamine oxidase activity in health and disease: a review.

    PubMed

    Sandler, M; Reveley, M A; Glover, V

    1981-03-01

    The most readily available source of monoamine oxidase in man is the platelet, although only the B form of the enzyme is represented in this site. Platelet activity is higher in women than in men. The enzyme activity is generally stable and is partly under genetic control. There is some evidence that individuals with low activity have a higher psychiatric morbidity than those with high activity. Despite some negative studies, the consensus of publication dealing with schizophrenia, migraine, and alcoholism find that mean platelet monoamine oxidase activity in the patient group is lower than in the controls. Values are raised in unipolar depression. Technical differences, or patient or control group heterogeneity, might well account for the absence of unanimity in the literature. A considerable degree of overlap between patient and control values, whatever the clinical diagnosis, appears to be the standard finding. Apart from these neuropsychiatric disturbances, platelet monoamine oxidase activity is raised in megaloblastic anaemia and reduced in iron deficiency anaemia. Although altered enzyme activity values may be linked to abnormal platelet populations in some of the haematological disorders discussed, in general the causes of abnormal platelet monoamine oxidase activity are unknown.

  8. Polyamines on the reproductive landscape.

    PubMed

    Lefèvre, Pavine L C; Palin, Marie-France; Murphy, Bruce D

    2011-10-01

    The polyamines are ubiquitous polycationic compounds. Over the past 40 yr, investigation has shown that some of these, namely spermine, spermidine, and putrescine, are essential to male and female reproductive processes and to embryo/fetal development. Indeed, their absence is characterized by infertility and arrest in embryogenesis. Mammals synthesize polyamines de novo from amino acids or import these compounds from the diet. Information collected recently has shown that polyamines are essential regulators of cell growth and gene expression, and they have been implicated in both mitosis and meiosis. In male reproduction, polyamine expression correlates with stages of spermatogenesis, and polyamines appear to function in promoting sperm motility. There is evidence for polyamine involvement in ovarian follicle development and ovulation in female mammals, and polyamine synthesis is required for steroidogenesis in the ovary. Studies of the embryo indicate a polyamine requirement that can be met from maternal sources before implantation, whereas elimination of polyamine synthesis abrogates embryo development at gastrulation. Polyamines play roles in embryo implantation, in decidualization, and in placental formation and function, and polyamine privation during gestation results in intrauterine growth retardation. Emerging information implicates dietary arginine and dietary polyamines as nutritional regulators of fertility. The mechanisms by which polyamines regulate these multiple and diverse processes are not yet well explored; thus, there is fertile ground for further productive investigation. PMID:21791568

  9. Spermidine oxidase-derived H₂O₂ regulates pollen plasma membrane hyperpolarization-activated Ca(2+) -permeable channels and pollen tube growth.

    PubMed

    Wu, Juyou; Shang, Zhonglin; Wu, Jun; Jiang, Xueting; Moschou, Panagiotis N; Sun, Wending; Roubelakis-Angelakis, Kalliopi A; Zhang, Shaoling

    2010-09-01

    Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca(2+) concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca(2+) current: the addition of Spd cannot induce the channel open probability increase in excised outside-out patches, indicating that the effect of Spd in the induction of Ca(2+) currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization-activated Ca(2+) -permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca(2+) -permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca(2+) -permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca(2+) influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd-derived H₂O₂ signals Ca(2+) influx, thereby regulating pollen tube growth.

  10. 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.

  11. 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.

  12. Use of polyamines as demulsifiers

    SciTech Connect

    Burkhardt, C.W.

    1983-10-25

    This invention relates to the use of polyamines and/or polyamine salts, preferably those having 20 or less amino groups, as demulsifiers. This invention is particularly useful in breaking emulsions formed in surfactant flooding of oil bearing subterranean formations.

  13. Fluorescence-activated sorting of rat hepatocytes based on their mixed function oxidase activities towards diethoxyfluorescein.

    PubMed Central

    White, I N; Green, M L; Legg, R F

    1987-01-01

    The formation of ethoxyfluorescein and fluorescein from diethoxyfluorescein by isolated rat hepatocytes has been used as a basis for separating such cells dependent on their mixed function oxidase activities by fluorescence-activated flow cytometry. Five equal fractions defined by computer-generated regions were isolated. Non-viable cells with low fluorescence (region 1) represented 10-15% of the population, while the remainder with higher mixed function oxidase activities (regions 2-5), were greater than 95% viable by Trypan Blue exclusion. In region 1, 30% of the viable cells were binucleate, 67% diploid while in region 5, 13% were binucleate and 69% tetraploid. At 3 h after sorting, following attachment to glass coverslips, exposure of cells to methyl methanesulphonate, retrorsine or norethindrone resulted in unscheduled DNA synthesis which was 2-fold higher in the tetraploid-rich region 5, while aflatoxin B1, benzo[a]pyrene or 2-acetylaminofluorene caused a 5-fold increase in unscheduled DNA synthesis in these cells, relative to the diploid-rich hepatocytes in region 2. Images Fig. 4. PMID:3689348

  14. Microglial NADPH oxidase activation mediates rod cell death in the retinal degeneration in rd mice.

    PubMed

    Zeng, H; Ding, M; Chen, X-X; Lu, Q

    2014-09-01

    Accumulating evidence supports that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes to microglia-mediated neurotoxicity in the CNS neurodegenerative diseases. Several studies, including ours, suggest that microglial activation is involved in the retinal degeneration in the animal models of retinitis pigmentosa (RP). In the present study, we investigated the activation of NADPH oxidase in the rod degeneration in rd mice and further explored its role in the microglia-mediated photoreceptor apoptosis. Expression of gp91phox protein, a major subunit of NAPDH oxidase in the whole retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16 and 18 was assessed by western blot analysis. Location of gp91phox in the rd retina at each age group and its cellular source were studied by immunohistochemical analysis and double labeling respectively. The generation of superoxide radicals in the rd retinas was demonstrated by intraperitoneal injection of hydroethidine. Apocynin was applied intraperitoneally in the rd mice from P8 to P14 to inhibit the activity of NAPDH oxidase and the outer nuclear layer (ONL) thickness was measured before and after apocynin treatment. Our results demonstrated that during the rod degenerative process, the expression of gp91phox started to increase in the outer part of rd retina at P10 and reached a peak at P14. Double labeling of gp91phox with CD11b showed co-localization of gp91phox in the retinal microglial cells. Increasing generation of superoxide radicals visualized by hydroethidine was noted at P8 and reached a peak at P14. Apocynin markedly reduced the production of superoxide radicals and preserved the rod cells. The results suggested that NADPH oxidase might play an important role in the rod degeneration in the rd mice. Inhibition of NAPDH oxidase could be a possible approach to treat RP in the early degenerative stage.

  15. 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

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

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    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.

  17. Activity of Monoamine Oxidase in the Nigrostriatal System at Presymptomatic and Early Symptomatic Stages of Parkinsonism in Mice.

    PubMed

    Khakimova, G R; Kozina, E A; Buneeva, O A; Aksenova, L N; Medvedev, A E; Ugryumov, M V

    2015-08-01

    Activities of monoamine oxidases A and B were examined on the models of presymptomatic and early symptomatic stages of Parkinson's disease developed in mice treated with MPTP, a specific neurotoxin affecting dopaminergic neurons. Activity of monoamine oxidases A, the key enzyme of dopamine degradation, is increased in neuronal somas during the symptomatic stage, and it is augmented in the axons during both stages. Neuronal activity of monoamine oxidases A is higher during the symptomatic stage than that during the presymptomatic stage, which can explain depletion of intercellular dopamine and appearance of motor disturbances. Activity of monoamine oxidase B in the striatum is reduced during the presymptomatic stage, but returns to the control level during the symptomatic stage. Variation in monoamine oxidase activity seems to reflect the compensatory mechanisms triggered in degrading nigrostriatal dopaminergic system.

  18. 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.

  19. Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

    PubMed

    Umezawa, Naoki; Horai, Yuhei; Imamura, Yuki; Kawakubo, Makoto; Nakahira, Mariko; Kato, Nobuki; Muramatsu, Akira; Yoshikawa, Yuko; Yoshikawa, Kenichi; Higuchi, Tsunehiko

    2015-08-17

    A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

  20. 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

  1. Artificial Warming and Rain Addition Increase Phenol Oxidase Activity in Arctic Soils

    NASA Astrophysics Data System (ADS)

    Kang, H.; Seo, J.; Jang, I.; Lee, Y. K.

    2014-12-01

    Artic tundra is one of the largest carbon stocks, of which amount is estimated up to 1,600 Pg. Global climate change models predict surface temperature rise and higher precipitation during summer in Arctic regions, raising concerns about faster decomposition of organic carbon and consequent releases of CO2, CH4 and DOC. Microorganisms are directly involved in decomposition process by releasing various extracellular enzymes. In particular, phenol oxidase was noted to play a key role because it is related to dynamics of highly recalcitrant carbon, which often represents a rate-limiting step of overall decomposition. In this study, we monitored phenol oxidase activity, hydrolases (β-glucosidase, cellobiohydrolase, N-acetylglucosaminidase and aminopeptidase), microbial abundance (qPCR) and chemical properties (δ13C and δ15N signatures) of tundra soils exposed to artificial warming and rain addition, by employing a passive chamber method in Cambridge Bay, Canada. Warming and rain addition combinedly increased phenol oxidase activity while no such changes were discernible for other hydrolases. Stable isotope signature indicates that warming induced water stress to the ecosystem and that nitrogen availability may be enhanced, which is partially responsible for the changes in enzyme activities. A short-term warming (2 years) may not accelerate mineralization of easily decomposable carbon, but may affect phenol oxidase which has the longer-term influence on recalcitrant carbon.

  2. 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

  3. 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.

  4. Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin.

    PubMed

    Abramovitch, D A; Marsh, D; Powell, G L

    1990-10-24

    Beef-heart cytochrome c oxidase lacking endogenous lipids can be prepared by cholate-mediated exchange with dimyristoylphosphatidylcholine (Powell, G. L., Knowles, P. F. and Marsh, D. (1985) Biochim. Biophys. Acta 816, 191-194). These preparations retained practically no endogenous cardiolipin (less than 0.19 mol cardiolipin per mol of oxidase) but in Tween 80 they retained unaltered electron transport activity. Resupplementation of the dimyristoylphosphatidylcholine-substituted cytochrome oxidase with cardiolipin and cardiolipin analogues with different numbers of acyl chains or with a methylated headgroup enhanced the activity of the reconstituted enzyme to an extent dependent on the structure of the cardiolipin derivative. The Eadie-Hofstee plot showed biphasic kinetic behavior for all reconstituted preparations, even those completely lacking cardiolipin. This biphasic substrate dependence of the kinetics was simulated using the model of Brzezinski, P. and Malmström, B. G. (Proc. Natl. Acad. Sci. USA 83 (1986) 4282-4286), which implicates two interconverting enzyme conformations in the proton transport step. The activation of cytochrome c oxidase by the cardiolipin analogues could be explained in terms of an electrostatic enhancement of the surface concentrations of both cytochrome c and protons, and a facilitated interconversion between the two enzyme conformations.

  5. Immunohistochemical Expression of Ornithine Decarboxylase, Diamine Oxidase, Putrescine, and Spermine in Normal Canine Enterocolic Mucosa, in Chronic Colitis, and in Colorectal Cancer

    PubMed Central

    Rossi, Giacomo; Cerquetella, Matteo; Pengo, Graziano; Mari, Subeide; Balint, Emilia; Bassotti, Gabrio; Manolescu, Nicolae

    2015-01-01

    We compared the immunohistochemical expression of putrescine (PUT), spermine (SPM), ornithine decarboxylase (ODC), and diamine oxidase (DAO) in bioptic samples of canine colonic mucosa with chronic inflammation (i.e., granulomatous colitis and lymphoplasmacytic colitis) or neoplasia. Single and total polyamines levels were significantly higher in neoplastic tissue than in normal samples. Samples with different degrees of inflammation showed a general decrease expression of ODC if compared to controls; SPM was practically not expressed in control samples and very low in samples with chronic-granulomatous inflammation. In carcinomatous samples, the ODC activity was higher with respect to controls and samples with inflammation. This is the first description of polyamines expression in dog colonic mucosa in normal and in different pathological conditions, suggesting that the balance between polyamine degradation and biosynthesis is evidently disengaged during neoplasia. PMID:26550563

  6. NADPH oxidase mediates β-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures

    PubMed Central

    Serrano, Faridis; Chang, Angela; Hernandez, Caterina; Pautler, Robia G; Sweatt, J David; Klann, Eric

    2009-01-01

    Background Previous studies have shown that beta amyloid (Aβ) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Aβ (1-42) or nicotine. Results We observed that Aβ and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Aβ and nicotine were blocked by inhibitors of NADPH oxidase. Conclusion Our findings indicate that NADPH oxidase-dependent redox signaling is required for Aβ-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimer's disease. PMID:19804648

  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. 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

  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

    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. 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

  12. 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

  13. Application of γ-aminobutyric acid demonstrates a protective role of polyamine and GABA metabolism in muskmelon seedlings under Ca(NO3)2 stress.

    PubMed

    Hu, Xiaohui; Xu, Zhiran; Xu, Weinan; Li, Jianming; Zhao, Ning; Zhou, Yue

    2015-07-01

    The effects of exogenous γ-aminobutyric acid (GABA) application on growth, polyamine and endogenous GABA metabolism in muskmelon leaves and roots were measured. Plants were treated under control or 80 mM Ca(NO3)2 stress conditions with or without foliar spraying 50 mM GABA. Ca(NO3)2 stress significantly suppressed seedling growth and GABA transaminase activity, and enhanced glutamate decarboxylase (GAD) activity and endogenous GABA levels. Polyamine (PA) biosynthesis and degradation capacity increased in parallel with increasing GAD activity. Exogenous GABA application effectively alleviated the growth inhibition caused by Ca(NO3)2 stress, and significantly enhanced the activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO). Exogenous GABA also significantly reduced the accumulation of free putrescine (Put) and increased the levels of free spermidine (Spd) and spermine (Spm) in leaves, which improved the capacity for polyamine biosynthesis. Application of exogenous GABA under Ca(NO3)2 stress enables the plants to maintain a higher ratio of free Spd and free Spm with respect to free Put. Our data suggest that exogenous GABA has an important role in improving muskmelon seedling tolerance to Ca(NO3)2 stress by improving biosynthesis of PAs and GABA, and by preventing PA degradation. There is a potential positive feedback mechanism that results from higher endogenous GABA content and the combined effects of Ca(NO3)2 stress and exogenous GABA, which coordinately alleviate Ca(NO3)2 stress injury by enhancing PA biosynthesis and converting free Put to an insoluble bound PA form, and reduce PA degradation in muskmelon seedlings.

  14. Pentamidine exerts in vitro and in vivo anti Trypanosoma cruzi activity and inhibits the polyamine transport in Trypanosoma cruzi.

    PubMed

    Díaz, María V; Miranda, Mariana R; Campos-Estrada, Carolina; Reigada, Chantal; Maya, Juan D; Pereira, Claudio A; López-Muñoz, Rodrigo

    2014-06-01

    Pentamidine is an antiprotozoal and fungicide drug used in the treatment of leishmaniasis and African trypanosomiasis. Despite its extensive use as antiparasitic drug, little evidence exists about the effect of pentamidine in Trypanosoma cruzi, the etiological agent of Chagas' disease. Recent studies have shown that pentamidine blocks a polyamine transporter present in Leishmania major; consequently, its might also block these transporters in T. cruzi. Considering that T. cruzi lacks the ability to synthesize putrescine de novo, the inhibition of polyamine transport can bring a new therapeutic target against the parasite. In this work, we show that pentamidine decreases, not only the viability of T. cruzi trypomastigotes, but also the parasite burden of infected cells. In T. cruzi-infected mice pentamidine decreases the inflammation and parasite burden in hearts from infected mice. The treatment also decreases parasitemia, resulting in an increased survival rate. In addition, pentamidine strongly inhibits the putrescine and spermidine transport in T. cruzi epimastigotes and amastigotes. Thus, this study points to reevaluate the utility of pentamidine and introduce evidence of a potential new action mechanism. In the quest of new therapeutic strategies against Chagas disease, the extensive use of pentamidine in human has led to a well-known clinical profile, which could be an advantage over newly synthesized molecules that require more comprehensive trials prior to their clinical use.

  15. Catechol oxidase activity of di-Cu2+-substituted aminopeptidase from Streptomyces griseus.

    PubMed

    da Silva, Giordano F Z; Ming, Li-June

    2005-11-30

    Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (kcat/Km) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities. PMID:16305209

  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. 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

  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.

  19. Wound healing response and xylem differentiation in tobacco plants over-expressing a fungal endopolygalacturonase is mediated by copper amine oxidase activity.

    PubMed

    Cona, Alessandra; Tisi, Alessandra; Ghuge, Sandip Annasaheb; Franchi, Stefano; De Lorenzo, Giulia; Angelini, Riccardo

    2014-09-01

    In this work, we have investigated the involvement of copper amine oxidase (CuAO; EC 1.4.3.21) in wound healing and xylem differentiation of Nicotiana tabacum plants over-expressing a fungal endopolygalacturonase (PG plants), which show constitutively activated defence responses. In petioles and stems of PG plants, we found higher CuAO activity and lower polyamine (PA) levels, particularly putrescine (Put), with respect to wild-type (WT) plants. Upon wounding, a more intense autofluorescence of cell wall phenolics was observed in correspondence of wound surface, extending to epidermis and cortical parenchima only in PG plants. This response was mostly dependent on CuAO activity, as suggested by the reversion of autofluorescence upon supply of 2-bromoethylamine (2-BrEt), a CuAO specific inhibitor. Moreover, in unwounded plants, histochemical analysis revealed a tissue-specific expression of the enzyme in the vascular cambium and neighboring derivative cells of both petioles and stems of PG plants, whereas the corresponding WT tissues appeared unstained or faintly stained. A higher histochemical CuAO activity was also observed in xylem cells of PG plants as compared to WT xylem tissues suggesting a peculiar role of CuAO activity in xylem differentiation in PG plants. Indeed, roots of PG plants exhibited early xylem differentiation, a phenotype consistent with both the higher CuAO and the lower Put levels observed and supported by the 2-BrEt-mediated reversion of early root xylem differentiation and H2O2 accumulation. These results strongly support the relevance of PA-catabolism derived H2O2 in defence responses, such as those signaled by a compromised status of cell wall pectin integrity.

  20. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

    PubMed

    Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

    2016-10-01

    Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process. PMID:27295021

  1. A perspective of polyamine metabolism.

    PubMed Central

    Wallace, Heather M; Fraser, Alison V; Hughes, Alun

    2003-01-01

    Polyamines are essential for the growth and function of normal cells. They interact with various macromolecules, both electrostatically and covalently and, as a consequence, have a variety of cellular effects. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homoeostasis argue that these amines are critical to cell survival. The regulation of polyamine content within cells occurs at several levels, including transcription and translation. In addition, novel features such as the +1 frameshift required for antizyme production and the rapid turnover of several of the enzymes involved in the pathway make the regulation of polyamine metabolism a fascinating subject. The link between polyamine content and human disease is unequivocal, and significant success has been obtained in the treatment of a number of parasitic infections. Targeting the polyamine pathway as a means of treating cancer has met with limited success, although the development of drugs such as DFMO (alpha-difluoromethylornithine), a rationally designed anticancer agent, has revolutionized our understanding of polyamine function in cell growth and provided 'proof of concept' that influencing polyamine metabolism and content within tumour cells will prevent tumour growth. The more recent development of the polyamine analogues has been pivotal in advancing our understanding of the necessity to deplete all three polyamines to induce apoptosis in tumour cells. The current thinking is that the polyamine inhibitors/analogues may also be useful agents in the chemoprevention of cancer and, in this area, we may yet see a revival of DFMO. The future will be in adopting a functional genomics approach to identifying polyamine-regulated genes linked to either carcinogenesis or apoptosis. PMID:13678416

  2. 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

    2016-03-02

    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.

  3. 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

  4. Polyamine metabolism in flax in response to treatment with pathogenic and non-pathogenic Fusarium strains.

    PubMed

    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

  5. 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.

  6. [Effect of lectins from Azospirillum brasilense to peroxidase and oxalate oxidase activity regulation in wheat roots].

    PubMed

    Alen'kina, S A; Nikitina, V E

    2010-01-01

    Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability oflectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect oflectin in under 10 min in a concentration of 10 microg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

  7. MYB8 Controls Inducible Phenolamide Levels by Activating Three Novel Hydroxycinnamoyl-Coenzyme A:Polyamine Transferases in Nicotiana attenuata[W][OA

    PubMed Central

    Onkokesung, Nawaporn; Gaquerel, Emmanuel; Kotkar, Hemlata; Kaur, Harleen; Baldwin, Ian T.; Galis, Ivan

    2012-01-01

    A large number of plants accumulate N-acylated polyamines (phenolamides [PAs]) in response to biotic and/or abiotic stress conditions. In the native tobacco (Nicotiana attenuata), the accumulation of two major PAs, caffeoylputrescine and dicaffeoylspermidine (DCS), after herbivore attack is known to be controlled by a key transcription factor, MYB8. Using a broadly targeted metabolomics approach, we show that a much larger spectrum of PAs composed of hydroxycinnamic acids and two polyamines, putrescine and spermidine, is regulated by this transcription factor. We cloned several novel MYB8-regulated genes, annotated as putative acyltransferases, and analyzed their function. One of the novel acyltransferases (AT1) is shown to encode a hydroxycinnamoyl-coenzyme A:putrescine acyltransferase responsible for caffeoylputrescine biosynthesis in tobacco. Another gene (acyltransferase DH29), specific for spermidine conjugation, mediates the initial acylation step in DCS formation. Although this enzyme was not able to perform the second acylation toward DCS biosynthesis, another acyltransferase gene, CV86, proposed to act on monoacylated spermidines, was isolated and partially characterized. The activation of MYB8 in response to herbivore attack and associated signals required the activity of LIPOXYGENASE3, a gene involved in jasmonic acid (JA) biosynthesis in N. attenuata. These new results allow us to reconstruct a complete branch in JA signaling that defends N. attenuata plants against herbivores: JA via MYB8’s transcriptional control of AT1 and DH29 genes controls the entire branch of PA biosynthesis, which allows N. attenuata to mount a chemically diverse (and likely efficient) defense shield against herbivores. PMID:22082505

  8. Determination of Diamine Oxidase in Lentil Seedlings by Enzymic Activity and Immunoreactivity

    PubMed Central

    Federico, Rodolfo; Angelini, Riccardo; Cesta, Alberinda; Pini, Carlo

    1985-01-01

    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 125I-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

  9. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    PubMed

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

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

    SciTech Connect

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

    2010-09-07

    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 (Dubois and Klinman (2006) Biochemistry 45, 3178), 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.

  11. Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase.

    PubMed

    Queisser, Nina; Schupp, Nicole; Stopper, Helga; Schinzel, Reinhard; Oteiza, Patricia I

    2011-12-01

    Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.

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

    PubMed

    Pils, D; Schmetterer, G

    2001-09-25

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

  13. Synthetic models of the active site of catechol oxidase: mechanistic studies.

    PubMed

    Koval, Iryna A; Gamez, Patrick; Belle, Catherine; Selmeczi, Katalin; Reedijk, Jan

    2006-09-01

    The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented. PMID:16936929

  14. Electrochemistry and chemiluminescence techniques compared in the detection of NADPH oxidase activity in phagocyte cells.

    PubMed

    Ashkenazi, A; Abu-Rabeah, K; Marks, R S

    2009-02-15

    Several methodologies have been used in clinical chemistry for real-time assessment of NADPH oxidase primary product superoxide anion which dismutases to hydrogen peroxide. Among these methodologies, isoluminol chemiluminescence (CL) is considered to be one of the more sensitive and reliable techniques for the assessment of NADPH oxidase activity in neutrophils. The electrochemical technique was recently designed and also applied for real-time detection of NADPH oxidase activity in neutrophils but its reliability and sensitivity has not been investigated so far. In this study, isoluminol CL and electrochemical techniques were investigated and compared by monitoring the generation of superoxide and hydrogen peroxide in both PLB 985 cell line differentiated into neutrophil-like cells and human neutrophils. The electrochemical technique was shown to be as sensitive as that of CL and able to detect the reactive oxygen species (ROS) release of as low as 500 cells. Thus, the electrochemical technique could be used as an alternative to optical techniques for the evaluation of extracellular ROS in phagocyte cells.

  15. 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

  16. Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

    PubMed

    Bacchi, C J; Lane, S; Weiss, L M; Yarlett, N; Takvorian, P; Cali, A; Wittner, M

    2001-01-01

    Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

  17. 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).

  18. Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

    PubMed

    Brekhman, Vera; Lugassie, Jennie; Zaffryar-Eilot, Shelly; Sabo, Edmond; Kessler, Ofra; Smith, Victoria; Golding, Hana; Neufeld, Gera

    2011-01-01

    Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

  19. A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

    PubMed

    Djajanegara, I; Holtzapffel, R; Finnegan, P M; Hoefnagel, M H; Berthold, D A; Wiskich, J T; Day, D A

    1999-07-01

    The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.

  20. Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

    NASA Astrophysics Data System (ADS)

    Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

    2013-11-01

    Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

  1. Plasma xanthine oxidase activity and lipid hydroperoxide levels in preterm infants.

    PubMed

    Supnet, M C; David-Cu, R; Walther, F J

    1994-09-01

    Ischemia-reperfusion injury may affect morbidity and mortality in preterm and asphyxiated term infants. Reoxygenation of hypoxic tissues leads to the formation of free oxygen radicals by xanthine oxidase that may induce lipid peroxidation, enzyme inhibition, and DNA strand breakage. We measured arterial cord blood samples from 36 healthy term infants for baseline values and arterial blood sampled at 1 and 4 h after birth from 45 preterm infants admitted for intensive care for serial estimates of plasma xanthine oxidase activity and lipid hydroperoxide levels. Mean +/- SEM plasma xanthine oxidase activity in cord blood of term infants was 2.3 +/- 0.4 mU/mL and lipid hydroperoxide levels were 2.6 +/- 0.3 nmol/mL. Eighteen of the 45 preterm infants met the criteria defining poor outcome (poor outcome group) and had lower umbilical arterial pH and base excess than the 27 preterm infants in the control group. Mean plasma xanthine oxidase activity increased from 2.7 +/- 0.4 at 1 h to 4.7 +/- 0.6 mU/mL at 4 h of age (p < 0.001) in the poor outcome group and decreased from 2.1 +/- 0.3 to 1.1 +/- 0.2 mU/mL (p = 0.004) in the control group. Lipid hydroperoxide levels in the poor outcome group increased from 2.8 +/- 0.6 nmol/mL at 1 h to 4.3 +/- 0.6 nmol/mL at 4 h of age (p < 0.001) and decreased from 2.1 +/- 0.6 to 1.6 +/- 0.2 nmol/mL (p = 0.008) in the control group. At 4 h of age, xanthine oxidase activity and lipid hydroperoxide levels were significantly higher in the poor outcome group than in the controls (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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

  3. Effect of heat stress on polyamine metabolism in proline-over-producing tobacco plants.

    PubMed

    Cvikrová, Milena; Gemperlová, Lenka; Dobrá, Jana; Martincová, Olga; Prásil, Ilja T; Gubis, Jozef; Vanková, Radomira

    2012-01-01

    The effect of heat stress on the accumulation of proline and on the level of polyamines (PAs) in tobacco plants was investigated. Responses to heat stress were compared in the upper and lower leaves and roots of tobacco plants that constitutively over-express a modified gene for the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CSF129A) and in the corresponding wild-type. In the initial phases of heat stress (after 2h at 40°C), the accumulation of proline increased in the wild type but slightly decreased in the transformants. The response to heat stress in proline-over-producing tobacco plants involved a transient increase in the levels of free and conjugated putrescine (Put) and in the levels of free spermidine (Spd), norspermidine (N-Spd) and spermine (Spm) after a 2-h lag phase, which correlated with stimulation of the activity of the corresponding biosynthetic enzymes. Diamine oxidase (DAO) activity increased in both plant genotypes, most significantly in the leaves of WT plants. Polyamine oxidase (PAO) activity increased in the roots of WT plants and decreased in the leaves and roots of the transformants. After 6h of heat stress, proline accumulation was observed in the transformants, especially in the lower leaves; much more modest increase was observed in the WT plants. A decrease in the levels of free and conjugated Put coincided with down-regulation of the activity of ornithine decarboxylase and marked stimulation of DAO activity in the leaves and roots of the transformants. PAO activity increased in the roots of the transformants but decreased in the leaves. Conversely, in WT tobacco subjected to 6h of heat stress, slight increases in free and conjugated PA levels were observed and the activity of DAO only increased in the roots; PAO activity did not change from the value observed during the initial phase of heat stress. 6 Hours' heat stress had no effect on the level of malondialdehyde (MDA; a product of lipid peroxidation), in

  4. Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity.

    PubMed

    Grönberg, L; Slotte, J P

    1990-04-01

    The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.

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

    PubMed

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems.

  6. Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

    SciTech Connect

    Ahluwalia, Jatinder

    2008-01-11

    Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

  7. Catalase, carbonic anhydrase and xanthine oxidase activities in patients with mycosis fungoides.

    PubMed

    Cengiz, Fatma Pelin; Beyaztas, Serap; Gokce, Basak; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-04-01

    Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma. In several studies the relationship between catalase (CAT), human cytosolic carbonic anhydrases (CA; hCA-I and hCA-II) and xanthine oxidase (XO) enzyme activities have been investigated in various types of cancers but carbonic anhydrase, catalase and xanthine oxidase activities in patients with MF have not been previously reported. Therefore, in this preliminary study we aim to investigate CAT, CA and XO activities in patients with MF. This study enrolled 32 patients with MF and 26 healthy controls. According to the results, CA and CAT activities were significantly lower in patients with mycosis fungoides than controls (p < 0.001) (p < 0.001). There was no significant difference in XO activity between patient and control group (p = 0.601). Within these findings, we believe these enzyme activity levels might be a potentially important finding as an additional diagnostic biochemical tool for MF.

  8. 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.

  9. Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿

    PubMed Central

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

    2011-01-01

    The Gram-negative intracellular pathogen Legionella pneumophilareplicates 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. pneumophilaexpressing a translocation reporter consisting of the Bordetella pertussisadenylate 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 SPE2induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophilamay require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophilareplication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophilaproliferation. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin.

    PubMed

    Campello, S; Beltramini, M; Giordano, G; Di Muro, P; Marino, S M; Bubacco, L

    2008-03-15

    The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly alpha-helical domain that directly binds the copper ions of the reaction center and a beta-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol. PMID:18237542

  16. Comparative role of polyamines in division and plastid differentiation of Euglena gracilis.

    PubMed

    Schuber, F; Aleksijevic, A; Blée, E

    1981-07-01

    Regulation of polyamine biosynthesis during growth and differentiation of Euglena gracilis was investigated. Increased activity of L-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotrophically or photoautotrophically grown cells. In photoautotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition form quiescence to active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decarboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena i.e. a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. Alpha-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.

  17. The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity

    PubMed Central

    Mandell, Gerald L.; Rubin, Walter; Hook, Edward W.

    1970-01-01

    Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN. Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and

  18. Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions.

    PubMed

    Ghuge, Sandip A; Tisi, Alessandra; Carucci, Andrea; Rodrigues-Pousada, Renato A; Franchi, Stefano; Tavladoraki, Paraskevi; Angelini, Riccardo; Cona, Alessandra

    2015-01-01

    Polyamines (PAs) are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs), including copper amine oxidases (CuAOs) and flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). The biologically-active hydrogen peroxide (H₂O₂) is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H₂O₂ biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H₂O₂ derived from PA oxidation in xylem tissue maturation under stress-simulated conditions. PMID:27135338

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

    PubMed

    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

    2015-09-01

    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.

  20. 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

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

    PubMed Central

    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

  2. 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

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

    PubMed

    Sluse, F E; Jarmuszkiewicz, W

    2000-03-01

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

  4. 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.

  5. 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.

  6. Polyamines and their derivatives as modulators in growth and differentiation.

    PubMed Central

    Canellakis, Z. N.; Marsh, L. L.; Bondy, P. K.

    1989-01-01

    The polyamines and their derivatives are essential for life in eukaryotic and most prokaryotic cells, but their exact role in preserving cell function is not clear. These polyamines provide endogenous cations and thus participate in regulation of the intracellular pH; in addition, polyamine derivatives modulate cell growth and differentiation. The naturally occurring monoacetyl derivatives can induce increased activity of ornithine decarboxylase, the first enzyme in polyamine synthesis, and thus produce positive feedback to their production. The diacetyl derivatives of putrescine and of the synthetic analogue, 1,6-diaminohexane, induce differentiation and inhibit growth in many types of cells in vitro. In addition, they inhibit the proliferative and secretory response of normal B lymphocytes to B-cell mitogens and reduce production of antibodies in vitro. They also inhibit the proliferation of chronic lymphocytic leukemia cells (a B-lymphocyte leukemia). The parent polyamines are post-translational modifiers of proteins, and hypusine, a derivative of spermidine, is a covalently bound constituent of the eukaryotic protein synthetic initiation factor, eIF-4D. Although these various actions do not at present fall into a coherent pattern, they clearly indicate that polyamines and their derivatives play an important part in modulating cell proliferation and differentiation. PMID:2697982

  7. 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

  8. 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

  9. 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

  10. Dinuclear copper complexes with imidazole derivative ligands: a theoretical study related to catechol oxidase activity.

    PubMed

    Martínez, Ana; Membrillo, Ingrid; Ugalde-Saldívar, Victor M; Gasque, Laura

    2012-07-19

    Catechol oxidase is a very important and interesting metalloprotein. In spite of the efforts to understand the reaction mechanism of this protein, there are important questions that remain unanswered concerning the catalytic mechanism of this enzyme. In this article, dinuclear copper compounds are used as biomimetic models of catechol oxidase to study plausible reaction paths. These dinuclear copper(II) complexes have distant metal centers (of 7.5 Å approximately) and superior catalytic activity to that of many dicopper complexes with shorter Cu-Cu distances. One mononuclear copper(II) complex is also analyzed in this investigation in order to see the influence of the two metal centers in the catalytic activity. Density functional theory calculations were performed to obtain optimized structures, vertical ionization energies, vertical electron affinities, the electrodonating power (ω(-)), the electroaccepting power (ω(+)) and the energy difference of several reaction paths. The K(M) experimental results that were previously reported compare well with the electroaccepting power (ω(+)) of the copper compounds that are included in this article, indicating that this index is useful for the interpretation of the electron transfer capacity and therefore the catalytic activity. The catechol moiety coordinates to only one Cu ion, but two metal atoms are needed in order to have a good electron acceptor capacity of the biomimetic models.

  11. 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.

  12. 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...

  13. D1-like receptors regulate NADPH oxidase activity and subunit expression in lipid raft microdomains of renal proximal tubule cells.

    PubMed

    Li, Hewang; Han, Weixing; Villar, Van Anthony M; Keever, Lindsay B; Lu, Quansheng; Hopfer, Ulrich; Quinn, Mark T; Felder, Robin A; Jose, Pedro A; Yu, Peiying

    2009-06-01

    NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats. PMID:19380616

  14. 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.

  15. 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

  16. NADPH oxidases promote apoptosis by activating ZNRF1 ubiquitin ligase in neurons treated with an exogenously applied oxidant

    PubMed Central

    Wakatsuki, Shuji; Araki, Toshiyuki

    2016-01-01

    ABSTRACT Reactive oxygen species (ROS) play an important role in causing neuronal death in a number of neurological disorders. We recently reported that ROS serve as a signal to activate neuronal apoptosis and axonal degeneration by activating ZNRF1 (zinc- and RING-finger 1), a ubiquitin ligase that targets AKT for proteasomal degradation in neurons. In the present study, we showed that the NADPH oxidase family of molecules is required for ZNRF1 activation by epidermal growth factor receptor (EGFR)-dependent phosphorylation in response to axonal injury. We herein demonstrate that NADPH oxidases promote apoptosis by activating ZNRF1, even in neurons treated with an exogenously applied oxidant. These results suggest an important role for NADPH oxidase in the initiation/promotion of neuronal degeneration by increasing ROS in close proximity to protein machineries, including those for ZNRF1 and EGFR, thereby promoting neuronal degeneration. PMID:27195063

  17. Identification of a mammalian vesicular polyamine transporter.

    PubMed

    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).

  18. Polyamines Can Increase Resistance of Neisseria gonorrhoeae to Mediators of the Innate Human Host Defense ▿

    PubMed Central

    Goytia, Maira; Shafer, William M.

    2010-01-01

    Polyamines are biogenic polycationic molecules involved in key cellular functions. Extracellular polyamines found in bodily fluids or laboratory media can be imported by bacteria or bind to negatively charged bacterial surface structures, where they can impair binding of antimicrobials. We hypothesized that the presence of polyamines in fluids that bathe urogenital mucosal surfaces could alter the susceptibility of the sexually transmitted strict human pathogen Neisseria gonorrhoeae to mediators of the innate host defense. Herein we report that polyamines can significantly increase gonococcal resistance to two structurally diverse cationic antimicrobial peptides (polymyxin B and LL-37) but not to antibiotics that exert activity in the cytosol or periplasm (e.g., ciprofloxacin, spectinomycin, or penicillin). The capacity of polyamines to increase gonococcal resistance to cationic antimicrobial peptides was dose dependent, correlated with the degree of cationicity, independent of a polyamine transport system involving the polyamine permeases PotH and PotI, and was reversible. In addition, we found that polyamines increase gonococcal resistance to complement-mediated killing by normal human serum. We propose that polyamines in genital mucosal fluids may enhance gonococcal survival during infection by reducing bacterial susceptibility to host-derived antimicrobials that function in innate host defense. PMID:20439477

  19. 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

  20. 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

  1. Solvent exchangeable protons and the activation of molecular oxygen: the galactose oxidase reaction.

    PubMed

    Kosman, D J; Driscoll, J J

    1988-01-01

    The reduction of O2 to H2O2 requires two protons as well as two electrons. Thus, activation of dioxygen reasonably may involve either general or specific acid catalysis. Consequently, the reduction of O2 to H2O2 could exhibit a kinetic solvent isotope effect (KSIE). The reaction catalyzed by the mononuclear Cu(II) enzyme, galactose oxidase does exhibit a KSIE (+1.55). The pL-rate profile exhibits an alkaline shift in D2O which can be attributed to the differential partitioning of H+ versus D+ between bulk water and a metal-bound H2O (delta pKa = +0.19). A variety of spectral evidence places an equatorial, Cu(II)-liganded water molecule at the active site of galactose oxidase. The analysis of the KSIE data is detailed and the potential generality of the function of such metal-bound H2O at other type 2 Cu(II) sites is discussed.

  2. 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.

  3. Activities of dl-α-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model▿

    PubMed Central

    Yarlett, Nigel; Waters, W. Ray; Harp, James A.; Wannemuehler, Michael J.; Morada, Mary; Bellcastro, Josephine; Upton, Steve J.; Marton, Laurence J.; Frydman, Benjamin J.

    2007-01-01

    The in vivo effectiveness of a series of conformationally restricted polyamine analogues alone and selected members in combination with dl-α-difluoromethylarginine against Cryptosporidium parvum infection in a T-cell receptor alpha-deficient mouse model was tested. Polyamine analogues were selected from the extended bis(ethyl)-sym-homospermidine or bis(ethyl)-spermine backbone having cis or trans double bonds at the center of the molecule. The cis isomers were found to have significantly greater efficacy in both preventing and curing infection in a mouse model than the trans polyamine analogues when tested in a T-cell receptor alpha-deficient mouse model. When tested in combination with dl-α-difluoromethylarginine, the cis-restricted analogues were found to be more effective in preventing oocyst shedding. This study demonstrates the potential of polyamine analogues as anticryptosporidial agents and highlights the presence of multiple points in polyamine synthesis by this parasite that are susceptible to inhibition resulting in growth inhibition. PMID:17242149

  4. 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

  5. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    PubMed

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling. PMID:21327819

  6. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    PubMed

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

  7. 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.

  8. Synthesis of some novel hydrazone and 2-pyrazoline derivatives: monoamine oxidase inhibitory activities and docking studies.

    PubMed

    Evranos-Aksöz, Begüm; Yabanoğlu-Çiftçi, Samiye; Uçar, Gülberk; Yelekçi, Kemal; Ertan, Rahmiye

    2014-08-01

    A novel series of 2-pyrazoline and hydrazone derivatives were synthesized and investigated for their human monoamine oxidase (hMAO) inhibitory activity. All compounds inhibited the hMAO isoforms (MAO-A or MAO-B) competitively and reversibly. With the exception of 5i, which was a selective MAO-B inhibitor, all derivatives inhibited hMAO-A potently and selectively. According to the experimental Ki values, compounds 6e and 6h exhibited the highest inhibitory activity towards the hMAO-A, whereas compound 5j, which carries a bromine atom at R(4) of the A ring of the pyrazoline, appeared to be the most selective MAO-A inhibitor. Tested compounds were docked computationally into the active site of the hMAO-A and hMAO-B isozymes. The computationally obtained results were in good agreement with the corresponding experimental values.

  9. Cytochrome oxidase activity is increased in +/Lc Purkinje cells destined to die.

    PubMed

    Vogel, M W; Fan, H; Sydnor, J; Guidetti, P

    2001-10-01

    +/Lc Purkinje cells degenerate postnatally because of a gain-of-function mutation in the delta2 glutamate receptor (Grid2) that causes a constitutive Na+ current leak. The effect of the resulting chronic depolarization on Purkinje cell metabolism was investigated by measuring levels of cytochrome oxidase (COX) activity in Purkinje cell dendrites using quantitative densitometry. Analysis of wild type controls and +/Lc mutants at P10, P15 and P25 showed that levels of COX activity were significantly increased above control levels by P15 and continued to increase through P25. The increase in COX activity is likely to reflect an increase in oxidative phosphorylation to accommodate the energy demands of removing excess Na+ and Ca2+ entering the Purkinje cells in response to the Grid2 leak current.

  10. 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

  11. (/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.

  12. A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells.

    PubMed

    Wagner, A M

    1995-07-17

    Incubation of Petunia hybrida cells with H2O2 leads to an increase in alternative oxidase activity measured after 24 h. This increased activity is accompanied by an increase in alternative oxidase protein. A model is presented for the regulation of alternative oxidase protein synthesis in which active oxygen species and especially H2O2 play a crucial role as second messengers in the signal transducing pathway from the mitochondria to the nucleus. It is proposed that also the induction of the alternative oxidase by salicylic acid is mediated via H2O2.

  13. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    PubMed

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-01

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

  14. 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

  15. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays.

    PubMed

    Hayat, Akhtar; Cunningham, Jessica; Bulbul, Gonca; Andreescu, Silvana

    2015-07-23

    Nanomaterial-based enzyme mimics have attracted considerable interest in chemical analysis as alternative catalysts to natural enzymes. However, the conditions in which such particles can replace biological catalysts and their selectivity and reactivity profiles are not well defined. This work explored the oxidase like properties of nanoceria particles in the development of colorimetric assays for the detection of dopamine and catechol. Selectivity of the system with respect to several phenolic compounds, the effect of interferences and real sample analysis are discussed. The conditions of use such as buffer composition, selectivity, pH, reaction time and particle type are defined. Detection limits of 1.5 and 0.2μM were obtained with nanoceria for dopamine and catechol. The same assay could be used as a general sensing platform for the detection of other phenolics. However, the sensitivity of the method varies significantly with the particle type, buffer composition, pH and with the structure of the phenolic compound. The results demonstrate that nanoceria particles can be used for the development of cost effective and sensitive methods for the detection of these compounds. However, the selection of the particle system and experimental conditions is critical for achieving high sensitivity. Recommendations are provided on the selection of the particle system and reaction conditions to maximize the oxidase like activity of nanoceria. PMID:26231899

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

    PubMed

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

    2015-12-16

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

  17. 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

  18. Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

    PubMed Central

    Speed, R R; Winkler, H H

    1990-01-01

    Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14CO2 released per h per 10(8) rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine. PMID:2120188

  19. 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

  20. 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...

  1. Polyamine Metabolism Changes in Psoriasis

    PubMed Central

    Broshtilova, Valentina; Lozanov, Valentina; Miteva, Ljubka

    2013-01-01

    Introduction: Polyamines – putrescine, spermidine and spermine are polycationic compounds ubiquitous for all living organisms. They are essential for the cell growth and differentiation, the control of cell cycle progress, apoptosis, and cancerogenesis. Accumulated scientific evidence suggests the central role of polyamines in the process of keratinocytic proliferation, differentiation, and regulation. Objective: To elucidate the polyamine metabolic changes that occur in benign keratinocytic proliferation. Fifty eight patients were enrolled in the study, 31 with plaque-form of psoriasis vulgaris, which had been referred to as a model of benign keratinocytic proliferation, and 27-healthy controls. Materials and Methods: An original, innovative chromatographic method was used to detect the levels of putrescine, spermidine, and spermine in all skin samples. Results: Were significantly proven (P < 0.05). No difference was found between the polyamines levels of non-lesional psoriatic skin and healthy controls. Psoriatic lesions showed a two-time higher concentration of all polyamines in lesional, compared to non-lesional skin. Spermine had the highest concentration and highest proliferation trend, which demonstrated the importance of propylamine synthesis in the pathogenesis of psoriasis. Spermine highest concentrations suggested the leading role of adenosine methionine decarboxylase (AMDC) in the pathogenesis of benign keratinocytic proliferations. Conclusions: Non-lesional skin in psoriatic patients did not show latent changes in polyamine metabolism. Psoriatic lesions demontrated two-time higher levels of the most essential biogenic polyamines compared to healthy controls. The highest level of spermine proved the crucial role of AMDC in the polyamine metabolism changes in psoriasis. Future therapeutic approaches should be focused on reduction of exogenic spermine intake, utilizing new spermine blockers, and synthesis of AMDC inhibitors. PMID:23919004

  2. Longan seed extract reduces hyperuricemia via modulating urate transporters and suppressing xanthine oxidase activity.

    PubMed

    Hou, Chien-Wei; Lee, Ying-Chung; Hung, Hsiao-Fang; Fu, Hua-Wen; Jeng, Kee-Ching

    2012-01-01

    Hyperuricemia causes gouty arthritis, kidney disease, heart disease, and other diseases. Xanthine oxidase (XOD) and urate transporters play important roles in urate homeostasis. Numerous plants have been identified as XOD inhibitors. Longan seeds are known to contain high levels of polyphenols such as corilagin, gallic acid and ellagic acid. We examined the effect of longan seed extract on XOD inhibition and urate transporters GLUT1 and GLUT9 using both in vitro and in vivo assays. The results showed that dried longan seed extract (LSE) and its active components inhibited XOD dose-dependently in vitro. LSE inhibited uric acid production and XOD activity in normal liver cells (clone-9 cells) and was not cytotoxic under the concentration of 200 μg/ml. For the in vivo study, Sprague-Dawley (SD) rats were given intraperitoneally for thirty minutes with or without allopurinol (a XOD inhibitor, 3.5 mg/kg) or LSE (80 mg/kg) and then injected intraperitioneally with 250 mg/kg of oxonic acid and 300 mg/kg of hypoxanthine intragastrically. LSE was able to reduce serum uric acid level and XOD activity in hyperuricemic rats. However, LSE or allopurinol did not inhibit the liver XOD activities. On the other hand, GLUT1 protein was suppressed in kidney and GLUT9 was induced in liver from experimental rats and LSE or allopurinol decreased GLUT9 but increased GLUT1 protein level in the liver and kidney, respectively. These results confirmed the claimed effect of longan seeds on gout and other complications and suggested that its urate reducing effect might be due to modulation of urate transporters and inhibition of circulating xanthine oxidase.

  3. 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.

  4. Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.

    PubMed

    Rodriguez, Hector M; Vaysberg, Maria; Mikels, Amanda; McCauley, Scott; Velayo, Arleene C; Garcia, Carlos; Smith, Victoria

    2010-07-01

    In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

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

    PubMed

    Banerjee, Soumyabrata; Poddar, Mrinal K

    2015-03-01

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

  6. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    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 lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  7. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    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 lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. PMID:24594397

  8. 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.

  9. 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

  10. 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

  11. Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities.

    PubMed

    Birkholtz, Lyn-Marie; Williams, Marni; Niemand, Jandeli; Louw, Abraham I; Persson, Lo; Heby, Olle

    2011-09-01

    New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.

  12. Polyphenols decreased liver NADPH oxidase activity, increased muscle mitochondrial biogenesis and decreased gastrocnemius age-dependent autophagy in aged rats.

    PubMed

    Laurent, Caroline; Chabi, Beatrice; Fouret, Gilles; Py, Guillaume; Sairafi, Badie; Elong, Cecile; Gaillet, Sylvie; Cristol, Jean Paul; Coudray, Charles; Feillet-Coudray, Christine

    2012-09-01

    This study explored major systems of reactive oxygen species (ROS) production and their consequences on oxidative stress, mitochondriogenesis and muscle metabolism in aged rats, and evaluated the efficiency of 30-day oral supplementation with a moderate dose of a red grape polyphenol extract (RGPE) on these parameters. In the liver of aged rats, NADPH oxidase activity was increased and mitochondrial respiratory chain complex activities were altered, while xanthine oxidase activity remained unchanged. In muscles, only mitochondrial activity was modified with aging. The oral intake of RGPE decreased liver NADPH oxidase activity in the aged rats without affecting global oxidative stress, suggesting that NADPH oxidase was probably not the dominant detrimental source of production of O(2)·(-) in the liver. Interestingly, RGPE supplementation increased mitochondrial biogenesis and improved antioxidant status in the gastrocnemius of aged rats, while it had no significant effect in soleus. RGPE supplementation also decreased age-dependent autophagy in gastrocnemius of aged rats. These results extended existing findings on the beneficial effects of RGPE on mitochondriogenesis and muscle metabolism in aged rats.

  13. Effects of fructooligosaccharides on cecum polyamine concentration and gut maturation in early-weaned piglets

    PubMed Central

    Sabater-Molina, María; Larqué, Elvira; Torrella, Francisco; Plaza, Javier; Ramis, Guillermo; Zamora, Salvador

    2011-01-01

    Polyamines are molecules involved in cell growth and differentiation and are produced by bacterial metabolism. However, their production and effects by the microbiota selected by fructooligosaccharides consumption are controversial. In this study, we investigated the influence of supplementation of fructooligosaccharides on the cecal polyamine production by the microflora selected, and its effect on gut maturation in newborn piglets. Twenty piglets were fed a control formula (n = 10) or a formula supplemented with fructooligosaccharides (8 g/l) (n = 10) for 13 days. Colony-forming unit’s count of cecal content was done in different media. Several intestinal development parameters were measured as well as the polyamine concentration in the cecal mucosa and cecal content. A dose-dependent study on in vitro polyamine production by fructooligosaccharides addition to the isolated cecal content was performed. Bifidogenic activity of fructooligosaccharides increased polyamine concentration in the cecal content, mainly putrescine, with no beneficial effect on gut maturation. Bifidobacterium spp. were able to produce polyamines, but they were not the most significant bacterial producer of polyamines in the cecum of piglets fed fructooligosaccharides. Bifidogenic activity of fructooligosaccharides did not lead to an increase in gut maturation in piglets of 15 days of age although polyamines were increased in the cecal content. PMID:21562644

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. Activation of lactoperoxidase system in milk by glucose oxidase immobilized in electrospun polylactide microfibers.

    PubMed

    Zhou, Y; Lim, L-T

    2009-03-01

    In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX-containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N-dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX-in-PLA fibers (1100 +/- 400 nm diameter) was more than 19 times higher than that of the GOX-in-PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN(-) than direct activation using H(2)O(2). Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.

  19. Activation of lactoperoxidase system in milk by glucose oxidase immobilized in electrospun polylactide microfibers.

    PubMed

    Zhou, Y; Lim, L-T

    2009-03-01

    In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX-containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N-dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX-in-PLA fibers (1100 +/- 400 nm diameter) was more than 19 times higher than that of the GOX-in-PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN(-) than direct activation using H(2)O(2). Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk. PMID:19323732

  20. Chronic effect of insulin on monoamine oxidase and antioxidant enzyme activities in the rat brainstem.

    PubMed

    Radojićić, R; Cvijić, G; Djordjević, J; Djurasević, S; Davidović, V

    1997-06-01

    It was shown that hydrogen peroxide (H2O2) is a possible intracellular second messenger in specific insulin action. Because its concentration in the cell depends on the activity of both antioxidant enzymes and monoamine oxidase (MAO), we studied the influence of different insulin doses (0.4 and 4.0 IU/kg body mass, i.p., daily injected over 3 days) on the activity of MAO, types A and B, copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase in the rat brainstem. Chronic insulin treatment significantly increased Vmax of MAO-A and B activities (P < 0.05, P < 0.025, respectively) independent of the dose applied. CuZnSOD activity was also increased (P < 0.025), but only when higher dose of hormone was injected. However, insulin had the opposite effect on MnSOD and catalase causing a decrease in their activities (P < 0.005). The observed changes in the activities of the enzymes studied are possible compensations that potentially maintain an optimal H2O2 level in the brainstem, which might be important for insulin action.

  1. 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.

  2. 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

  3. Polyphenol oxidase activity as a potential intrinsic index of adequate thermal pasteurization of apple cider.

    PubMed

    Chen, L; Ingham, B H; Ingham, S C

    2004-05-01

    In response to increasing concerns about microbial safety of apple cider, the U.S. Food and Drug Administration has mandated treatment of cider sufficient for a 5-log reduction of the target pathogen. Pasteurization has been suggested as the treatment most likely to achieve a 5-log reduction, with Escherichia coli O157:H7 as the target pathogen. Regulators and processors need a reliable method for verifying pasteurization, and apple cider polyphenol oxidase (PPO) activity was studied as a potential intrinsic index for thermal pasteurization. The effect of pasteurization conditions and apple cider properties on PPO activity and survival of three pathogens (E. coli O157:H7, Salmonella, and Listeria monocytogenes) was studied using a Box-Behnken response surface design. Factors considered in the design were pasteurization conditions, i.e., hold temperature (60, 68, and 76 degrees C), preheat time (10, 20, 30 s), and hold time (0, 15, 30 s), pH, and sugar content ((o)Brix) of apple cider. Response surface contour plots were constructed to illustrate the effect of these factors on PPO activity and pathogen survival. Reduction in PPO activity of at least 50% was equivalent to a 5-log reduction in E. coli O157:H7 or L. monocytogenes for cider at pH 3.7 and 12.5 (o)Brix. Further studies, however, are needed to verify the relationship between PPO activity and pathogen reduction in cider with various pH and (o)Brix values.

  4. 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.

  5. 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

  6. 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

  7. Reduced mitochondria cytochrome oxidase activity in adult children of mothers with Alzheimer's disease.

    PubMed

    Mosconi, Lisa; de Leon, Mony; Murray, John; E, Lezi; Lu, Jianghua; Javier, Elizabeth; McHugh, Pauline; Swerdlow, Russell H

    2011-01-01

    Biomarker studies demonstrate inheritance of glucose hypometabolism and increased amyloid-β deposition in adult offspring of mothers, but not fathers, affected by late-onset Alzheimer's disease (LOAD). The underlying genetic mechanisms are unknown. We investigated whether cognitively normal (NL) individuals with a maternal history of LOAD (MH) have reduced platelet mitochondrial cytochrome oxidase activity (COX, electron transport chain complex IV) compared to those with paternal (PH) or negative family history (NH). Thirty-six consecutive NL individuals (age 55 ± 15 y, range 27-71 y, 56% female, CDR = 0, MMSE ≥28, 28% APOE-4 carriers), including 12 NH, 12 PH, and 12 MH, received a blood draw to measure platelet mitochondrial COX activity. Citrate synthase activity (CS) was measured as a reference. Groups were comparable for clinical and neuropsychological measures. We found that after correcting for CS, COX activity was reduced by 29% in MH compared to NH, and by 30% in MH compared to PH (p ≤ 0.006). Results remained significant controlling for age, gender, education, and APOE. No differences were found between PH and NH. COX measures discriminated MH from the other groups with accuracy ≥75%, and relative risk ≥3 (p ≤ 0.005). Among NL with LOAD-parents, only those with MH showed reduced COX activity in platelet mitochondria compared to PH and NH. The association between maternal history of LOAD and systemic COX reductions suggests transmission via mitochondrial DNA, which is exclusively maternally inherited in humans.

  8. The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus.

    PubMed

    Semeykina, A L; Skulachev, V P; Verkhovskaya, M L; Bulygina, E S; Chumakov, K M

    1989-08-15

    +-motive terminal oxidase activity. PMID:2776760

  9. 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

  10. 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

  11. 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.

  12. Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells.

    PubMed

    Itoh, Tomohiro; Terazawa, Riyako; Kojima, Keitaro; Nakane, Keita; Deguchi, Takashi; Ando, Masashi; Tsukamasa, Yasuyuki; Ito, Masafumi; Nozawa, Yoshinori

    2011-09-01

    This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells. PMID:21682664

  13. 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.

  14. Monoamine oxidase (MAO) inhibitory activity: 3-phenylcoumarins versus 4-hydroxy-3-phenylcoumarins.

    PubMed

    Delogu, Giovanna L; Serra, Silvia; Quezada, Elias; Uriarte, Eugenio; Vilar, Santiago; Tatonetti, Nicholas P; Viña, Dolores

    2014-08-01

    Monoamine oxidase (MAO) is a useful target in the treatment of neurodegenerative diseases and depressive disorders. Both isoforms, MAO-A and MAO-B, are known to play critical roles in disease progression, and as such, the identification of novel, potent and selective inhibitors is an important research goal. Here, two series of 3-phenylcoumarin derivatives were synthesized and evaluated against MAO-A and MAO-B. Most of the compounds tested acted preferentially on MAO-B, with IC50 values in the micromolar to nanomolar range. Only 6-chloro-4-hydroxy-3-(2'-hydroxyphenyl)coumarin exhibited activity against the MAO-A isoform, while still retaining good selectivity for MAO-B. 6-Chloro-3-phenylcoumarins unsubstituted at the 4 position were found to be more active as MAO-B inhibitors than the corresponding 4-hydroxylated coumarins. For 4-unsubstituted coumarins, meta and para positions on the 3-phenyl ring seem to be the most favorable for substitution. Molecular docking simulations were used to explain the observed hMAO-B structure-activity relationships for this type of compound. 6-Chloro-3-(3'-methoxyphenyl)coumarin was the most active compound identified (IC50=0.001 μM) and is several times more potent and selective than the reference compound, R-(-)-deprenyl hydrochloride. This compound represents a novel tool for the further investigation of the therapeutic potential of MAO-B inhibitors.

  15. Prehepatic portal hypertension induces alterations in cytochrome oxidase activity in the rat adrenal gland.

    PubMed

    López, Laudino; Aller, Maria-Angeles; Miranda, Ruben; Sánchez-Patán, Fernando; Nava, Maria-Paz; Arias, Jaime; Arias, Jorge-Luis

    2006-01-01

    One approach to assess neuroendocrine response to portal hypertension in short-term portal vein-stenosed rats consists in studying metabolic and functional activity patterns in adrenal glands using mitochondrial enzyme cytochrome c oxidase (COX) as a histochemical marker. Male Wistar rats were divided into two groups: a control group (Group I; n = 8), in which the animals did not undergo any operative intervention, and a triple calibrated portal vein stenosis group (TPVS) (Group II; n = 7). The sections of suprarenal glands were histochemically stained for COX and the optical densitometry was measured by a computer image analyzer attached to a microscope. In TPVS rats, COX activity in the adrenal gland cortex is lower than in control rats and affects the fascicular (52.30, 47.16-60.98, vs. 67.12, 60.31-73.89, p = .002), glomerular (49.68, 46.19-53.56 vs. 70.47, 64.64-73.51, p < .001), and reticular (47.35, 35.63-54.39, vs. 55.37, 49.76-58.97; p < .05) layers. In contrast, COX activity in the adrenal gland medulla is similar in TPVS rats and in control rats (29.91, 29.54-31.18, vs. 29.67, 28.95-30.23). The changes in adrenocortical COX activity in short-term-TPVS rats could constitute a pathogenic factor for both splanchnic and systemic hyperdynamic circulations, described in this experimental model of prehepatic portal hypertension.

  16. 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.

  17. 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

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

    PubMed

    Calabriso, Nadia; Massaro, Marika; Scoditti, Egeria; D'Amore, Simona; Gnoni, Antonio; Pellegrino, Mariangela; Storelli, Carlo; De Caterina, Raffaele; Palasciano, Giuseppe; Carluccio, Maria Annunziata

    2016-02-01

    Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1-10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.

  19. 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

  20. Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

    PubMed

    Mu, D; Janes, S M; Smith, A J; Brown, D E; Dooley, D M; Klinman, J P

    1992-04-25

    The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

  1. 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

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

    PubMed

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

    2016-05-05

    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.

  3. Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene

    SciTech Connect

    Virbasius, J.V.; Scarpulla, R.C. )

    1991-11-01

    A mutational analysis of the rat cytochrome c oxidase subunit IV (RCO4) promoter region revealed the presence of a major control element consisting of a tandemly repeated pair of binding sites for a nuclear factor from HeLa cells. This factor was designated NRF-2 (nuclear respiratory factor 2) because a functional recognition site was also found in the human ATP synthase {beta}-subunit gene. Deletion or site-directed point mutations of the NRF-2 binding sites in the RCO4 promoter resulted in substantial loss of transcriptional activity, and synthetic oligomers of the NRF-2 binding sites from both genes stimulated a heterologous promoter when cloned in cis. NRF-2 binding a transcriptional activation required a purine-rich core sequence, GGAA. This motif is characteristic of the recognition site for a family of activators referred to as ETS domain proteins because of the similarity within their DNA-binding domains to the ets-1 proto-oncogene product. NRF-2 recognized an authentic Ets-1 site within the Moloney murine sarcoma virus long terminal repeat, and this site was able to compete for NRF-2 binding to the RCO4 promoter sequence. However, in contrast to Ets-1, which appears to be exclusive to lymphoid tissues, NRF-2 has the broad tissue distribution expected of a regulator of respiratory chain expression.

  4. Cluster B personality disorders are associated with allelic variation of monoamine oxidase A activity.

    PubMed

    Jacob, Christian P; Müller, Johannes; Schmidt, Michael; Hohenberger, Katrin; Gutknecht, Lise; Reif, Andreas; Schmidtke, Armin; Mössner, Rainald; Lesch, Klaus Peter

    2005-09-01

    Genetic variants of the monoamine oxidase A (MAOA) have been associated with aggression-, anxiety-, and addiction-related behavior in several nonclinical and clinical populations. Here, we investigated the influence of allelic variation of MAOA activity on aggression-related personality traits and disease risk in patients with personality disorders. Personality disorders were diagnosed with the Structured Clinical Interview of DSM-IV and were allocated to cluster A, B, and C. Personality features were assessed by the revised NEO Personality Inventory and the Tridimensional Personality Questionnaire. The genotype of the MAOA gene-linked polymorphic region (MAOA-LPR) was determined in 566 patients with personality disorders and in 281 healthy controls. MAOA genotype was significantly associated with cluster B personality disorders (chi2=7.77, p=0.005, df=1) but not with cluster C personality disorders. In total, 26.0% of cluster B patients were hemi- or homozygous for the low-activity variant of the MAOA genotype, compared to 16.4% in the control group. Associations between MAOA variants and personality domains related to impulsivity and aggressiveness were inconsistent. Our findings further support the notion that allelic variation of MAOA activity contributes modestly to the balance of hyper- (impulsive-aggressive) and hyporeactive (anxious-depressive) traits.

  5. Activation of defense mechanism in wheat by polyphenol oxidase from aphid saliva.

    PubMed

    Ma, Rui; Chen, Ju-Lian; Cheng, Deng-Fa; Sun, Jing-Rui

    2010-02-24

    The saliva of two cereal aphids, Sitobion avenae and Schizaphis graminum in third-instar nymphs, was collected after 24 h of feeding by 30 aphids, separately, on artificial diet sachets, and the salivary enzymes were determined. The result showed that polyphenol oxidase (PPO) existed in the saliva of both aphid species, and the enzymatic activities were 6.2 x 10(-3) U/g for S. avenae and 2.37 x 10(-1) U/g for S. graminum, revealing a 38-fold higher activity in the saliva of S. graminum than in the saliva of S. avenae. It was speculated that the higher PPO activity in S. graminum saliva was a contributing factor to the light yellow spot left on the feeding site of the wheat leaf by S. graminum; no such spot was left by S. avenae. After treatment of a wheat seedling with the saliva of S. avenae and S. graminum and PPO at the concentration of aphid saliva, transcript profiling data showed that aphid saliva and PPO significantly induced expression of the genes aos and fps. Because genes aos and fps encode the key enzymes in the defense signal pathways jasmonic acid and terpene signal pathways, respectively, it was deduced that PPO from aphid saliva, as the main elicitor, triggers an appropriate defense response in wheat through jasmonic acid and terpene signal pathways. PMID:20112908

  6. 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

  7. Reduced activity of monoamine oxidase in the rat brain following repeated nandrolone decanoate administration.

    PubMed

    Birgner, Carolina; Kindlundh-Högberg, Anna M S; Oreland, Lars; Alsiö, Johan; Lindblom, Jonas; Schiöth, Helgi B; Bergström, Lena

    2008-07-11

    Anabolic androgenic steroids (AAS) are known as doping agents within sports and body-building, but are currently also abused by other groups in society in order to promote increased courage and aggression. We previously showed that 14 days of daily intramuscular injections of the AAS nandrolone decanoate (15 mg/kg) reduced the extracellular levels of the dopaminergic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens shell using microdialysis. The aim of the present study was to investigate whether the same dose regimen of nandrolone decanoate may affect the activities of the dopamine-metabolizing enzymes monoamine oxidases A and B (MAO-A and MAO-B). A radiometric assay was used to determine the activities of MAO-A and MAO-B in rat brain tissues after 14 days of daily i.m. nandrolone decanoate injections at the doses 3 and 15 mg/kg. Gene transcript contents of MAO-A, MAO-B and cathecol-O-methyltransferase (COMT) were measured with quantitative real-time reverse transcription PCR. 3 mg/kg of nandrolone decanoate significantly reduced the activity of both MAO-A and -B in the caudate putamen. 15 mg/kg of nandrolone decanoate significantly reduced the activity of MAO-A in the amygdala and increased the gene transcript level of MAO-B in the substantia nigra. In conclusion, imbalanced MAO activities may contribute to explain the impulsive and aggressive behaviour often described in AAS abusers. The reduced MAO activities observed are in line with our previously presented findings of decreased extracellular levels of DOPAC and HVA in the rat brain, indicating decreased monoaminergic activity following repeated AAS administration. PMID:18539264

  8. 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

  9. 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.

  10. The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity.

    PubMed

    Lim, Yong-beom; Mays, Charles E; Kim, Younghwan; Titlow, William B; Ryou, Chongsuk

    2010-03-01

    Branched polyamines are effective in inhibiting prions in a cationic surface charge density dependent manner. However, toxicity associated with branched polyamines, in general, often hampers the successful application of the compounds to treat prion diseases. Here, we report that constitutively maintained cationic properties in branched polyamines reduced the intrinsic toxicity of the compounds while retaining the anti-prion activities. In prion-infected neuroblastoma cells, quaternization of amines in polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimers markedly increased the nontoxic concentration ranges of the compounds and still supported, albeit reduced, an appreciable level of anti-prion activity in clearing prions from the infected cells. Furthermore, quaternized PEI was able to degrade prions at acidic pH conditions and inhibit the in vitro prion propagation facilitated by conversion of the normal prion protein isoform to its misfolded counterpart, although such activities were decreased by quaternization. Quaternized PAMAM was least effective in degrading prions but efficiently inhibited prion conversion with the same efficacy as unmodified PAMAM. Our results suggest that quaternization represents an effective strategy for developing nontoxic branched polyamines with potent anti-prion activity. This study highlights the importance of polyamine structural control for developing polyamine-based anti-prion agents and understanding of an action mechanism of quaternized branched polyamines.

  11. 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.

  12. 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

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

    PubMed Central

    Cederbaum, Arthur I.

    2014-01-01

    The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed. PMID:25454746

  16. Potent and Selective Monoamine Oxidase-B Inhibitory Activity: Fluoro- vs. Trifluoromethyl-4-hydroxylated Chalcone Derivatives.

    PubMed

    Mathew, Bijo; Mathew, Githa Elizabeth; Uçar, Gülberk; Baysal, Ipek; Suresh, Jerad; Mathew, Sincy; Haridas, Abitha; Jayaprakash, Venkatesan

    2016-08-01

    For various neurodegenerative disorders like Alzheimer's and Parkinson's diseases, selective and reversible MAO-B inhibitors have a great therapeutic value. In our previous study, we have shown that a series of methoxylated chalcones with F functional group exhibited high binding affinity toward human monoamine oxidase-B (hMAO-B). In continuation of our earlier study and to extend the understanding of the structure-activity relationships, a series of five new chalcones were studied for their inhibition of hMAO. The results demonstrated that these compounds are reversible and selective hMAO-B inhibitors with a competitive mode of inhibition. The most active compound, (2E)-1-(4-hydroxyphenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one, exhibited a Ki value of 0.33 ± 0.01 μm toward hMAO-B with a selectivity index of 26.36. A molecular docking study revealed that the presence of a H-bond network in hydroxylated chalcone with the N(5) atom of FAD is crucial for MAO-B selectivity and potency.

  17. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  18. Lysyl oxidase: Influence of dietary copper on accumulation land functional activity in rat skin

    SciTech Connect

    Romero-Chapman, N.; Tinker, D.; Uriu-Hare, J.; Keen C.L.; Gacheru, S.; Rucker, R.B. )

    1991-03-11

    Lysyl oxidase (Lys. Ox.) functions extracellularly and catalyzes the oxidative deamination of peptidyl lysine in collagen and elastin. Lys. Ox. was purified 150- to 175-fold from urea extracts of rat skin and uteri. Both {sup {minus}}40 and {sup {minus}}32 kDa polypeptide chains could be isolated from rat skin with apparent Lys. Ox. activity. Antibodies raised in chickens against the {sup {minus}}40 kDa form of Lys. Ox. detected the {sup {minus}}32 kDa form in immunoblots. Consequently, it is inferred that the {sup {minus}}32 kDa form of Lys. Ox. is processed from the {sup {minus}}40 kDa form of the enzyme. Antibodies were also used to prepare antirat Lys. Ox. affinity columns to separate Lys. Ox. from other proteins. Sixteen hours after an oral dose of Cu-67, about 6-8% of the Cu-67 was incorporated into rat skin was found in associated with Lys. Ox. The Lys. Ox. concentration in rat skin was 2.5 to 7.5 nmoles (determined by enzyme liked immunosorption assays). Changing the Cu status by feeding a diet low in Cu did not influence Lys. Ox. accumulation or the % age of Cu-67 in skin as Lys. Ox. However, Lys. Ox. function activity was one-third normal values in rats deprived of Cu.

  19. 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

  20. 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

  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. Enzymatic activity of cholesterol oxidase immobilized onto polymer nanoparticles mediated by Congo red.

    PubMed

    Silva, Rubens A; Carmona-Ribeiro, Ana Maria; Petri, Denise F S

    2013-10-01

    Poly(ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized by means of dynamic light scattering (DLS), zeta (ζ) potential measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption of Congo red (CR) onto PS/PEG particles was evidenced by the decrease of ζ potential values and increase in the particles mean diameter in comparison to bare particles. Cholesterol oxidase (ChOx), the main enzyme in the oxidation of cholesterol, adsorbed onto PS/PEG and PS/PEG/CR particles, as revealed by the increase in the particles mean size and spectrophotometry. The enzymatic activity of free and immobilized ChOx was determined as a function of time by means of a coupled reaction with horseradish peroxidase. The activity of free ChOx decreased with time, while the activity of immobilized ChOx increased with time; after 1h reaction the latter was half of the former. Freeze-drying the ChOx covered PS/PEG/CR particles allowed their storage for at least one month under room conditions without loss of enzymatic activity. Conjugation effects between CR and ChOx or cholesterol evidenced by circular dichroism and spectrophotometry rendered a conformational state of ChOx, such that the enzymatic action was favored. ChOx adsorbed onto PS/PEG presents no enzymatic activity, probably due to ChOx denaturation or unfavorable orientation. Freeze-dried and freshly prepared dispersions of ChOx immobilized onto PS/PEG/CR particles yielded linear response in the cholesterol concentration range of 100mgdL(-1) (lowest limit of normal blood concentration) to 300mgdL(-1) (high risk level).

  4. A high-performance-liquid-chromatographic method for the assay of coproporphyrinogen oxidase activity in rat liver.

    PubMed Central

    Li, F; Lim, C K; Peters, T J

    1986-01-01

    An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy. PMID:3814086

  5. 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

  6. 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

  7. 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

  8. 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

  9. High alternative oxidase activity in cold soils and its implication to the Dole Effect

    NASA Astrophysics Data System (ADS)

    Angert, Alon; Rodeghiero, Mirco; Griffin, Kevin

    2012-08-01

    Variations in the Dole Effect, which have been used to infer past changes in biospheric productivity, are strongly affected by isotopic discrimination in soil respiration. Respiration through the alternative oxidase (AOX) pathway is associated with a higher discrimination than the one associated with the “normal” dark respiration pathway (the cytochrome pathway, COX). However, observations of O2 discrimination and AOX activity in undisturbed natural environments are scarce. In the current study we measured the O2 concentration and stable isotopes in the root zone of tundra, boreal forest and alpine forest soils. To estimate the discrimination from this data, we have performed O2 diffusion experiments in gamma-sterilized soil columns, with varying soil clay content. The discrimination found in the diffusion experiments was independent of clay content, and the value found, 14 ± 2‰, is the same as the one for binary diffusion of O2 in N2, indicating no interaction between the O2 and clay particles. Based on the field and laboratory results, the respiratory discrimination in the soils studied is 15-31‰, with the higher values associated with colder soils. The high discrimination found for cold (<6°C) soils indicates that AOX is a major respiratory pathway in these soils. This relationship between soil temperature and discrimination can be used in future interpretations of Dole Effect variations.

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

    PubMed

    Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

    2014-12-01

    In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

  11. 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

  12. 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

  13. 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

  14. 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.

  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. 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.

  17. Polyamines may regulate S-phase progression but not the dynamic changes of chromatin during the cell cycle.

    PubMed

    Laitinen, J; Stenius, K; Eloranta, T O; Hölttä, E

    1998-02-01

    Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. PMID:9443076

  18. Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

    PubMed Central

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

    2014-01-01

    Introduction Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. Aims We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. Methods SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Main Outcome Measures Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Results Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Conclusion Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. PMID:24251665

  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. 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

  1. Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation.

    PubMed

    Tisi, Alessandra; Federico, Rodolfo; Moreno, Sandra; Lucretti, Sergio; Moschou, Panagiotis N; Roubelakis-Angelakis, Kalliopi A; Angelini, Riccardo; Cona, Alessandra

    2011-09-01

    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 G(1) and G(2) 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,N(1)-dimethylthiourea, a hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H(2)O(2) derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.

  2. 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

  3. 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.

  4. Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars.

    PubMed

    Chang, S; Tan, C; Frankel, E N; Barrett, D M

    2000-02-01

    The antioxidant potential of eight clingstone peach cultivars was investigated by determining phenolic compounds and inhibition of low-density lipoprotein (LDL) oxidation. Cultivars low in polyphenol oxidase (PPO) were also selected to minimize enzymatic browning. Inhibition of LDL oxidation varied from 17.0 to 37.1% in peach flesh extract, from 15.2 to 49.8% in whole peach extract, and from 18.2 to 48.1% in peel extract. Total phenols were 432.8-768.1 mg/kg in flesh extract, 483.3-803.0 mg/kg in whole extract, and 910.9-1922.9 mg/kg in peel extract. The correlation coefficient between relative LDL antioxidant activity and concentration of total phenols was 0.76. Peel PPO activity was higher than flesh activity in most cultivars. The lowest PPO and specific activities were found in the Walgant cultivar, followed by Kakamas and 18-8-23. These three cultivars combine the desirable characteristics of strong antioxidant activity, low PPO activity, and lower susceptibility to browning reactions.

  5. 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

  6. Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site.

    PubMed

    Hoeser, Jo; Hong, Sangjin; Gehmann, Gerfried; Gennis, Robert B; Friedrich, Thorsten

    2014-05-01

    Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

  7. Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity.

    PubMed

    Song, Ju Dong; Lee, Sang Kwon; Park, Si Eun; Kim, Kang Mi; Kim, Koanhoi; Park, Yeong Min; Park, Young Chul

    2011-11-01

    Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

  8. 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.

  9. 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

  10. 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.

  11. Pulsed EPR Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

    PubMed Central

    Klein, Eric L.; Belaidi, Abdel Ali; Raitsimring, Arnold M.; Davis, Amanda C.; Krämer, Tobias; Astashkin, Andrei V.; Neese, Frank; Schwarz, Günter; Enemark, John H.

    2014-01-01

    Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of the density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO variant are dominated by the ‘inter-doublet’ transition arising from the strong nuclear quadrupole interaction, as also occurs for the 33S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al., Inorg. Chem. 2012, 51, 1408 – 1418]. The estimated experimental hfi and nqi parameters for 33S (aiso = 3 MHz and e2Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two 33S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V). PMID:24387640

  12. 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

  13. 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

  14. 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.

  15. The crosslinking of polysaccharides with polyamines and dextran-polyallylamine antibacterial hydrogels.

    PubMed

    O'Connor, Naphtali A; Abugharbieh, Ahmad; Yasmeen, Farzana; Buabeng, Emmanuel; Mathew, Steve; Samaroo, Diana; Cheng, Hai-Ping

    2015-01-01

    A facile modular approach to rapidly prepare pH-responsive hydrogels by crosslinking polysaccharides with polyamines is demonstrated. Hydrogels are prepared by first reacting the less reactive polysaccharides with the cross-linker epichlorohydrin and completed by the addition of polyamines. The crosslinking of polysaccharides with polyamines provides a facile method for incorporating functionality into polysaccharide based hydrogels. This process is demonstrated with the polysaccharides dextran, pullulan and carboxymethyl cellulose and with the polyamines polyallylamine and polyethylene imine. The hydrogels were characterized by FTIR and swelling studies, which showed pH-dependent swelling due to the presence of the polyamine. The hydrogels can also be tailored by varying the mass ratio between the polysaccharide and polyamine. Absorption studies of organic analytes showed the polyamine content affecting the uptake of a charged substrate (methylene blue) and no effect on a neutral substrate (6-methyl coumarin). This synthetic method was also used to prepare hydrogels with antibacterial activity against E. coli and S. aureus by utilizing an amphiphilic polyallylamine. PMID:25128095

  16. 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

  17. Polyamine biosynthesis is critical for growth and differentiation of the pancreas.

    PubMed

    Mastracci, Teresa L; Robertson, Morgan A; Mirmira, Raghavendra G; Anderson, Ryan M

    2015-08-24

    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.

  18. The crosslinking of polysaccharides with polyamines and dextran-polyallylamine antibacterial hydrogels.

    PubMed

    O'Connor, Naphtali A; Abugharbieh, Ahmad; Yasmeen, Farzana; Buabeng, Emmanuel; Mathew, Steve; Samaroo, Diana; Cheng, Hai-Ping

    2015-01-01

    A facile modular approach to rapidly prepare pH-responsive hydrogels by crosslinking polysaccharides with polyamines is demonstrated. Hydrogels are prepared by first reacting the less reactive polysaccharides with the cross-linker epichlorohydrin and completed by the addition of polyamines. The crosslinking of polysaccharides with polyamines provides a facile method for incorporating functionality into polysaccharide based hydrogels. This process is demonstrated with the polysaccharides dextran, pullulan and carboxymethyl cellulose and with the polyamines polyallylamine and polyethylene imine. The hydrogels were characterized by FTIR and swelling studies, which showed pH-dependent swelling due to the presence of the polyamine. The hydrogels can also be tailored by varying the mass ratio between the polysaccharide and polyamine. Absorption studies of organic analytes showed the polyamine content affecting the uptake of a charged substrate (methylene blue) and no effect on a neutral substrate (6-methyl coumarin). This synthetic method was also used to prepare hydrogels with antibacterial activity against E. coli and S. aureus by utilizing an amphiphilic polyallylamine.

  19. 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

  20. Design, synthesis, and biological evaluation of semicarbazide-sensitive amine oxidase (SSAO) inhibitors with anti-inflammatory activity.

    PubMed

    Wang, Eric Y; Gao, Hongfeng; Salter-Cid, Luisa; Zhang, Jun; Huang, Li; Podar, Erika M; Miller, Andrew; Zhao, Jingjing; O'rourke, Anne; Linnik, Matthew D

    2006-04-01

    In an attempt to examine the effect of inhibition of semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6, also known as VAP-1) as a novel anti-inflammatory target, the structure/mechanism based design and synthesis of a series of novel hydrazino-containing small molecules are described. The in vitro biological results show that compounds 4a,c are highly potent SSAO inhibitors with notable selectivity toward SSAO over monoamine oxidases A and B (MAO-A and MAO-B). SAR studies based on compound 4c were performed, and the results are discussed. The most potent and selective compound, 4a (IC(50) = 2 nM), is an orally active, competitive, and apparently irreversible inhibitor of SSAO that is effective at reducing disease incidence and severity in an in vivo animal disease model of multiple sclerosis.

  1. 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

  2. Polyamine Metabolism in Potassium-Deficient Bacteria

    PubMed Central

    Rubenstein, Kenneth E.; Streibel, Ellen; Massey, Sarah; Lapi, Lillie; Cohen, Seymour S.

    1972-01-01

    The metabolism of polyamines was studied in K+-dependent strains of Escherichia coli. When these stringent organisms were in a medium containing Na+ instead of K+, protein synthesis was arrested, but synthesis of ribonucleic acid continued as it would in a relaxed organism. The Na+ medium inhibited synthesis of spermidine and S-adenosylmethionine. However, the synthesis of putrescine was accelerated at least five- to eightfold. Exogenous ornithine doubled even this rate of putrescine synthesis but did not increase the low level of putrescine synthesis in the K+ medium. In K+ or Na+ media, with or without 0.3 mm arginine, putrescine was derived almost entirely from ornithine via ornithine decarboxylase. Addition of spermidine (5 mm) to a Na+ culture markedly inhibited putrescine synthesis. The ornithine decarboxylase of an extract of a K−-dependent strain prepared at low ionic strength was separated from ribosomes, deoxyribonucleic acid, and associated polyamines by centrifugation, and from many ions by ultrafiltration and fractionation on Sephadex G-100. Addition of Na+ and K+ salts to 200 mm was markedly inhibitory. The combined reductions both in synthesis of the inhibitor spermidine and in intracellular ionic strength may explain the in vivo activation of this enzyme. PMID:4565534

  3. 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

  4. 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.

  5. A spectrophotometric assay for monoamine oxidase activity with 2, 4-dinitrophenylhydrazine as a derivatized reagent.

    PubMed

    Huang, Guili; Zhu, Fei; Chen, Yuhang; Chen, Shiqiang; Liu, Zhonghong; Li, Xin; Gan, Linlin; Zhang, Li; Yu, Yu

    2016-11-01

    A simple, rapid and reliable spectrophotometry was developed to determine monoamine oxidase (MAO). In this study, 2,4-dinitrophenylhydrazine (DNPH), a classic derivatizing reagent, was used to detect MAO-dependent aldehyde production; and traditional DNPH spectrophotometry was simplified. Benzylamine and serotonin oxidation were catalyzed by MAO-B and MAO-A, respectively, to aldehydes. These were derivatized with DNPH, and the corresponding quinones were further formed by adding NaOH. These DNPH derivatives with large conjugated structures were directly measured spectrophotometrically at 465 nm and 425 nm, without the need for precipitating, washing and suspending procedures. The addition of NaOH caused a red shift of the maximum absorption wavelength of these derivatives, which reduced the interference of free DNPH. MAO-B protein was as low as 47.5 μg in rat liver with correlation coefficients ranging within 0.995-0.999. This method is 2-3 times more sensitive than direct spectrophotometry. The detection of MAO inhibition through this method showed that IC50 values of rasagiline are 8.00 × 10(-9) M for MAO-B and 2.59 × 10(-7) M for MAO-A. These results are similar to the values obtained by direct spectrophotometry. Our study suggests that DNPH spectrophotometry is suitable to detect MAO activity, and has the potential for MAO inhibitor screening in the treatment of MAO-mediated diseases.

  6. Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling

    PubMed Central

    Lin, Fan; Ding, Haidong; Wang, Jinxiang; Zhang, Hong; Zhang, Aying; Zhang, Yun; Tan, Mingpu; Dong, Wen; Jiang, Mingyi

    2009-01-01

    In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling. PMID:19592501

  7. 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.

  8. 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

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

    PubMed

    Sergutina, A V; Rakhmanova, V I

    2016-06-01

    Monoamine oxidase activity was quantitatively assessed by cytochemical method in brain structures (layers III and V of the sensorimotor cortex, caudate nucleus, nucleus accumbens, hippocampal CA3 field) of rats of August line and Wistar population with high and low locomotor activity in the open fi eld test. Monoamine oxidase activity (substrate tryptamine) predominated in the nucleus accumbens of Wistar rats with high motor activity in comparison with rats with low locomotor activity. In August rats, enzyme activity (substrates tryptamine and serotonin) predominated in the hippocampus of animals with high motor activity. Comparison of August rats with low locomotor activity and Wistar rats with high motor activity (i.e. animals demonstrating maximum differences in motor function) revealed significantly higher activity of the enzyme (substrates tryptamine and serotonin) in the hippocampus of Wistar rats. The study demonstrates clear-cut morphochemical specificity of monoaminergic metabolism based on the differences in the cytochemical parameter "monoamine oxidase activity", in the studied brain structures, responsible for the formation and realization of goal-directed behavior in Wistar and August rats.

  10. The dual actions of Paederia scandens extract as a hypouricemic agent: xanthine oxidase inhibitory activity and uricosuric effect.

    PubMed

    Yan, Haiyan; Ma, Ying; Liu, Mei; Zhou, Lanlan

    2008-09-01

    Hyperuricemia is associated with a number of pathological conditions, such as gout. Lowering of elevated uric acid levels in the blood could be achieved by xanthine oxidase inhibitors and inhibitors of renal urate reabsorption. Some natural compounds isolated from herbs used in traditional Chinese medicine have been previously demonstrated to act as xanthine oxidase inhibitors. In the present investigation, Paederia scandens (Lour.) Merrill (Rubiaceae) extract (PSE; 4.5, 2.25, and 1.125 g/kg) orally for 14 days was demonstrated to possess in vivo potent hypouricemic activity in hyperuricemic rats pretreated with potassium oxonate. In addition, PSE was also demonstrated to be an inhibitor of xanthine oxidase. Lineweaver-Burk analysis of the enzyme kinetics indicated that the inhibition of PSE was of a mixed type. Using an oxonate-induced hyperuricemic rat model, PSE was indeed shown to exhibit uricosuric action in vivo, which could explain, at least in part, the observed hypouricemic effect of PSE in these rats. The potential application of this compound in the treatment of conditions associated with hyperuricemia is discussed.

  11. 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.

  12. 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

  13. Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

    SciTech Connect

    Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

    2010-10-01

    The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by {alpha}-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

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

    PubMed

    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. PMID:27417759

  15. 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. PMID:27417759

  16. Fulvene-5 inhibition of Nadph oxidases attenuates activation of epithelial sodium channels in A6 distal nephron cells.

    PubMed

    Trac, David; Liu, Bingchen; Pao, Alan C; Thomas, Sheela V; Park, Michael; Downs, Charles A; Ma, He-Ping; Helms, My N

    2013-10-01

    Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel antioxidant drugs, such as Nox4 inhibitor compounds, are being developed. There is, however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single-channel patch-clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04 ± 0.18 (means ± SE) to 0.25 ± 0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic cell stretch response of A6 cells, indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertension. PMID:23863470

  17. Antioxidant effect of imperatorin from Angelica dahurica in hypertension via inhibiting NADPH oxidase activation and MAPK pathway.

    PubMed

    Cao, Yanjun; Zhang, Yanmin; Wang, Nan; He, Langchong

    2014-08-01

    Imperatorin (IMP) is an active furocoumarin in the traditional Chinese medicine Angelica dahurica and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of IMP on blood pressure (BP) and antioxidant effects in spontaneously hypertensive rats (SHR) and human embryonic kidney 293 cells. SHR were administered IMP (6.25, 12.5, and 25 mg/kg/d) or tempol (18 mg/kg/d) daily by gavage for 12 weeks. Thiobarbituric acid-reactive substances, proteinuria levels, and superoxide dismutase activity were evaluated with commercial kits. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits of the renal cortical tissues were determined by reverse transcriptase polymerase chain reaction and Western blot. Twenty-four hour urinary 8-Iso-prostaglandin F2α was measured by enzyme linked immunosorbent assay. Systolic BP and diastolic BP were significantly reduced by treatment with IMP (6.25, 12.5, and 25 mg/kg/d) in SHR. Meanwhile, we found that renal cortical superoxide dismutase activities were significantly increased in IMP-treated groups. Renal cortical and urinary thiobarbituric acid-reactive substances' levels, the 24-hour urinary excretion of 8-Iso-prostaglandin F2α, and proteinuria in the IMP-treated group, were lower than SHR group. After that, we found the messenger RNA expressions and protein levels of NADPH oxidase subunits were markedly reduced after IMP treated in SHR. IMP also reduced the phosphorylation of protein kinase B, extracellular signal-regulated kinase1/2, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase in renal cortical in SHR. In addition, H2O2-induced ROS production in human embryonic kidney 293 cells was markedly attenuated by IMP. H2O2-induced activation of MAPK, protein kinase B, and expression of NADPH oxidase were also attenuated by pretreatment of IMP. In summary, IMP showed antihypertensive effect via prevention of renal injury not only by reducing NADPH

  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. 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

  20. 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

  1. 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.

  2. Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite.

    PubMed Central

    Fraser, Alison V; Woster, Patrick M; Wallace, Heather M

    2002-01-01

    Human promyelogenous leukaemic cells (HL-60) were treated with novel spermine analogue, ( S )- N (1)-(2-methyl-1-butyl)- N (11)-ethyl-4,8-diazaundecane (IPENSpm), and the effects on growth and intracellular polyamine metabolism were measured. IPENSpm was cytotoxic to these cells at concentrations greater than 2.5 microM. It induced apoptosis in a caspase-dependent manner and its toxicity profile was comparable with etoposide, a well-known anti-tumour agent and inducer of apoptosis. IPENSpm decreased intracellular polyamine content as a result of changes in ornithine decarboxylase activity and increases in spermidine/spermine N(1)-acetyltransferase and polyamine export. Analysis showed spermine and spermidine as the major intracellular polyamines, while putrescine and acetyl-polyamines were the main export compounds. IPENSpm used the polyamine transporter system for uptake and its accumulation in cells was prevented by polyamine transport inhibitors. IPENSpm can be classified as a polyamine anti-metabolite and it may be a promising new lead compound in terms of treatment of some human cancers. PMID:12086584

  3. 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

  4. Impaired Pulmonary NF-κB Activation in Response to Lipopolysaccharide in NADPH Oxidase-Deficient Mice

    PubMed Central

    Koay, M. Audrey; Christman, John W.; Segal, Brahm H.; Venkatakrishnan, Annapurna; Blackwell, Thomas R.; Holland, Steven M.; Blackwell, Timothy S.

    2001-01-01

    Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-κB. We investigated the role of NADPH oxidase in the NF-κB activation pathway by utilizing knockout mice (p47phox−/−) lacking the p47phox component of NADPH oxidase. Wild-type (WT) controls and p47phox−/− mice were treated with intraperitoneal (i.p.) Escherichia coli lipopolysaccharide (LPS) (5 or 20 μg/g of body weight). LPS-induced NF-κB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox−/− mice 90 min after treatment with 20 but not 5 μg of i.p. LPS per g. In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox−/− mice compared to WT mice following treatment with aerosolized LPS. In contrast to NF-κB activation in p47phox−/− mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice. We conclude that LPS-induced NF-κB activation is deficient in the lungs of p47phox−/− mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response. PMID:11553535

  5. Xanthoangelol and 4-Hydroxyderricin Are the Major Active Principles of the Inhibitory Activities against Monoamine Oxidases on Angelica keiskei K

    PubMed Central

    Kim, Ji Ho; Son, Yeon Kyung; Kim, Gun Hee; Hwang, Keum Hee

    2013-01-01

    Monoamine oxidase inhibitors (MAOI) have been widely used as antidepressants. Recently, there has been renewed interest in MAO inhibitors. The activity-guided fractionation of extracts from Angelica keiskei Koidzumi (A. keiskei K.) led to the isolation of two prenylated chalcones, xanthoangelol and 4-hydroxyderricin and a flavonoid, cynaroside. These three isolated compounds are the major active ingredients of A. keiskei K. to inhibit the MAOs and DBH activities. Xanthoangelol is a nonselective MAO inhibitor, and a potent dopamine β-hydroxylase (DBH) inhibitor. IC50 values of xanthoangelol to MAO-A and MAO-B were calculated to be 43.4 μM, and 43.9 μM. These values were very similar to iproniazid, which is a nonselective MAO inhibitor used as a drug against depression. The IC50 values of iproniazid were 37 μM, and 42.5 μM in our parallel examination. Moreover, IC50 value of xanthoangelol to DBH was calculated 0.52 μM. 4-Hydroxyderricin is a potent selective MAO-B inhibitor and also mildly inhibits DBH activity. The IC50 value of 4-hydroxyderricin to MAO-B was calculated to be 3.43 μM and this value was higher than that of deprenyl (0.046 μM) used as a positive control for selective MAO-B inhibitor in our test. Cynaroside is a most potent DBH inhibitor. The IC50 value of cynaroside to DBH was calculated at 0.0410 μM. Results of this study suggest that the two prenylated chalcones, xanthoangelol and 4-hydroxyderricin isolated from A. keiskei K., are expected for potent candidates for development of combined antidepressant drug. A. keiskei K. will be an excellent new bio-functional food material that has the combined antidepressant effect. PMID:24265870

  6. Xanthoangelol and 4-Hydroxyderricin Are the Major Active Principles of the Inhibitory Activities against Monoamine Oxidases on Angelica keiskei K.

    PubMed

    Kim, Ji Ho; Son, Yeon Kyung; Kim, Gun Hee; Hwang, Keum Hee

    2013-05-30

    Monoamine oxidase inhibitors (MAOI) have been widely used as antidepressants. Recently, there has been renewed interest in MAO inhibitors. The activity-guided fractionation of extracts from Angelica keiskei Koidzumi (A. keiskei K.) led to the isolation of two prenylated chalcones, xanthoangelol and 4-hydroxyderricin and a flavonoid, cynaroside. These three isolated compounds are the major active ingredients of A. keiskei K. to inhibit the MAOs and DBH activities. Xanthoangelol is a nonselective MAO inhibitor, and a potent dopamine β-hydroxylase (DBH) inhibitor. IC50 values of xanthoangelol to MAO-A and MAO-B were calculated to be 43.4 μM, and 43.9 μM. These values were very similar to iproniazid, which is a nonselective MAO inhibitor used as a drug against depression. The IC50 values of iproniazid were 37 μM, and 42.5 μM in our parallel examination. Moreover, IC50 value of xanthoangelol to DBH was calculated 0.52 μM. 4-Hydroxyderricin is a potent selective MAO-B inhibitor and also mildly inhibits DBH activity. The IC50 value of 4-hydroxyderricin to MAO-B was calculated to be 3.43 μM and this value was higher than that of deprenyl (0.046 μM) used as a positive control for selective MAO-B inhibitor in our test. Cynaroside is a most potent DBH inhibitor. The IC50 value of cynaroside to DBH was calculated at 0.0410 μM. Results of this study suggest that the two prenylated chalcones, xanthoangelol and 4-hydroxyderricin isolated from A. keiskei K., are expected for potent candidates for development of combined antidepressant drug. A. keiskei K. will be an excellent new bio-functional food material that has the combined antidepressant effect.

  7. Xanthoangelol and 4-Hydroxyderricin Are the Major Active Principles of the Inhibitory Activities against Monoamine Oxidases on Angelica keiskei K.

    PubMed

    Kim, Ji Ho; Son, Yeon Kyung; Kim, Gun Hee; Hwang, Keum Hee

    2013-05-30

    Monoamine oxidase inhibitors (MAOI) have been widely used as antidepressants. Recently, there has been renewed interest in MAO inhibitors. The activity-guided fractionation of extracts from Angelica keiskei Koidzumi (A. keiskei K.) led to the isolation of two prenylated chalcones, xanthoangelol and 4-hydroxyderricin and a flavonoid, cynaroside. These three isolated compounds are the major active ingredients of A. keiskei K. to inhibit the MAOs and DBH activities. Xanthoangelol is a nonselective MAO inhibitor, and a potent dopamine β-hydroxylase (DBH) inhibitor. IC50 values of xanthoangelol to MAO-A and MAO-B were calculated to be 43.4 μM, and 43.9 μM. These values were very similar to iproniazid, which is a nonselective MAO inhibitor used as a drug against depression. The IC50 values of iproniazid were 37 μM, and 42.5 μM in our parallel examination. Moreover, IC50 value of xanthoangelol to DBH was calculated 0.52 μM. 4-Hydroxyderricin is a potent selective MAO-B inhibitor and also mildly inhibits DBH activity. The IC50 value of 4-hydroxyderricin to MAO-B was calculated to be 3.43 μM and this value was higher than that of deprenyl (0.046 μM) used as a positive control for selective MAO-B inhibitor in our test. Cynaroside is a most potent DBH inhibitor. The IC50 value of cynaroside to DBH was calculated at 0.0410 μM. Results of this study suggest that the two prenylated chalcones, xanthoangelol and 4-hydroxyderricin isolated from A. keiskei K., are expected for potent candidates for development of combined antidepressant drug. A. keiskei K. will be an excellent new bio-functional food material that has the combined antidepressant effect. PMID:24265870

  8. Electrochemical activity of glucose oxidase on a poly(ionic liquid) - Au nanoparticle composite.

    SciTech Connect

    Lee, S.; Ringstrand, B. S.; Stone, D. A.; Firestone, M. A.

    2012-01-01

    Glucose oxidase (GOx) adsorbed on an ionic liquid-derived polymer containing internally organized columns of Au nanoparticles exhibits direct electron transfer and bioelectrocatalytic properties towards the oxidation of glucose. The cationic poly(ionic liquid) provides an ideal substrate for the electrostatic immobilization of GOx. The encapsulated Au nanoparticles serve to both promote the direct electron transfer with the recessed enzyme redox centers and impart electronic conduction to the composite, allowing it to function as an electrode for electrochemical detection.

  9. [Effect of centrophenoxine, piracetam and aniracetam on the monoamine oxidase activity in different brain structures of rats].

    PubMed

    Stancheva, S L; Alova, L G

    1988-01-01

    In vitro studies of effects of some nootropic drugs (centrophenoxine, piracetam and aniracetam) on monoamine oxidase (MAO) activity in the rat striatum and hypothalamus, using tyramine, serotonin and beta-phenylethylamine as substrates, were carried out. At all concentrations used (5.10(-5)-1.10(-3) M) centrophenoxine inhibited total MAO, MAO A and MAO B in both brain structures. Piracetam activated striatal and hypothalamic total MAO, hypothalamic MAO A and MAO B but exerted a pronounced inhibitory effect on MAO A and MAO B activity in the striatum. Aniracetam inhibited total MAO and MAO A in both brain structures but activated striatal and hypothalamic MAO B. The different effects of centrophenoxine, piracetam and aniracetam on MAO activity in the brain structures support the view for the independent mode of action of nootropic drugs in spite of their similar molecular and metabolic activity.

  10. Polyamine analysis by LC-MS.

    PubMed

    Häkkinen, Merja R

    2011-01-01

    This chapter describes a protocol to analyze polyamines without any derivatization steps utilizing LC-MS/MS. Polyamines are separated by reversed phase LC prior MS analysis using heptafluorobutyric acid as MS compatible volatile ion-pairing agent, and selective and sensitive MS detection is performed using MS/MS in selected reaction monitoring mode.

  11. Polyamine intake, dietary pattern, and cardiovascular disease.

    PubMed

    Soda, Kuniyasu

    2010-09-01

    In addition to general lifestyle, a number of foods and dietary patterns, such as the Mediterranean diet (MD), are associated with lower incidences of chronic, age-related diseases, and mortality. We have shown that increased polyamine intake decreases age-associated pathology and increases longevity in mice. Several foods in the MD, such as fruits and legumes, are foods containing high amount of polyamines. Among age-associated conditions, cardiovascular diseases (CVD) are the leading cause of mortality worldwide, and individuals who adhere to a MD have a lower incidence of CVD. The possible contribution of increased polyamine intake to CVD prevention is discussed in this manuscript. Polyamines from food are distributed to all organs and tissues, and long-term intake increases polyamine concentration in blood. Because most polyamines are associated with red and white blood cells, they act to suppress synthesis of pro-inflammatory cytokines and of leukocyte function-associated antigen-1. Foods with anti-inflammatory properties such as n-3 polyunsaturated fatty acids are known to help prevent CVD. Additionally, suppression of de novo polyamine synthesis results from increased polyamines intake, normally synthesized from arginine. This in turn increases availability of arginine for synthesis of nitric oxide, which plays an important role in preserving normal vascular physiology.

  12. Cross-linking of structural proteins in ageing skin: an in situ assay for the detection of amine oxidase activity.

    PubMed

    Langton, Abigail K; Griffiths, Christopher E M; Sherratt, Michael J; Watson, Rachel E B

    2013-02-01

    With increasing age, dynamic tissues such as lungs, blood vessels and skin lose their ability to both deform and recoil, culminating in tissue stiffening. This loss of tissue elasticity, which profoundly impacts tissue function and thus morbidity, may be due not only to changes in the relative abundance of key extracellular matrix proteins within tissues but also to their accumulation of post-translational modifications. Whilst to date attention has focussed primarily on the age-related non-enzymatic formation of advanced glycation end products, the accumulation of pathological enzyme-mediated cross-links may also lead to age-related tissue stiffening. The lysyl oxidase (LOX) family of enzymes are constitutively expressed in adult tissues and are known to drive the catalysis of cross-links in both fibrillar collagens and elastin. Although immunochemical approaches are commonly used to localise the inactive pro-enzyme of LOX, and biochemical methods are employed to quantify activity in homogenised tissue, they do not allow for the in situ localisation of the enzyme. Thus, we have developed a novel assay to both detect and localise LOX enzyme activity in situ. LOX family members are amine oxidases and this assay uses the principle that an amine substrate in the presence of this class of enzyme will be oxidised to an aldehyde and hydrogen peroxide (H2O2). In turn, H2O2, when combined with luminol and horseradish peroxidase, will produce a light-emitting reaction that can be detected by film autoradiography. The development of a technique to localise specific amine oxidase activity in tissue sections may provide crucial additional information on the exact role played by this class of enzymes in mediating age-related tissue stiffening.

  13. 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.

  14. 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.

  15. Design, synthesis of novel pyranotriazolopyrimidines and evaluation of their anti-soybean lipoxygenase, anti-xanthine oxidase, and cytotoxic activities.

    PubMed

    Saïd, Abderrahim Ben; Romdhane, Anis; Elie, Nicolas; Touboul, David; Jannet, Hichem Ben; Bouajila, Jalloul

    2016-12-01

    The synthesis of 14-(aryl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-yl) acetamidoximes 2a-e has been accomplished by reaction of 2-acetonitrile derivatives 1a-e with hydroxylamine. Cyclocondensation reaction of precursors 2a-e with some elctrophilic species such as ethylorthoformate, acetic anhydride, and methyl-acetoacetate provided the new oxadiazole derivatives 3a-e, 4a-e, and 5a-e, respectively. On the other hand, the reaction of precursors 2a-e with 2-chloropropanoyl chloride afforded the new acetimidamides 6a-e which evolve under reflux of toluene to the new oxadiazoles 7a-e. The synthetic compounds were screened for their anti-xanthine oxidase, anti-soybean lipoxygenase, and cytotoxic activities. Moderate to weak xanthine oxidase and soybean lipoxygenase inhibitions were obtained but significant cytotoxic activities were noted. The most cytotoxic activities were recorded mainly (i) 5a was the most active (IC50 = 4.0 μM) and selective against MCF-7 and (ii) 2a was cytotoxic against the four cell lines with selectivity for MCF-7 and OVCAR-3 (IC50 = 17 and 12 μM, respectively) while 2e is highly selective against OVCAR-3 (IC50 = 10 μM).

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. Flexibility and enzyme activity of NADH oxidase from Thermus thermophilus in the presence of monovalent cations of Hofmeister series.

    PubMed

    Tóth, Kamil; Sedlák, Erik; Sprinzl, Mathias; Zoldák, Gabriel

    2008-05-01

    Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na(+), Gdm(+), NH(4)(+), Li(+), K(+) and Cs(+)) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis-Menten constants for NADH, accessibility of the active site as reflected by Stern-Volmer constants and activity of NOX at high cation concentrations (1-2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465-476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters. PMID:18339331

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

    PubMed

    Popiolek, Michael; Ross, John F; Charych, Erik; Chanda, Pranab; Gundelfinger, Eckart D; Moss, Stephen J; Brandon, Nicholas J; Pausch, Mark H

    2011-08-19

    Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.

  7. A single amino acid substitution confers high cinchonidine oxidation activity comparable with that of rabbit to monkey aldehyde oxidase 1.

    PubMed

    Fukiya, Kensuke; Itoh, Kunio; Yamaguchi, Satoshi; Kishiba, Akiko; Adachi, Mayuko; Watanabe, Nobuaki; Tanaka, Yorihisa

    2010-02-01

    Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.

  8. 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

  9. 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

  10. 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

  11. Antioxidant activities and xanthine oxidase inhibitory effects of extracts and main polyphenolic compounds obtained from Geranium sibiricum L.

    PubMed

    Wu, Nan; Zu, Yuangang; Fu, Yujie; Kong, Yu; Zhao, Jintong; Li, Xiaojuan; Li, Ji; Wink, Michael; Efferth, Thomas

    2010-04-28

    The antioxidant capacity and xanthine oxidase inhibitory effects of extracts and main polyphenolic compounds of Geranium sibiricum were studied in the present work. The antioxidant capacity was evaluated by ferric reducing antioxidant power, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, superoxide radical scavenging, nitric oxide scavenging, beta-carotene-linoleic acid bleaching, and reducing power assays. Among the extracts and four fractions, the ethyl acetate fraction showed the highest phenolic content (425.36 +/- 9.70 mg of gallic acid equivalent/g extracts) and the best antioxidant activity. The IC(50) values of the ethyl acetate fraction were 0.93, 3.32, 2.06, 2.66, and 1.64 microg/mL in the DPPH radical scavenging, superoxide radical scavenging, nitric oxide scavenging, beta-carotene-linoleic acid bleaching, and reducing power assays, respectively. Of the polyphenolic compounds separated from the ethyl acetate fraction, geraniin showed a higher activity than corilagin and gallic acid. The IC(50) values ranged from 0.87 to 2.53 microM, which were even lower than the positive control (except for allopurinol). All test samples except for the petroleum ether fraction showed xanthine oxidase inhibitory effects. We conclude that G. sibiricum represents a valuable natural antioxidant source and is potentially applicable in the healthy food industry.

  12. 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.

  13. Polyamines and abiotic stress tolerance in plants.

    PubMed

    Gill, Sarvajeet Singh; Tuteja, Narendra

    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.

  14. 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

  15. Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

    PubMed Central

    Pick, Edgar

    2014-01-01

    The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

  16. Heredity mode of genetic polymorphism in aldehyde oxidase activity in Donryu strain rats.

    PubMed

    Adachi, M; Itoh, K; Abe, H; Tanaka, Y

    2008-01-01

    Donryu strain rats show genetic polymorphisms in the aldehyde oxidase gene, resulting in the phenotypic expression of ultrarapid metabolizers with homozygous nucleotide sequences (337G, 2604C), extensive metabolizers with heterozygous nucleotide sequences (377G/A, 2604C/T), and poor metabolizers with homozygous nucleotide sequences (377A, 2604T). In the mating experiments the ratio of the number of ultrarapid metabolizers, extensive metabolizers, and poor metabolizers rats in the F1 generation from the heterozygous F0 extensive metabolizers male and female rats was roughly 0.6 : 1.5 : 1, and the ratio converged to approximately 1 : 2 : 1 in the F2 generation from the heterozygous F1 extensive metabolizers male and female rats. On the contrary, all the F2 generation from homozygous F1 ultrarapid metabolizers male and female rats or from homozygous F1 poor metabolizers male and female rats had the ultrarapid metabolizers or the poor metabolizers genotypes and phenotypes. The genotypes completely agreed with the phenotypes in all individuals of F0, F1, and F2 generations. The results indicate that the genetic polymorphism of aldehyde oxidase in Donryu strain rats obeys Mendelian heredity. The reason for a low ratio of the ultrarapid metabolizers rats in the commercially available Donryu strain rats - not more than several per cent - compared with the ratio expected from the Mendelian rule is unknown.

  17. 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

  18. 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.

  19. Some aspects of the modular organization of the primary visual cortex of the cat: patterns of cytochrome oxidase activity.

    PubMed

    Merkul'eva, N S; Makarov, F N

    2008-10-01

    The distribution of the enzyme cytochrome oxidase (CO) in continuous series of parasagittal sections from field 17 and frontal sections of the dorsal nucleus of the lateral geniculate body (LGB) from normal kittens and adult cats was studied. In all cats apart from neonates, layer IV showed regularly alternating areas with above-background levels of CO activity ("spots"). There was a significant increase in the contrast of the "spots" from days 13 to 21, which was followed by a significant decrease from days 48 to 93. These changes coincided with ontogenetic changes in the level of visual system plasticity. There were no differences in CO activity between layers A and A1 of the dorsal nucleus of the LGB. It is suggested that the non-uniform distribution of the level of functional activity of neurons in field 17 reflects the formation of columnar cortical structures during the critical period of postnatal ontogenesis.

  20. 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.

  1. Preparation and biological assessment of hydroxycinnamic acid amides of polyamines.

    PubMed

    Fixon-Owoo, Solomon; Levasseur, Frédéric; Williams, Keith; Sabado, Thomas N; Lowe, Mike; Klose, Markus; Joffre Mercier, A; Fields, Paul; Atkinson, Jeffrey

    2003-06-01

    Many plants contain hydroxycinnamic acid conjugates of polyamines that are remarkably similar in general structure to the acylated polyamines found in spider and wasp toxins. In an effort to determine whether these compounds might play a role in the chemical defense of plants against arthropod pests we synthesized a variety of analogues of the coumaric (4-hydroxycinnamic) acid conjugates of di-, tri-, and tetraamines using common protection and acylation strategies. N(1)- and N(8)-coumaroyl spermidine were tested in feeding trials with insect larvae including the European corn borer (Ostrinia nubilalis), the tobacco budworm (Heliothis verescens) and the oblique banded leaf roller (Choristoneura rosaceana). Antifeedant assays with the rice weevil Sitophilus oryzae were also performed. Neither the naturally occurring coumaric acid conjugates of polyamines nor their analogues showed notable toxicity towards insects, despite precautions to maintain these easily oxidized materials in the wet diet. However, more direct bioassays of these compounds on glutamate dependent neuroreceptors including the deep abdominal extensor muscles of crayfish, or mammalian NMDA, delta2, and AMPA receptors, clearly showed that these compounds were inhibitory. N(1)-Coumaoryl spermine, a dodecyl and a cyclohexyl analogue were especially active at NMDA NR1/NR2B receptors. The latter had an IC(50) of 300 microM in the crayfish. N(1)-Coumaroyl spermine had an IC(50) in the crayfish preparation of 70-300 microM and against the mammalian NR1/NR2B receptor of 38 nM. Structure-activity variations show similar trends of length and hydrophobicity as has been seen previously with analogues of spider toxins. We conclude from this work that while the coumaric acid polyamine conjugates are active when directly applied to neuroreceptors, they show no overt toxicity when ingested by insect larvae.

  2. 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

  3. Current Status of the Polyamine Research Field

    PubMed Central

    Pegg, Anthony E.; Casero, Robert A.

    2013-01-01

    This chapter provides an overview of the polyamine field and introduces the 32 other chapters that make up this volume. These chapters provide a wide range of methods, advice, and background relevant to studies of the function of polyamines, the regulation of their content, their role in disease, and the therapeutic potential of drugs targeting polyamine content and function. The methodology provided in this new volume will enable laboratories already working in this area to expand their experimental techniques and facilitate the entry of additional workers into this rapidly expanding field. PMID:21318864

  4. 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.

  5. 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

  6. 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

  7. Flavonoids from Sideritis Species: Human Monoamine Oxidase (hMAO) Inhibitory Activities, Molecular Docking Studies and Crystal Structure of Xanthomicrol.

    PubMed

    Turkmenoglu, Fatma Pinar; Baysal, İpek; Ciftci-Yabanoglu, Samiye; Yelekci, Kemal; Temel, Hamdi; Paşa, Salih; Ezer, Nurten; Çalış, İhsan; Ucar, Gulberk

    2015-04-23

    The inhibitory effects of flavonoids on monoamine oxidases (MAOs) have attracted great interest since alterations in monoaminergic transmission are reported to be related to neurodegenerative diseases such as Parkinson's and Alzheimer's diseases and psychiatric disorders such as depression and anxiety, thus MAOs may be considered as targets for the treatment of these multi-factorial diseases. In the present study, four Sideritis flavonoids, xanthomicrol (1), isoscutellarein 7-O-[6'''-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside (2), isoscutellarein 7-O-[6'''-O-acetyl-β-D-allopyranosyl-(1→2)]-6''-O-acetyl-β-D-glucopyranoside (3) and salvigenin (4) were docked computationally into the active site of the human monoamine oxidase isoforms (hMAO-A and hMAO-B) and were also investigated for their hMAO inhibitory potencies using recombinant hMAO isoenzymes. The flavonoids inhibited hMAO-A selectively and reversibly in a competitive mode. Salvigenin (4) was found to be the most potent hMAO-A inhibitor, while xanthomicrol (1) appeared as the most selective hMAO-A inhibitor. The computationally obtained results were in good agreement with the corresponding experimental values. In addition, the x-ray structure of xanthomicrol (1) has been shown. The current work warrants further preclinical studies to assess the potential of xanthomicrol (1) and salvigenin (4) as new selective and reversible hMAO-A inhibitors for the treatment of depression and anxiety.

  8. 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

  9. Polyamines in Relation to Growth in Carrot Cell Cultures 1

    PubMed Central

    Fallon, Kevin M.; Phillips, Richard

    1988-01-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID:16666271

  10. Polyamines in relation to growth in carrot cell cultures.

    PubMed

    Fallon, K M; Phillips, R

    1988-09-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

  11. Intracellular sources of ornithine for polyamine synthesis in endothelial cells.

    PubMed

    Li, Hui; Meininger, Cynthia J; Bazer, Fuller W; Wu, Guoyao

    2016-10-01

    Polyamines are essential for proliferation of endothelial cells (EC) and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using N(ω)-hydroxy-nor-L-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10 µM for intact EC. Nor-NOHA (0.5 mM) alone inhibited arginase activity in EC by 98 %, increased total cellular concentrations of arginine by 14 %, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74 %, respectively. Arginine and glutamine contributed to 73 and 26 % of the ornithine produced by EC, respectively. Gabaculine (1 mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42 %, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5 mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines.  These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.

  12. [L-Lysine-α-Oxidase in vitro Activity in Experiments on Models of Viruses Sindbis, Forest-Spring Encephalitis, Western Nile, Tyaginya and Dhori].

    PubMed

    Smirnova, I P; Larichev, V F; Shneider, Yu A

    2015-01-01

    The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses. PMID:26415376

  13. Concentration of biologically active polyamines in rabbit meat, liver and kidney after slaughter and their changes during meat storage and cooking.

    PubMed

    Dadáková, Eva; Pelikánová, Tamara; Kalač, Pavel

    2012-03-01

    The concentration of putrescine (PUT), spermidine (SPD) and spermine (SPM) was determined in chilled meat and kidneys of 18 rabbits and in liver of 12 animals 24h after slaughter. Very low PUT concentrations were detected only in kidneys. Mean SPD levels were 2.2, 2.2, 61.7 and 32.7mgkg(-1) in saddle, leg, liver and kidneys, respectively. The respective SPM concentrations were 14.7, 8.0, 115 and 88.4mgkg(-1). SPD and SPM losses of about one third of the initial levels were apparent in saddles stored at -18°C for 8months. Losses of both polyamines of about 15-20% of the initial concentrations were found in saddles stored aerobically at +2°C for up to 9days. Stewing of saddles caused significant SPD and SPM losses of about 20-25%, while upon roasting and pan-roasting without oil a decrease of about 50% of the initial concentration was observed.

  14. Potent inhibitory effects of N-aryl S-alkylthiocarbamate derivatives on the dopa oxidase activity of mushroom tyrosinase.

    PubMed

    Lee, Kun Ho; Koketsu, Mamoru; Choi, Sang Yoon; Lee, Kang Jin; Lee, Pyeongjae; Ishihara, Hideharu; Kim, Sun Yeou

    2005-07-01

    This study reports the potent inhibitory effect of N-aryl S-alkylthiocarbamate derivatives on mushroom tyrosinase (MT) activity. N-Aryl S-alkylthiocarbamate derivatives were found to exhibit a potent inhibitory effect on the dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Most of the N-aryl S-alkylthiocarbamate derivatives (compounds from A to J) exhibited higher inhibitory effects than kojic acid (IC50=318 microM), a well known tyrosinase inhibitor. Tyrosinase was the most inhibited by S-phenetyl N-phenylthiocarbamate (compound E, IC50=7.25 microM), and this inhibition was 44 times stronger than that of kojic acid. Compound E exhibited 95.0% of inhibition at 100 microM. A kinetic study of MT inhibition by compound E using the Lineweaver-Burk plots analysis was performed. And the kinetics profiles observed suggest that compound E competitively inhibits MT.

  15. 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

  16. The antioxidant activity of soursop decreases the expression of a member of the NADPH oxidase family.

    PubMed

    Zamudio-Cuevas, Y; Díaz-Sobac, R; Vázquez-Luna, A; Landa-Solís, C; Cruz-Ramos, M; Santamaría-Olmedo, M; Martínez-Flores, K; Fuentes-Gómez, A J; López-Reyes, A

    2014-02-01

    Cellular oxidative stress produced by an increase in free radicals is one of the factors that promote the development of chronic degenerative diseases; therefore, consuming natural antioxidants helps minimize their negative effects. This study evaluated the cytotoxicity of the soursop extract (Annona muricata), its cytoprotective capacity against oxidative stress induced by hydrogen peroxide, the inhibitory potential of reactive oxygen species (ROS), the molecular mechanism of its antioxidant action, and its capacity to repair cellular damage in the fibroblast cell line. The soursop extract proved not to be cytotoxic in fibroblast cultures and showed cytoprotective capacity against hydrogen peroxide-induced stress; in cell culture it reduced the generation of ROS significantly by inhibiting a sub-unit of the NADPH oxidase enzyme (p47phox). The soursop extract can prevent damage caused by cellular oxidants.

  17. Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors.

    PubMed

    Watanabe, Bunta; Ichiyanagi, Atsushi; Hirokawa, Kozo; Gomi, Keiko; Nakatsu, Toru; Kato, Hiroaki; Kajiyama, Naoki

    2015-09-15

    Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 μM were obtained for 1a-c, respectively.

  18. Polyamines and cancer: Implications for chemoprevention and chemotherapy

    PubMed Central

    Nowotarski, Shannon L.; Woster, Patrick M.; Casero, Robert A.

    2013-01-01

    Polyamines are small organic cations that are essential for normal cell growth and development in eukaryotes. Under normal physiological conditions, intracellular polyamine concentrations are tightly regulated through a dynamic network of biosynthetic and catabolic enzymes and a poorly characterized transport system. This precise regulation ensures that the intracellular concentration of polyamines is maintained within strictly controlled limits. It has frequently been observed that the metabolism of, and the requirement for, polyamines in tumours is frequently dysregulated. Elevated levels of polyamines have been associated with breast, colon, lung, prostate, and skin cancers, and altered levels of the rate limiting enzymes in both biosynthesis and catabolism have been observed. Based on these observations and the absolute requirement for polyamines in tumour growth, the polyamine pathway is a rational target for chemoprevention and chemotherapeutics. Here we describe the recent advances made in the polyamine field and focus on the roles of polyamines and polyamine metabolism in neoplasia through a discussion of the current animal models for the polyamine pathway, chemotherapeutic strategies that target the polyamine pathway, chemotherapeutic clinical trials for polyamine pathway specific drugs, and ongoing clinical trials targeting polyamine biosynthesis. PMID:23432971

  19. 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

  20. Stress and polyamine metabolism in fungi.

    PubMed

    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.

  1. Polyamines in cell walls of chlorococcalean microalgae.

    PubMed

    Burczyk, Jan; Zych, Maria; Ioannidis, Nikolaos E; Kotzabasis, Kiriakos

    2014-01-01

    Biotechnology of microalgae represents a very attractive alternative as a source of energy and substances of high value when compared with plant cultivation. Cell walls of green microalgae have an extraordinary chemical and mechanical resistance and may impede some steps in the biotechnological/industrial exploitation of algae. The aim of the present contribution was to check the presence of polyamines in the cell walls of chlorococcalean green microalgae. Polyamines are nitrogenous compounds synthesized normally in cells and may affect the properties of the cell wall. Our work included strains either forming or not forming the polymer algaenan, allowing us to conclude that algaenan is not a prerequisite for the presence of polyamines in the cell walls. Polyamines were detected in isolated cell walls of Scenedesmus obliquus, Chlorella fusca, Chlorella saccharophila, and Chlorella vulgaris. Their concentration in isolated cell walls ranged between 0.4 and 8.4 nmol/mg dry weight. PMID:24772826

  2. Stress and Polyamine Metabolism in Fungi

    NASA Astrophysics Data System (ADS)

    Valdés-Santiago, Laura; Ruiz-Herrera, José

    2013-12-01

    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.

  3. Correlation of endogenous free polyamine levels with root nodule senescence in different genotypes in Vigna mungo L.

    PubMed

    Lahiri, Kajari; Chattopadhyay, Soumen; Ghosh, Bharati

    2004-05-01

    Endogenous free polyamines, nitrogenase (EC 1.1.8.6.1, acetylene reduction), and leghaemoglobin (pyridine-hemochrome assay) levels were compared among five genotypes of developing Vigna root nodules grown under field conditions. Nitrogenase activity and leghaemoglobin level attained a peak at the flowering stage and gradually declined thereafter. Individual and total polyamine also followed the same pattern. Ranking on the basis of legume yield and other morphometric attributes was PDU-2 > UH-28 > UH-82 > T-9 > Sardhomash. Except spermine, the levels of putrescine, spermidine, and total polyamine showed significant differences (p<0.05) amongst the genotypes, particularly from flowering to mid-pod development stage. Genotype, development stage, and their interaction between the two had significant (p<0.01) effects on individual as well as total polyamines. Moreover, significant high linear correlations were found between total free polyamine and putrescine with conventional nodule senescence marker like nitrogenase (R2 = 0.94 and R2 = 0.92, respectively). Putrescine had an overall positive correlation with high legume yield. The results strongly suggest a relationship between polyamine and nodule senescence. Endogenous free polyamine and putrescine may be considered as genotypic markers for nodule senescence in field grown V. mungo. It is suggested that the flowering stage is more suitable for selection.

  4. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

    PubMed

    Liu, Susan B; Ikenaga, Naoki; Peng, Zhen-Wei; Sverdlov, Deanna Y; Greenstein, Andrew; Smith, Victoria; Schuppan, Detlef; Popov, Yury

    2016-04-01

    Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversalin vivousing chronic administration of irreversible LOX inhibitor β-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl4)-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl4withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its resolution.-Liu, S. B., Ikenaga, N., Peng, Z.-W., Sverdlov, D. Y., Greenstein, A., Smith, V., Schuppan, D., Popov, Y. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

  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. 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

  7. 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.

  8. 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

  9. Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation.

    PubMed

    Burtin, D; Martin-Tanguy, J; Paynot, M; Rossin, N

    1989-01-01

    We studied the effects of dl-alpha-difluoromethylarginine (DFMA) and dl-alpha-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.

  10. Effects of the Suicide Inhibitors of Arginine and Ornithine Decarboxylase Activities on Organogenesis, Growth, Free Polyamine and Hydroxycinnamoyl Putrescine Levels in Leaf Explants of Nicotiana Xanthi n.c. Cultivated in Vitro in a Medium Producing Callus Formation

    PubMed Central

    Burtin, Daniel; Martin-Tanguy, Josette; Paynot, Michel; Rossin, Nadia

    1989-01-01

    We studied the effects of dl-α-difluoromethylarginine (DFMA) and dl-α-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines. Images Figure 1 Figure 2 PMID:16666499

  11. Exploring polyamine biosynthetic diversity through comparative and functional genomics.

    PubMed

    Michael, Anthony J

    2011-01-01

    The existence of multiple, alternative pathways for polyamine biosynthesis, and the presence of alternative polyamine structural analogs, is an indication of the physiological importance of polyamines and their long evolutionary history. Polyamine biosynthesis is modular: diamines are synthesized directly or indirectly from amino acids, and triamines are synthesized from diamines by transfer of aminopropyl, carboxyaminopropyl, or aminobutyl groups to the diamine. Diversification of polyamine biosynthesis has depended on gene duplication and functional divergence, on gene fusion, and on horizontal gene transfer. Four examples of polyamine biosynthetic diversification are presented here with a discussion of methodological and conceptual approaches for identification of new pathways.

  12. Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity.

    PubMed Central

    Umbach, A. L.; Siedow, J. N.

    1993-01-01

    Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis. PMID:12231983

  13. [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.

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

    PubMed

    Cai, Lu; Wang, Chao; Huo, Xiao-Kui; Dong, Pei-Pei; Zhang, Bao-Jing; Zhang, Hou-Li; Huang, Shan-Shan; Zhang, Bo; Yu, Sheng-Ming; Zhong, Ming; Ma, Xiao-Chi

    2016-01-01

    Phyllodium pulchellum (P. pulchellum) is a folk medicine with a significant number of bioactivities. The aim of this study was to investigate the effects displayed by alkaloids fractions, isolated from the roots of P. pulchellum, on neurotransmitters monoamine levels and on monoamine oxidase (MAO) activity. Six alkaloids, which had indolealkylamine or β-carboline skeleton, were obtained by chromatographic technologies and identified by spectroscopic methods such as NMR and MS. After treatment with alkaloids of P. pulchellum, the reduction of DA levels (54.55%) and 5-HT levels (35.01%) in rat brain was observed by HPLC-FLD. The effect of alkaloids on the monoamines metabolism was mainly related to MAO inhibition, characterized by IC50 values of 37.35 ± 6.41 and 126.53 ± 5.39 μg/mL for MAO-A and MAO-B, respectively. The acute toxicity indicated that P. pulchellum extract was nontoxic. PMID:27195015

  15. 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

  16. [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

  17. 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.

  18. An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells.

    PubMed

    Lai, Chung-Fang; Seshadri, Venkat; Huang, Kane; Shao, Jian-Su; Cai, Jun; Vattikuti, Radhika; Schumacher, Arwyn; Loewy, Arleen P; Denhardt, David T; Rittling, Susan R; Towler, Dwight A

    2006-06-23

    Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid

  19. Polyamines are essential for virulence in Salmonella enterica serovar Gallinarum despite evolutionary decay of polyamine biosynthesis genes.

    PubMed

    Schroll, Casper; Christensen, Jens P; Christensen, Henrik; Pors, Susanne E; Thorndahl, Lotte; Jensen, Peter R; Olsen, John E; Jelsbak, Lotte

    2014-05-14

    Serovars of Salmonella enterica exhibit different host-specificities where some have broad host-ranges and others, like S. Gallinarum and S. Typhi, are host-specific for poultry and humans, respectively. With the recent availability of whole genome sequences it has been reported that host-specificity coincides with accumulation of pseudogenes, indicating adaptation of host-restricted serovars to their narrow niches. Polyamines are small cationic amines and in Salmonella they can be synthesized through two alternative pathways directly from l-ornithine to putrescine and from l-arginine via agmatine to putrescine. The first pathway is not active in S. Gallinarum and S. Typhi, and this prompted us to investigate the importance of polyamines for virulence in S. Gallinarum. Bioinformatic analysis of all sequenced genomes of Salmonella revealed that pseudogene formation of the speC gene was exclusive for S. Typhi and S. Gallinarum and happened through independent events. The remaining polyamine biosynthesis pathway was found to be essential for oral infection with S. Gallinarum since single and double mutants in speB and speE, encoding the pathways from agmatine to putrescine and from putrescine to spermidine, were attenuated. In contrast, speB was dispensable after intraperitoneal challenge, suggesting that putrescine was less important for the systemic phase of the disease. In support of this hypothesis, a ΔspeE;ΔpotCD mutant, unable to synthesize and import spermidine, but with retained ability to import and synthesize putrescine, was attenuated after intraperitoneal infection. We therefore conclude that polyamines are essential for virulence of S. Gallinarum. Furthermore, our results point to distinct roles for putrescine and spermidine during systemic infection.

  20. 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.