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  1. Respiratory Muscle Training Improves Diaphragm Citrate Synthase Activity and Hemodynamic Function in Rats with Heart Failure.

    Science.gov (United States)

    Jaenisch, Rodrigo Boemo; Bertagnolli, Mariane; Borghi-Silva, Audrey; Arena, Ross; Lago, Pedro Dal

    2017-01-01

    Enhanced respiratory muscle strength in patients with heart failure positively alters the clinical trajectory of heart failure. In an experimental model, respiratory muscle training in rats with heart failure has been shown to improve cardiopulmonary function through mechanisms yet to be entirely elucidated. The present report aimed to evaluate the respiratory muscle training effects in diaphragm citrate synthase activity and hemodynamic function in rats with heart failure. Wistar rats were divided into four experimental groups: sedentary sham (Sed-Sham, n=8), trained sham (RMT-Sham, n=8), sedentary heart failure (Sed-HF, n=7) and trained heart failure (RMT-HF, n=7). The animals were submitted to a RMT protocol performed 30 minutes a day, 5 days/week, for 6 weeks. In rats with heart failure, respiratory muscle training decreased pulmonary congestion and right ventricular hypertrophy. Deleterious alterations in left ventricular pressures, as well as left ventricular contractility and relaxation, were assuaged by respiratory muscle training in heart failure rats. Citrate synthase activity, which was significantly reduced in heart failure rats, was preserved by respiratory muscle training. Additionally, a negative correlation was found between citrate synthase and left ventricular end diastolic pressure and positive correlation was found between citrate synthase and left ventricular systolic pressure. Respiratory muscle training produces beneficial adaptations in the diaphragmatic musculature, which is linked to improvements in left ventricular hemodynamics and blood pressure in heart failure rats. The RMT-induced improvements in cardiac architecture and the oxidative capacity of the diaphragm may improve the clinical trajectory of patients with heart failure.

  2. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  3. Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

    Science.gov (United States)

    Muhitch, Michael J.

    1988-01-01

    Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID:16665871

  4. Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.

    OpenAIRE

    Homann, M J; Henry, S A; Carman, G M

    1985-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.

  5. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    Science.gov (United States)

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly. Copyright © 2016. Published by Elsevier Inc.

  6. Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

    Science.gov (United States)

    Chaves, Julie E; Rueda-Romero, Paloma; Kirst, Henning; Melis, Anastasios

    2017-12-15

    Efforts to heterologously produce quantities of isoprene hydrocarbons (C 5 H 8 ) renewably from CO 2 and H 2 O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific-activity of the unfused cpcB-IspS control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g -1 in the unfused cpcB-IspS control to 5.4 mg g -1 in the cpcB*L7*IspS strain, a 27-fold improvement.

  7. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content......, whereas impaired insulin activation of muscle glycogen synthase represents a consistent, molecular defect found in both type 2 diabetic and high-risk individuals. Despite several studies of the insulin signaling pathway believed to mediate dephosphorylation and hence activation of glycogen synthase......, the molecular mechanisms responsible for this defect remain unknown. Recently, the use of phospho-specific antibodies in human diabetic muscle has revealed hyperphosphorylation of glycogen synthase at sites not regulated by the classical insulin signaling pathway. In addition, novel approaches such as gene...

  8. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  9. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    International Nuclear Information System (INIS)

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality. Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein

  10. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate...

  11. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  12. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    Science.gov (United States)

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  13. Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

    Directory of Open Access Journals (Sweden)

    Alistair K Brown

    2009-07-01

    Full Text Available There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml and against Mycobacterium tuberculosis (MIC = 12 microg/ml. Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

  14. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    Science.gov (United States)

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  15. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  16. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal...... and sex showed no such changes. Our data support the hypothesis that NO is a pathogenic factor in MS, and that NOS IR is strongly expressed in brain regions appearing normal by MRI...

  17. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  18. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ∊

    International Nuclear Information System (INIS)

    Roy, Ankoor; Hutcheon, Marcus L.; Duncan, Thomas M.; Cingolani, Gino

    2012-01-01

    A phosphomimetic mutation in subunit ∊ dramatically increases reproducibility for crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) (subunit composition α 3 β 3 γ∊). Diffraction data were collected to ∼3.15 Å resolution using synchrotron radiation. The bacterial ATP synthase (F O F 1 ) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F O F 1 represents nature’s smallest rotary motor, composed of a membrane-embedded proton transporter (F O ) and a peripheral catalytic complex (F 1 ). The ATPase activity of isolated F 1 is fully expressed by the α 3 β 3 γ ‘core’, whereas single δ and ∊ subunits are required for structural and functional coupling of E. coli F 1 to F O . In contrast to mitochondrial F 1 -ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F 1 -ATPase catalytic core. Destabilizing the compact conformation of ∊’s C-terminal domain with a phosphomimetic mutation (∊S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F 1 that diffract to ∼3.15 Å resolution

  19. Regulation of expression, activity and localization of fungal chitin synthases

    Science.gov (United States)

    Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen R.; Steinbach, William J.

    2013-01-01

    The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. PMID:21526913

  20. A thiazolidinedione improves in vivo insulin action on skeletal muscle glycogen synthase in insulin-resistant monkeys.

    Science.gov (United States)

    Ortmeyer, H K; Bodkin, N L; Haney, J; Yoshioka, S; Horikoshi, H; Hansen, B C

    2000-01-01

    Thiazolidinediones (TZD) have been shown to have anti-diabetic effects including the ability to decrease fasting hyperglycemia and hyperinsulinemia, increase insulin-mediated glucose disposal rate (M) and decrease hepatic glucose production, but the mechanisms of action are not well established. To determine whether a TZD (R-102380, Sankyo Company Ltd., Tokyo, Japan) could improve insulin action on skeletal muscle glycogen synthase (GS), the rate-limiting enzyme in glycogen synthesis, 4 insulin-resistant obese monkeys were given 1 mg/kg/day R-102380 p.o. for a 6-week period. Skeletal muscle GS activity and glucose 6-phosphate (G6P) content were compared between pre-dosing and dosing periods before and during the maximal insulin-stimulation of a euglycemic hyperinsulinemic clamp. Compared to pre-dosing, insulin-stimulated GS activity and G6P content were increased by this TZD: GS independent activity (p = 0.02), GS total activity (p = 0.005), GS fractional activity (p = 0.06) and G6P content (p = 0.02). The change in GS activity induced by in vivo insulin (insulin-stimulated minus basal) was also increased by this TZD: GS independent activity (p = 0.03) and GS fractional activity (p = 0.04). We conclude that the TZD R-102380 improves insulin action at the skeletal muscle in part by increasing the activity of glycogen synthase. This improvement in insulin sensitivity may be a key factor in the anti-diabetic effect of the thiazolidinedione class of agents.

  1. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.; (Vanderbilt); (UAB); (Brown); (Swansea)

    2010-01-08

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 {angstrom}) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 {angstrom}). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 {alpha}-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an {alpha}-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

  2. Mutation of Cellulose Synthase Gene Improves the Nutritive Value of Rice Straw

    Directory of Open Access Journals (Sweden)

    Yanjing Su

    2012-06-01

    Full Text Available Rice straw is an important roughage resource for ruminants in many rice-producing countries. In this study, a rice brittle mutant (BM, mutation in OsCesA4, encoding cellulose synthase and its wild type (WT were employed to investigate the effects of a cellulose synthase gene mutation on rice straw morphological fractions, chemical composition, stem histological structure and in situ digestibility. The morphological fractions investigation showed that BM had a higher leaf sheath proportion (43.70% vs 38.21%, p0.05 was detected in neutral detergent fiber (NDFom and ADL contents for both strains. Histological structure observation indicated that BM stems had fewer sclerenchyma cells and a thinner sclerenchyma cell wall than WT. The results of in situ digestion showed that BM had higher DM, NDFom, cellulose and hemicellulose disappearance at 24 or 48 h of incubation (p<0.05. The effective digestibility of BM rice straw DM and NDFom was greater than that of WT (31.4% vs 26.7% for DM, 29.1% vs 24.3% for NDFom, p<0.05, but the rate of digestion of the slowly digested fraction of BM rice straw DM and NDF was decreased. These results indicated that the mutation in the cellulose synthase gene could improve the nutritive value of rice straw for ruminants.

  3. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)

    2011-08-17

    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  4. Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

    Science.gov (United States)

    Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong

    2017-05-01

    S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

    Science.gov (United States)

    Shi-Jean S. Sung; W.J. Sheih; D.R. Geiger; C.C. Black

    1994-01-01

    Activities of sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with 14C-import, elongation and dry weight accumulation. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the...

  6. Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

    Science.gov (United States)

    Yeo, Yun-Soo; Nybo, S Eric; Chittiboyina, Amar G; Weerasooriya, Aruna D; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A; Coates, Robert M; Watt, David S; Chappell, Joe

    2013-02-01

    Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

  7. Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis*

    Science.gov (United States)

    Yeo, Yun-Soo; Nybo, S. Eric; Chittiboyina, Amar G.; Weerasooriya, Aruna D.; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C. Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A. Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A.; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A.; Coates, Robert M.; Watt, David S.; Chappell, Joe

    2013-01-01

    Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes. PMID:23243312

  8. Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate.

    Science.gov (United States)

    Edgar, Steven; Li, Fu-Shuang; Qiao, Kangjian; Weng, Jing-Ke; Stephanopoulos, Gregory

    2017-02-17

    Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5α-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors.

  9. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

    DEFF Research Database (Denmark)

    Maile, C A; Hingst, Janne Rasmuss; Mahalingan, K K

    2017-01-01

    BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical and reco...

  10. Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli.

    Science.gov (United States)

    Picaud, Sarah; Olsson, Mikael E; Brodelius, Peter E

    2007-01-01

    Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.

  11. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  12. Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Cho, Eun Jung; Park, Myoung Soo; Kim, Sahng Seop; Kang, Gun; Choi, Sunga; Lee, Yoo Rhan; Chang, Seok Jong; Lee, Kwon Ho; Lee, Sang Do; Park, Jin Bong

    2011-01-01

    Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100µg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10µg/ml with an ED50 value of 2µg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium. PMID:22359471

  13. Antioxidant and nitric oxide synthase activation properties of Ganoderma applanatum.

    Science.gov (United States)

    Acharya, Krishnendu; Yonzone, Parinita; Rai, Manjula; Rupa, Acharya

    2005-10-01

    In vitro evaluation of antioxidant activities of Ganoderma applanatum showed significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with standard drug catechin. IC50 values of crude, boiled and ethanolic extracts of G. applanatum were 604.8, 624 and 267 microg/ml, respectively in case of hydroxyl radical scavenging activity, and 441, 520.5 and 166.16 microg/ml, respectively in case of lipid peroxidation. Furthermore, crude, boiled and ethanolic extracts also increased significantly nitric oxide production (156.67, 121.88 and 742 pmole/mg dry wt/hr, respectively) over the control. The results of present investigation revealed that G. applanatum have potential therapeutic use.

  14. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte

    2014-01-01

    with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. RESULTS: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed...... with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ˜50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL...... to development of PME....

  15. Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase

    OpenAIRE

    1995-01-01

    Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from non...

  16. Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients

    Science.gov (United States)

    2014-01-01

    normal values after treatment with zinc sulfate. Volume of semen, progressive sperm motility percentage and total normal sperm count were increased after zinc supplementation. Conclusions Treatment of asthenospermic patients with zinc supplementation leads to restored peroxynitrite levels, arginase activity and NO synthase activity to normal values and gives a statistically significant improvement of semen parameters compared with controls. Trial registration ClinicalTrials.gov identifier: NCT01684059 PMID:24383664

  17. CDBG Public Improvements Activity

    Data.gov (United States)

    Department of Housing and Urban Development — CDBG activity related to public improvements, including senior centers, youth centers, parks, street improvements, water/sewer improvements, child care centers, fire...

  18. Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity

    DEFF Research Database (Denmark)

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva

    2005-01-01

    GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) doma...... with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase domain......GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain...... of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position...

  19. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    Energy Technology Data Exchange (ETDEWEB)

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: tmiller2@utep.edu [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  20. Unchanged gene expression of glycogen synthase in muscle from patients with NIDDM following sulphonylurea-induced improvement of glycaemic control

    DEFF Research Database (Denmark)

    Vestergaard, H; Lund, S; Bjørbaek, C

    1995-01-01

    We have previously shown that the mRNA expression of muscle glycogen synthase is decreased in non-insulin-dependent diabetic (NIDDM) patients; the objective of the present protocol was to examine whether the gene expression of muscle glycogen synthase in NIDDM is affected by chronic sulphonylurea...... treatment. Ten obese patients with NIDDM were studied before and after 8 weeks of treatment with a weight-maintaining diet in combination with the sulphonylurea gliclazide. Gliclazide treatment was associated with significant reductions in HbA1C (p=0.001) and fasting plasma glucose (p=0.005) as well...... metabolism (p=0.02) was demonstrated in teh gliclazide-treated patients when compared to pre-treatment values. In biopsies obtained from vastus lateralis muscle during insulin infusion, the half-maximal activation of glycogen synthase was achieved at a significantly lower concentration of the allosteric...

  1. Rosiglitazone Affects Nitric Oxide Synthases and Improves Renal Outcome in a Rat Model of Severe Ischemia/Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Boris Betz

    2012-01-01

    Full Text Available Background. Nitric oxide (NO-signal transduction plays an important role in renal ischemia/reperfusion (I/R injury. NO produced by endothelial NO-synthase (eNOS has protective functions whereas NO from inducible NO-synthase (iNOS induces impairment. Rosiglitazone (RGZ, a peroxisome proliferator-activated receptor (PPAR-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg was administered i.p. to SD-rats (f subjected to bilateral renal ischemia (60 min. Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NOx-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3 was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion and reduces histomorphological injury. Additionally, RGZ reduces NOx plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.

  2. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    Science.gov (United States)

    Haagsma, Anna C; Driessen, Nicole N; Hahn, Marc-Manuel; Lill, Holger; Bald, Dirk

    2010-12-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Energy Technology Data Exchange (ETDEWEB)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  4. Glycogen synthase kinase-3β activity and cognitive functioning in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Miskowiak, Kamilla Woznica; Jacoby, Anne Sophie

    2018-01-01

    Cognitive deficits are common in patients with bipolar disorder (BD) in remission and may be associated with glycogen synthase kinase-3 (GSK-3) activity, which is inhibited by lithium. GSK-3 may be a relevant treatment target for interventions tailored at cognitive disturbances in BD...... ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β...... but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6-12 month follow-up study, GSK- 3β activity...

  5. Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution

    Directory of Open Access Journals (Sweden)

    Silva José Carlos da

    2003-01-01

    Full Text Available One management practice of which the efficiency has not yet been scientifically tested is spraying coffee plants with diluted sucrose solutions as a source of carbon for the plant. This paper evaluates the effect of foliar spraying with sugar on the endogenous level of carbohydrates and on the activities of invertase and sucrose synthase in coffee (Coffea arabica L. seedlings with reduced (low and high (normal levels of carbon reserve. The concentrations used were 0.5 and 1.0% sucrose, and water as a control. The use of sucrose at 1.0% caused an increase in the concentration of total soluble sugars in depauperate plants, as well as increased the activity of the following enzymes: cell wall and vacuole acid invertase, neutral cytosol invertase and sucrose synthase. In plants with high level of carbon reserve, no increments in total soluble sugar levels or in enzymatic activity were observed. Regardless of treatments or plants physiological state, no differences in transpiration or stomatal conductance were observed, demonstrating the stomatal control of transpiration. Photosynthesis was stimulated with the use of 0.5 and 1.0 % sucrose only in depauperate plants. Coffee seedling spraying with sucrose is only efficient for depauperate plants, at the concentration of 1.0%.

  6. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  7. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  8. Dual regulation of muscle glycogen synthase during exercise by activation and compartmentalization

    DEFF Research Database (Denmark)

    Prats, Clara; Helge, Jørn W; Nordby, Pernille

    2009-01-01

    Glycogen synthase (GS) is considered the rate-limiting enzyme in glycogenesis but still today there is a lack of understanding on its regulation. We have previously shown phosphorylation-dependent GS intracellular redistribution at the start of glycogen re-synthesis in rabbit skeletal muscle (Prats......, C., Cadefau, J. A., Cussó, R., Qvortrup, K., Nielsen, J. N., Wojtaszewki, J. F., Wojtaszewki, J. F., Hardie, D. G., Stewart, G., Hansen, B. F., and Ploug, T. (2005) J. Biol. Chem. 280, 23165-23172). In the present study we investigate the regulation of human muscle GS activity by glycogen, exercise......, and insulin. Using immunocytochemistry we investigate the existence and relevance of GS intracellular compartmentalization during exercise and during glycogen re-synthesis. The results show that GS intrinsic activity is strongly dependent on glycogen levels and that such regulation involves associated...

  9. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization

    NARCIS (Netherlands)

    Dong, L.; Miettinen, K.; Verstappen, F.W.A.; Voster, A.; Jongsma, M.A.; Memelink, J.; Krol, van der S.; Bouwmeester, H.J.

    2013-01-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and

  10. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet; O'Neil-Johnson, Mark; Starks, Courtney; Hamberger, Björn; Bohlmann, Jörg

    2014-09-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual βα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  11. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    Science.gov (United States)

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Activated astrocytes induce nitric oxide synthase-2 in cerebral endothelium via tumor necrosis factor alpha.

    Science.gov (United States)

    Shafer, R A; Murphy, S

    1997-12-01

    Astrocytes under pathological conditions become activated and produce a variety of cytokines and low molecular weight signal molecules. Previously we demonstrated that activated astrocytes release nitric oxide which can downregulate the expression of nitric oxide synthase (NOS)-2 in co-cultured cerebral endothelium, and also release a transcriptionally regulated factor that can induce NOS-2 expression in endothelium (Borgerding and Murphy: J Neurochem 65:1342, 1995). The activity of this NOS-2-inducing factor was impeded by inhibitors of tyrosine kinases and NF-kappaB activation. Tumor necrosis factor (TNF alpha) alone, or in combination with IL-6, induced NOS-2 expression in endothelial cells. A neutralizing antibody against TNF alpha attenuated the NOS-2 expression in endothelial cells exposed to activated astrocytes. These results imply that cytokine-activated astrocytes release TNF alpha which can induce NOS-2 expression in endothelium and suggest that activated astrocytes within the CNS may induce expression of NOS-2 in cells of the adjacent microvasculature. The ensuing alterations in blood-brain barrier properties may be either beneficial or detrimental.

  13. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

    Science.gov (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

    2002-11-01

    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  14. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  15. Improved pestalotiollide B production by deleting competing polyketide synthase genes in Pestalotiopsis microspora.

    Science.gov (United States)

    Chen, Longfei; Li, Yingying; Zhang, Qian; Wang, Dan; Akhberdi, Oren; Wei, Dongsheng; Pan, Jiao; Zhu, Xudong

    2017-02-01

    Pestalotiollide B, an analog of dibenzodioxocinones which are inhibitors of cholesterol ester transfer proteins, is produced by Pestalotiopsis microspora NK17. To increase the production of pestalotiollide B, we attempted to eliminate competing polyketide products by deleting the genes responsible for their biosynthesis. We successfully deleted 41 out of 48 putative polyketide synthases (PKSs) in the genome of NK17. Nine of the 41 PKS deleted strains had significant increased production of pestalotiollide B (P polyketides.

  16. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    Science.gov (United States)

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

  17. Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

    Science.gov (United States)

    Ferro, Ana Margarida; Ramos, Patrícia; Guerra, Ângela; Parreira, Paula; Brás, Teresa; Guerreiro, Olinda; Jerónimo, Eliana; Capel, Carmen; Capel, Juan; Yuste-Lisbona, Fernando J; Duarte, Maria F; Lozano, Rafael; Oliveira, M Margarida; Gonçalves, Sónia

    2018-04-01

    Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

  18. Effect of hydrogen peroxide on rabbit urinary bladder citrate synthase activity in the presence and absence of a grape suspension

    Directory of Open Access Journals (Sweden)

    Vijay Venugopal

    2010-12-01

    Full Text Available PURPOSE: The etiology of obstructive bladder dysfunction includes free radical damage to mitochondria. Feeding rabbits a standardized grape suspension protects the ability of the bladder to contract and empty in part by preventing mitochondrial damage, thus maintaining smooth muscle and mucosal metabolism. The objective of the current study is to determine the direct effect of this grape suspension on the response of mitochondria to the oxidative effects of hydrogen peroxide. MATERIALS AND METHODS: Six male rabbits were anesthetized with sodium pentobarbital and the bladders excised. Four full thickness strips were obtained for contractile studies and the balance separated into smooth muscle and mucosa compartments by blunt dissection. The effect of hydrogen peroxide on the contractile response to field stimulation was quantitated. Each tissue was homogenized and the effects of increasing concentrations of hydrogen peroxide in the presence and absence of grape suspension on citrate synthase activity was determined. RESULTS: Citrate synthase activity was significantly higher in the mucosa than in the muscle. The grape suspension had no effect on control citrate synthase activity. However, the grape suspension provided significant protection of both smooth muscle and mucosal citrate synthase activity. CONCLUSIONS: These studies support the conclusion that the grape suspension provides direct protection of mitochondrial function.

  19. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E

    2006-01-01

    Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may re...

  20. Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

    DEFF Research Database (Denmark)

    Ørskov, Lotte; Bak, Jens Friis; Abildgaard, Ulrik

    1996-01-01

    The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU.kg-1. min-1) euglycaemia followed by: 1)240 min of graded ...

  1. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  2. Role of sphingomyelin synthase in controlling the antimicrobial activity of neutrophils against Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Asfia Qureshi

    2010-12-01

    Full Text Available The key host cellular pathway(s necessary to control the infection caused by inhalation of the environmental fungal pathogen Cryptococcus neoformans are still largely unknown. Here we have identified that the sphingolipid pathway in neutrophils is required for them to exert their killing activity on the fungus. In particular, using both pharmacological and genetic approaches, we show that inhibition of sphingomyelin synthase (SMS activity profoundly impairs the killing ability of neutrophils by preventing the extracellular release of an antifungal factor(s. We next found that inhibition of protein kinase D (PKD, which controls vesicular sorting and secretion and is regulated by diacylglycerol (DAG produced by SMS, totally blocks the extracellular killing activity of neutrophils against C. neoformans. The expression of SMS genes, SMS activity and the levels of the lipids regulated by SMS (namely sphingomyelin (SM and DAG are up-regulated during neutrophil differentiation. Finally, tissue imaging of lungs infected with C. neoformans using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS, revealed that specific SM species are associated with neutrophil infiltration at the site of the infection. This study establishes a key role for SMS in the regulation of the killing activity of neutrophils against C. neoformans through a DAG-PKD dependent mechanism, and provides, for the first time, new insights into the protective role of host sphingolipids against a fungal infection.

  3. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  4. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    Science.gov (United States)

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-11-13

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  5. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    DEFF Research Database (Denmark)

    Vigelsø, Andreas; Andersen, Nynne B; Dela, Flemming

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and chan......Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity...... and changes in CS activity is often assumed. However, this relationship and absolute values of CS and maximal oxygen uptake (V.O2max) has never been assessed across different studies. A systematic PubMed search on literature published from 1983 to 2013 was performed. The search profile included: citrate...... and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P

  6. Glycogen synthase kinase-3β haploinsufficiency lengthens the circadian locomotor activity period in mice.

    Science.gov (United States)

    Lavoie, Joëlle; Hébert, Marc; Beaulieu, Jean-Martin

    2013-09-15

    The mood stabiliser drug lithium has been reported to impact circadian rhythms in vertebrates. Among several putative therapeutic molecular targets, direct inhibition of glycogen synthase kinase-3 beta (GSK3β) by lithium has been proposed to underlie its effects on circadian physiology. Here we study the effect of GSK3β haploinsufficiency on the circadian locomotor activity in mice during a free-running period in comparison to wildtype littermates (WT). Mice were housed individually to record their circadian wheel running activity and were entrained to a 12h light/12h dark cycle for 14 days and then placed under constant darkness for 14 days to allow free-running. During the free-running phase, the circadian locomotor activity period of GSK3β(+/-) was significantly lengthened (23.83±0.05h) when compared to the WT mice (23.54±0.10h; p=0.0374). No significant difference in locomotor activity was observed. Knowing that GSK3β interacts with most of the core clock components, these data suggest that GSK3β acts as a critical intrinsic regulator of the circadian clock and plays an important role in regulating its period in response to lithium treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Purification of soluble cytochrome b5 as a component of the reductive activation of porcine methionine synthase.

    Science.gov (United States)

    Chen, Z; Banerjee, R

    1998-10-02

    In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B12 cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b5. It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b5 reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b5 as a member of the reductive activation system for methionine synthase describes a function for this protein in non-erythrocyte cells. In erythrocytes, soluble cytochrome b5 functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.

  8. [Antibacterial activity of Porphyra spp. epiphytic bacteria and polyketide synthase I gene screening].

    Science.gov (United States)

    Fang, Wenya; Yang, Rui; Zhu, Peng; Shan, Yuanyuan; Yan, Xiaojun

    2009-02-01

    Based on the antibacterial analysis, we screened Polyketide synthase (PKS I) gene from the epiphytic bacteria of Porphyra spp., in order to obtain the PKS I positive strains and detect the potential connection between the PKS pathway and the antibacterial mechanisms. A total of 31 bacteria with broad-spectrum antibacterial activity were screened by agar-screening methods. The 16S rDNA and the Ketosynthase gene were amplified from the genome DNA of these bacteria, which were cloned into pMD19-T vector for sequencing analysis. Porphyra spp. epiphytic bacteria showed broad-spectrum antibacterial activity. Three PKS I positive epiphytic bacteria were obtained from Wenzhou rotten Porphyra spp. samples which had high antibacterial activity. The BLAST results indicated that the Ketosynthase fragments of PKS I from the strains of WPhG3, WPySwl and WPySw2 shared highest similarity (98%, 99%, 98%) to the strains of Bacillus subtilis subsp. Subtilis str. 168 (NP_389602), Bacillus subtilis (ABR19776) and Aspergillus carbonarius (AAZ99721), respectively. Furthermore, the phylogenetic analysis based on 16S rDNA sequences indicated that they belonged to the genus of Bacillus. The flora of Porphyra spp. epiphytic bacteria was complex, which regulated the phycosphere in many ways. The PKS I pathway might be a performance of antibacterial function of Bacillus from Wenzhou rotten samples.

  9. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  10. Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2008-03-01

    Full Text Available Abstract Background β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha. Results The P. chrysogenum pcbC gene encoding isopenicillin N synthase (IPNS was successfully expressed in H. polymorpha, but the protein produced was unstable and inactive when the host was grown at its optimal growth temperature (37°C. Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25°C or 30°C. Furthermore, IPNS produced at these lower cultivation temperatures was functionally active. Localization experiments demonstrated that, like in P. chrysogenum, in H. polymorpha IPNS is located in the cytosol. Conclusion In P. chrysogenum, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies. In this study, we focus on the cytosolic enzyme IPNS. Our data show that high amounts of functionally active IPNS enzyme can be produced in the heterologous host during cultivation at 25°C, the optimal growth temperature for P. chrysogenum. This is a new step forward in the metabolic reprogramming of H. polymorpha to produce penicillin.

  11. Impact of Environmental Thermal Stimulation on Activation of Hypothalamic Neuronal Nitric Oxide Synthase during the Prenatal Ontogenesis in Muscovy Ducks

    Directory of Open Access Journals (Sweden)

    Valery Dunai

    2012-01-01

    Full Text Available The aim of the study is to investigate the influence of prenatal temperature stimulation on neuronal NO synthase (nNOS expression in the anterior hypothalamus of Muscovy duck embryos. Experiments were performed on embryonic day (E E20, E23, E28, and E33 using histochemistry for identification of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d as marker of NOS-containing neurons. Until the experiments, all duck embryos were incubated under standard temperature conditions (37.5∘C. During 3 hours before the start of the experiments, one group was incubated at 37.5∘C (control group, the second was warm-experienced at 39∘C, and the third was cold-experienced at 34∘C. In normal and warm-incubated duck embryos, nNOS activity could be first detected on E23. Particularly, after cold stimulation, a significant increase in nNOS activity was found in all embryos investigated even on day 20. Warm stimulation obviously induces the opposite effect, but at later embryonic age (E33. It can be concluded that probably in late-term bird embryos NO acts as a mediator of the neuronal cold pathway in the anterior hypothalamus, which might be improved by prenatal cold stimulation.

  12. Impact of environmental thermal stimulation on activation of hypothalamic neuronal nitric oxide synthase during the prenatal ontogenesis in Muscovy ducks.

    Science.gov (United States)

    Dunai, Valery; Tzschentke, Barbara

    2012-01-01

    The aim of the study is to investigate the influence of prenatal temperature stimulation on neuronal NO synthase (nNOS) expression in the anterior hypothalamus of Muscovy duck embryos. Experiments were performed on embryonic day (E) E20, E23, E28, and E33 using histochemistry for identification of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as marker of NOS-containing neurons. Until the experiments, all duck embryos were incubated under standard temperature conditions (37.5°C). During 3 hours before the start of the experiments, one group was incubated at 37.5°C (control group), the second was warm-experienced at 39°C, and the third was cold-experienced at 34°C. In normal and warm-incubated duck embryos, nNOS activity could be first detected on E23. Particularly, after cold stimulation, a significant increase in nNOS activity was found in all embryos investigated even on day 20. Warm stimulation obviously induces the opposite effect, but at later embryonic age (E33). It can be concluded that probably in late-term bird embryos NO acts as a mediator of the neuronal cold pathway in the anterior hypothalamus, which might be improved by prenatal cold stimulation.

  13. Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

    Directory of Open Access Journals (Sweden)

    George P. Lisi

    2018-02-01

    Full Text Available The enzyme imidazole glycerol phosphate synthase (IGPS is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosylformimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR stimulates millisecond (ms timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent.

  14. Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

    Science.gov (United States)

    Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

    2013-08-21

    The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

  15. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  16. Reconstitution of Staphylococcus aureus Lipoteichoic Acid Synthase Activity Identifies Congo Red as a Selective Inhibitor.

    Science.gov (United States)

    Vickery, Christopher R; Wood, B McKay; Morris, Heidi G; Losick, Richard; Walker, Suzanne

    2018-01-24

    Lipoteichoic acid (LTA) is an anionic surface polymer that is essential for normal growth of Staphylococcus aureus, making the LTA polymerase, LTA synthase (LtaS), a proposed drug target for combating Staphylococcal infections. LtaS is a polytopic membrane protein with five membrane-spanning helices and an extracellular domain, and it uses phosphatidylglycerol to assemble a glycerol phosphate chain on a glycosylated diacylglycerol membrane anchor. We report here the first reconstitution of LtaS polymerization activity and show that the azo dye Congo red inhibits this enzyme both in vitro and in cells. Related azo dyes and the previously reported LtaS inhibitor 1771 have weak or no in vitro inhibitory activity. Synthetic lethality with mutant strains known to be nonviable in the absence of LTA confirms selective inhibition by Congo red. As the only validated LtaS inhibitor, Congo red can serve as a probe to understand how inhibiting lipoteichoic acid biosynthesis affects cell physiology and may also guide the discovery of more potent inhibitors for use in treating S. aureus infections.

  17. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Andersen, Nynne Bjerre; Dela, Flemming

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and chan......Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity......, synthase, human, skeletal, muscle, training, not electrical stimulation, not in-vitro, not rats. Studies that reported changes in CS activity and V.O2max were included. Different training types and subject populations were analyzed independently to assess correlation between relative changes in V.O2max.......4). Training induced changes in whole body oxidative capacity is matched by changes in muscle CS activity in a nearly 1:1 relationship. Absolute values of CS across different studies cannot be compared unless a standardized analytical method is used by all laboratories...

  18. Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

    Science.gov (United States)

    Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

    2006-09-01

    Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  19. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Directory of Open Access Journals (Sweden)

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  20. Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

    Science.gov (United States)

    Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

    2013-11-06

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

  1. Substitution of Two Active-Site Residues Alters C9-Hydroxylation in a Class II Diterpene Synthase.

    Science.gov (United States)

    Mafu, Sibongile; Fischer, Emil; Addison, J Bennett; Riberio Barbosana, Isabel; Zerbe, Philipp

    2016-12-14

    Diterpenes form a vast and diverse class of natural products of both ecological and economic importance. Class II diterpene synthase (diTPS) enzymes control the committed biosynthetic reactions underlying diterpene chemical diversity. Homology modelling with site-directed mutagenesis identified two active-site residues in the horehound (Marrubium vulgare) class II diTPS peregrinol diphosphate synthase (MvCPS1); residue substitutions abolished the unique MvCPS1-catalysed water-capture reaction at C9 and redirected enzyme activity toward formation of an alternative product, halima-5(10),13-dienyl diphosphate. These findings contributed new insight into the steric interactions that govern diTPS-catalysed regiospecific oxygenation reactions and highlight the feasibility of diTPS engineering to provide a broader spectrum of bioactive diterpene natural products. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

    Science.gov (United States)

    Gebril, Sayed; Seger, Mark; Villanueva, Fabiola Muro; Ortega, Jose Luis; Bagga, Suman; Sengupta-Gopalan, Champa

    2015-10-01

    Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

  3. Soluble Starch Synthase III-1 in Amylopectin Metabolism of Banana Fruit: Characterization, Expression, Enzyme Activity, and Functional Analyses

    Science.gov (United States)

    Miao, Hongxia; Sun, Peiguang; Liu, Qing; Jia, Caihong; Liu, Juhua; Hu, Wei; Jin, Zhiqiang; Xu, Biyu

    2017-01-01

    Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline during storage. The SS activity started to increase later than amylopectin and starch content. Secondly, four putative SS genes were cloned and characterized from banana fruit. Among them, MaSSIII-1 showed the highest expression in banana pulp during fruit development at transcriptional levels. Further Western blot analysis suggested that the protein was gradually increased during banana fruit development, but drastically reduced during storage. This expression pattern was highly consistent with changes in starch granules, amylopectin content, and SS activity at the late phase of banana fruit development. Lastly, overexpression of MaSSIII-1 in tomato plants distinctly changed the morphology of starch granules and significantly increased the total starch accumulation, amylopectin content, and SS activity at mature-green stage in comparison to wild-type. The findings demonstrated that MaSSIII-1 is a key gene expressed in banana fruit and responsible for the active amylopectin biosynthesis, this is the first report in a fresh fruit species. Such a finding may enable the development of molecular markers for banana breeding and genetic improvement of nutritional value and functional properties of banana fruit. PMID:28424724

  4. Soluble Starch Synthase III-1 in Amylopectin Metabolism of Banana Fruit: Characterization, Expression, Enzyme Activity, and Functional Analyses.

    Science.gov (United States)

    Miao, Hongxia; Sun, Peiguang; Liu, Qing; Jia, Caihong; Liu, Juhua; Hu, Wei; Jin, Zhiqiang; Xu, Biyu

    2017-01-01

    Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline during storage. The SS activity started to increase later than amylopectin and starch content. Secondly, four putative SS genes were cloned and characterized from banana fruit. Among them, MaSSIII-1 showed the highest expression in banana pulp during fruit development at transcriptional levels. Further Western blot analysis suggested that the protein was gradually increased during banana fruit development, but drastically reduced during storage. This expression pattern was highly consistent with changes in starch granules, amylopectin content, and SS activity at the late phase of banana fruit development. Lastly, overexpression of MaSSIII-1 in tomato plants distinctly changed the morphology of starch granules and significantly increased the total starch accumulation, amylopectin content, and SS activity at mature-green stage in comparison to wild-type. The findings demonstrated that MaSSIII-1 is a key gene expressed in banana fruit and responsible for the active amylopectin biosynthesis, this is the first report in a fresh fruit species. Such a finding may enable the development of molecular markers for banana breeding and genetic improvement of nutritional value and functional properties of banana fruit.

  5. Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Mogensen, Martin; Petersen, Ingrid

    2005-01-01

    of these metabolic malfunctions, we studied mitochondrial respiration, uncoupled respiration and oxidative enzyme activities (citrate synthase (CS), 3-hydroxy-acyl-CoA-dehydrogenase activity (HAD)) before and after acute exposure to insulin and/or palmitate in myotubes established from healthy lean and obese...... subjects. Co-incubation with palmitate (0.6 mM) for 4 h abolished the stimulatory effect of insulin on CS activity in non-diabetic myotubes. No differences were detected in mitochondrial respiration and HAD activity between myotubes from non-diabetic subjects and T2D patients, and none of these measures...

  6. β-Carboline harmine reverses the effects induced by stress on behaviour and citrate synthase activity in the rat prefrontal cortex.

    Science.gov (United States)

    Abelaira, Helena Mendes; Réus, Gislaine Zilli; Scaini, Giselli; Streck, Emilio Luiz; Crippa, José Alexandre; Quevedo, João

    2013-12-01

    The present study was aimed at evaluating the effects of the administration of β-carboline harmine on behaviour and citrate synthase activity in the brain of rats exposed to chronic mild stress (CMS) procedure. To this aim, after 40 days of exposure to CMS procedure, rats were treated with harmine (15 mg/kg/day) for 7 days, then memory, anhedonia and citrate synthase activity were assessed. Result Our findings demonstrated that stressed rats treated with saline increased the sucrose intake, and the stressed rats treated with harmine reversed this effect. Neither stress nor harmine treatment altered memory performance in rats. In addition, chronic stressful situations induced increase in citrate synthase activity in the prefrontal cortex, but not in the hippocampus and striatum. Treatment with harmine reversed the increase in citrate synthase activity in the prefrontal cortex. These findings support the hypothesis that harmine could be involved in controlling the energy metabolism.

  7. Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

    International Nuclear Information System (INIS)

    Miziorko, H.M.; Behnke, C.E.

    1985-01-01

    3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

  8. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein-protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

  10. Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

    NARCIS (Netherlands)

    Kol, Matthijs|info:eu-repo/dai/nl/25059434X; Panatala Narendranath, Radhakrishnan|info:eu-repo/dai/nl/315032162; Nordmann, Mirjana; Swart, Leoni; Van Suijlekom, Leonie; Cabukusta, Birol; Hilderink, Angelika; Grabietz, Tanja; Mina, John G M; Somerharju, Pentti; Korneev, Sergei; Tafesse, Fikadu G; Holthuis, Joost C.M.|info:eu-repo/dai/nl/153674520

    2017-01-01

    SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma

  11. Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle.

    Science.gov (United States)

    Wojtaszewski, Jørgen F P; Jørgensen, Sebastian B; Hellsten, Ylva; Hardie, D Grahame; Richter, Erik A

    2002-02-01

    5'-AMP-activated protein kinase (AMPK) functions as a metabolic switch in mammalian cells and can be artificially activated by 5-aminoimidazole-4-carboxamide (AICA)-riboside. AMPK activation during muscle contraction is dependent on muscle glycogen concentrations, but whether glycogen also modifies the activation of AMPK and its possible downstream effectors (glycogen synthase and glucose transport) by AICA-riboside in resting muscle is not known. Thus, we have altered muscle glycogen levels in rats by a combination of swimming exercise and diet and investigated the effects of AICA-riboside in the perfused rat hindlimb muscle. Two groups of rats, one with super-compensated muscle glycogen content (approximately 200-300% of normal; high glycogen [HG]) and one with moderately lowered muscle glycogen content (approximately 80% of normal; low glycogen [LG]), were generated. In both groups, the degree of activation of the alpha2 isoform of AMPK by AICA-riboside depended on muscle type (white gastrocnemius > red gastrocnemius > soleus). Basal and AICA-riboside-induced alpha2-AMPK activity were markedly lowered in the HG group (approximately 50%) compared with the LG group. Muscle 2-deoxyglucose uptake was also increased and glycogen synthase activity decreased by AICA-riboside. Especially in white gastrocnemius, these effects, as well as the absolute activity levels of AMPK-alpha2, were markedly reduced in the HG group compared with the LG group. The inactivation of glycogen synthase by AICA-riboside was accompanied by decreased gel mobility and was eliminated by protein phosphatase treatment. We conclude that acute AICA-riboside treatment leads to phosphorylation and deactivation of glycogen synthase in skeletal muscle. Although the data do not exclude a role of other kinases/phosphatases, they suggest that glycogen synthase may be a target for AMPK in vivo. Both basal and AICA-riboside-induced AMPK-alpha2 and glycogen synthase activities, as well as glucose transport

  12. Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

    DEFF Research Database (Denmark)

    Glintborg, Dorte; Højlund, Kurt; Andersen, Nicoline Resen

    2008-01-01

    . No significant abnormalities in GSK-3alpha or -3beta were found in PCOS subjects. Pioglitazone treatment improved insulin-stimulated glucose metabolism and GS activity in PCOS (all P ...CONTEXT: Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. SUBJECTS AND METHODS: We...... investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were...

  13. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

    Science.gov (United States)

    Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

    2011-07-01

    The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either

  14. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

    Science.gov (United States)

    Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

    2017-01-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

  15. Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex

    Directory of Open Access Journals (Sweden)

    Tatiane Marcusso Orsini

    2016-01-01

    Full Text Available Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO, favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy2SO3(NO]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell.

  16. Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans.

    Science.gov (United States)

    Pacheco-Moisés, Fermín; Minauro-Sanmiguel, Fernando; Bravo, Concepción; García, José J

    2002-08-01

    The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur J. Biochem. 267, 993-1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal delta pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed.

  17. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  18. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  19. Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-01-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. PMID:21948846

  20. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    Science.gov (United States)

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

  1. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.

    Science.gov (United States)

    Maile, C A; Hingst, J R; Mahalingan, K K; O'Reilly, A O; Cleasby, M E; Mickelson, J R; McCue, M E; Anderson, S M; Hurley, T D; Wojtaszewski, J F P; Piercy, R J

    2017-01-01

    Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower K m for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Fu Y

    2014-06-01

    Full Text Available Yunfeng Fu,1 Xinyu Wang,1 Xiaodong Cheng,1 Feng Ye,2 Xing Xie,1,2 Weiguo Lu1,2 1Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, 2Women's Reproduction and Health Laboratory of Zhejiang Province, Hangzhou, People's Republic of China Background: Glycogen synthase kinase-3 (GSK-3 plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/βTyr279/216 in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer. Methods: Immunohistochemistry was used to examine expression of GSK-3α/β and pGSK-3α/βTyr279/216 in 71 human epithelial ovarian cancer tissues and correlations between protein expression, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay following exposure of ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice. Results: The expression levels of GSK-3α/β and pGSK-3α/βTyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3α/β was more likely to be found in patients with advanced International Federation of Gynecology and Obstetrics (FIGO stages and high serum cancer antigen 125. Higher expression of pGSK-3α/βTyr279/216 was associated with advanced FIGO stages, residual tumor mass, high serum cancer antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan–Meier survival curves in patients with high expression of GSK-3α/β or pGSK-3α/βTyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3α/β expression, and pGSK-3α/βTyr279/216 expression were independent prognostic factors for overall

  3. Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity

    Directory of Open Access Journals (Sweden)

    Arsa Thammahong

    2017-04-01

    Full Text Available Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB, which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions.

  4. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    Science.gov (United States)

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-10-02

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  5. The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3a activity in diabetic myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael; Brusgaard, Klaus; Handberg, Aa.

    2004-01-01

    in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein...

  6. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu [Purdue University, 175 South University Street, West Lafayette, IN 47907-2063 (United States)

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  7. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles

    DEFF Research Database (Denmark)

    Kolnes, Anders J; Birk, Jesper Bratz; Eilertsen, Einar

    2015-01-01

    Adrenaline increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated...... in condition with decreased GS activation. Saline or adrenaline (0.02mg/100g rat) was injected subcutaneously in Wistar rats (~130 g) with low (24 h fasted), normal (normal diet) and high glycogen content (fasted-refed) and epitrochlearis muscles were removed after 3 h and incubated ex vivo eliminating...... adrenaline action. Adrenaline injection reduced glycogen content in epitrochlearis muscles with high (120.7±17.8 vs 204.6±14.5 mmol•kg(-1); p

  8. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    International Nuclear Information System (INIS)

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-01-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

  9. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

    Directory of Open Access Journals (Sweden)

    Sheo B Singh

    Full Text Available Platensimycin (PTM is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+ with high de novo lipogenesis (DNL tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG, reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans.We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1 inhibitor.The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO mice as well as non-human primates (NHPs. Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice.These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

  10. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    Science.gov (United States)

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

  11. Enhanced growth and improved vascular function in offspring from successive pregnancies in endothelial nitric oxide synthase knockout mice

    NARCIS (Netherlands)

    Longo, M; Jain, [No Value; Langenveld, J; Vedernikov, YP; Garfield, RE; Hankins, GDV; Anderson, GD; Saade, GR

    2004-01-01

    Objective: Transgenic mice that lack endothelial nitric oxide synthase have offspring with growth deficiency and abnormal vascular reactivity in later life. Our objective was to evaluate the role of parity in the modulation of the fetal programming of growth and vascular responses in these

  12. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe Varieties

    Directory of Open Access Journals (Sweden)

    Ehsan Karimi

    2012-11-01

    Full Text Available The effect of foliar application of salicylic acid (SA at different concentrations (10−3 M and 10−5 M was investigated on the production of secondary metabolites (flavonoids, chalcone synthase (CHS activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231 in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS enzyme activity (involving flavonoid synthesis and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10−5 M SA treatment. As the SA concentration was decreased from 10−3 M to 10−5 M, the free radical scavenging power (FRAP increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 μg mL−1, the DPPH antioxidant activity recorded the highest value of 58.30%–72.90% with the 10−5 M SA treatment followed by the 10−3 M SA (52.14%–63.66% treatment. The lowest value was recorded in the untreated control plants (42.5%–46.7%. These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10−5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of

  13. Glycogen content regulates insulin- but not contraction-mediated glycogen synthase activation in the rat slow-twitch soleus muscles.

    Science.gov (United States)

    Lai, Y-C; Lin, F-C; Jensen, J

    2009-10-01

    The aim of this study was to investigate the effect of glycogen content on glycogen synthase (GS) activation and phosphorylation in the slow-twitch soleus muscles after contraction, during insulin stimulation and when these two stimuli were combined. Glycogen content was manipulated in vivo with 24 h fasting and fasting followed by 24 h refeeding. Soleus strips were electrically stimulated for 30 min in vitro, and GS activation and phosphorylation were investigated after an additional 30 min incubation with or without insulin. Fasting reduced glycogen content in soleus muscle by 40% and refeeding enhanced by 40%, compared to rats with free access to chow. Insulin-stimulated GS fractional activity was inversely correlated with glycogen content (R = -0.95, P glycogen synthesis was also inversely correlated with glycogen content (R = -0.70, P glycogen content; rate of glycogen synthesis after contraction was also similar. After contraction, insulin additively increased GS activation at all glycogen contents. Group means of GS fractional activity was inversely correlated with GS Ser(641) (R = -0.93, P Glycogen content regulates insulin- but not contraction-stimulated GS activation and glycogen synthesis in soleus muscles. Furthermore, phosphorylation of GS Ser(641) and Ser(645,649,653,657) seems to regulate GS activity in soleus.

  14. Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.

    Science.gov (United States)

    Prosser, Ian; Altug, Iris G; Phillips, Andy L; König, Wilfried A; Bouwmeester, Harro J; Beale, Michael H

    2004-12-15

    The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant-insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D,E), Sc11 is unusual in that this motif occurs as (303)NDTYD. Mutagenesis of this motif to (303)DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.

  15. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    Science.gov (United States)

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction. Copyright © 2015 the American Physiological Society.

  16. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    Science.gov (United States)

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro.

  17. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    Science.gov (United States)

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  18. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

    Directory of Open Access Journals (Sweden)

    Hildgund Schrempf

    2010-09-01

    Full Text Available A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein, has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

  19. Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

    Science.gov (United States)

    SEPAROVIC, DUSKA; BREEN, PAUL; BOPPANA, NITHIN B.; VAN BUREN, ERIC; JOSEPH, NICHOLAS; KRAVEKA, JACQUELINE M.; RAHMANIYAN, MEHRDAD; LI, LI; GUDZ, TATYANA I.; BIELAWSKA, ALICJA; BAI, AIPING; BIELAWSKI, JACEK; PIERCE, JASON S.; KORBELIK, MLADEN

    2013-01-01

    Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of

  20. Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation.

    Directory of Open Access Journals (Sweden)

    Lisa A Ridnour

    Full Text Available Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1 has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

  1. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  2. Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development.

    Science.gov (United States)

    Corpas, Francisco J; Barroso, Juan B; Carreras, Alfonso; Valderrama, Raquel; Palma, José M; León, Ana M; Sandalio, Luisa M; del Río, Luis A

    2006-07-01

    Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from L-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.

  3. Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites.

    Science.gov (United States)

    Tarun, A S; Theologis, A

    1998-05-15

    The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.

  4. Motor activity improves temporal expectancy.

    Directory of Open Access Journals (Sweden)

    Lilian Fautrelle

    Full Text Available Certain brain areas involved in interval timing are also important in motor activity. This raises the possibility that motor activity might influence interval timing. To test this hypothesis, we assessed interval timing in healthy adults following different types of training. The pre- and post-training tasks consisted of a button press in response to the presentation of a rhythmic visual stimulus. Alterations in temporal expectancy were evaluated by measuring response times. Training consisted of responding to the visual presentation of regularly appearing stimuli by either: (1 pointing with a whole-body movement, (2 pointing only with the arm, (3 imagining pointing with a whole-body movement, (4 simply watching the stimulus presentation, (5 pointing with a whole-body movement in response to a target that appeared at irregular intervals (6 reading a newspaper. Participants performing a motor activity in response to the regular target showed significant improvements in judgment times compared to individuals with no associated motor activity. Individuals who only imagined pointing with a whole-body movement also showed significant improvements. No improvements were observed in the group that trained with a motor response to an irregular stimulus, hence eliminating the explanation that the improved temporal expectations of the other motor training groups was purely due to an improved motor capacity to press the response button. All groups performed a secondary task equally well, hence indicating that our results could not simply be attributed to differences in attention between the groups. Our results show that motor activity, even when it does not play a causal or corrective role, can lead to improved interval timing judgments.

  5. Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.

    Science.gov (United States)

    Chang, Albert S; Grant, Ruriko; Tomita, Hirofumi; Kim, Hyung-Suk; Smithies, Oliver; Kakoki, Masao

    2016-11-01

    Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

  6. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    Science.gov (United States)

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  8. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    Science.gov (United States)

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (parginase activity (pactivity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    International Nuclear Information System (INIS)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-01-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

  10. NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

    Science.gov (United States)

    Dunn, Michael F.

    2013-01-01

    crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

  11. Chamomile: an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity.

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-12-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.

  12. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ , the upstream kinase regulating NF-κ B/Rel activity, and degradation of inhibitory factor-κ B. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent. PMID:21042790

  13. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  14. PWR system reliability improvement activities

    International Nuclear Information System (INIS)

    Yoshikawa, Yuichiro

    1985-01-01

    In Japan lacking in energy resources, it is our basic energy policy to accelerate the development program of nuclear power, thereby reducing our dependence. As referred to in the foregoing, every effort has been exerted on our part to improve the PWR system reliability by dint of the so-called 'HOMEMADE' TQC activities, which is our brain-child as a result of applying to the energy industry the quality control philosophy developed in the field of manufacturing industry

  15. Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β

    Science.gov (United States)

    Zhou, Mingjie; Ren, Huanhuan; Wang, Wenjuan; Zheng, Qiusheng; Wang, Dong

    2015-01-01

    Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dt max) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dt max, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R. PMID:26265983

  16. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    Directory of Open Access Journals (Sweden)

    Shailendra P. Singh

    2015-08-01

    Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

  17. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles.

    Science.gov (United States)

    Kolnes, Anders J; Birk, Jesper B; Eilertsen, Einar; Stuenæs, Jorid T; Wojtaszewski, Jørgen F P; Jensen, Jørgen

    2015-02-01

    Epinephrine increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown, and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated in condition with decreased GS activation. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously in Wistar rats (∼130 g) with low (24-h-fasted), normal (normal diet), and high glycogen content (fasted-refed), and epitrochlearis muscles were removed after 3 h and incubated ex vivo, eliminating epinephrine action. Epinephrine injection reduced glycogen content in epitrochlearis muscles with high (120.7 ± 17.8 vs. 204.6 ± 14.5 mmol/kg, P muscles with low glycogen (90.0 ± 5.0 vs. 102.8 ± 7.8 mmol/kg, P = 0.17). In saline-injected rats, GS phosphorylation at sites 2+2a, 3a+3b, and 1b was higher and GS activity lower in muscles with high compared with low glycogen. GS sites 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where epinephrine decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where epinephrine injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independently of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection decreased glycogen content. In conclusion, epinephrine stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low, glycogen content. Epinephrine-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content. Copyright © 2015 the American Physiological Society.

  18. Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

    Science.gov (United States)

    Clevenger, Kenneth D; Ye, Rosa; Bok, Jin Woo; Thomas, Paul M; Islam, Md Nurul; Miley, Galen P; Robey, Matthew T; Chen, Cynthia; Yang, KaHoua; Swyers, Michael; Wu, Edward; Gao, Peng; Wu, Chengcang C; Keller, Nancy P; Kelleher, Neil L

    2018-03-20

    The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-C T and the internal C-domains as BenZ-C 1 and BenZ-C 2 . Unexpectedly, we also uncovered evidence suggesting BenY-C T or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal C T domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

  19. The Influence of Hyperoxia On Heat Shock Proteins Expression and Nitric Oxide Synthase Activity – the Review

    Directory of Open Access Journals (Sweden)

    Szyller Jakub

    2017-03-01

    Full Text Available Any stay in an environment with an increased oxygen content (a higher oxygen partial pressure, pO2 and an increased pressure (hyperbaric conditions leads to an intensification of oxidative stress. Reactive oxygen species (ROS damage the molecules of proteins, nucleic acids, cause lipid oxidation and are engaged in the development of numerous diseases, including diseases of the circulatory system, neurodegenerative diseases, etc. There are certain mechanisms of protection against unfavourable effects of oxidative stress. Enzymatic and non-enzymatic systems belong to them. The latter include, among others, heat shock proteins (HSP. Their precise role and mechanism of action have been a subject of intensive research conducted in recent years. Hyperoxia and hyperbaria also have an effect on the expression and activity of nitrogen oxide synthase (NOS. Its product - nitrogen oxide (NO can react with reactive oxygen species and contribute to the development of nitrosative stress. NOS occurs as isoforms in various tissues and exhibit different reactions to the discussed factors. The authors have prepared a brief review of research determining the effect of hyperoxia and hyperbaria on HSP expression and NOS activity.

  20. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. A novel pathway for inducible nitric-oxide synthase activation through inflammasomes.

    Science.gov (United States)

    Buzzo, Carina L; Campopiano, Julia C; Massis, Liliana M; Lage, Silvia L; Cassado, Alexandra A; Leme-Souza, Rafael; Cunha, Larissa D; Russo, Momtchilo; Zamboni, Dario S; Amarante-Mendes, Gustavo P; Bortoluci, Karina R

    2010-10-15

    Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1β and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

  2. A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes*

    Science.gov (United States)

    Buzzo, Carina L.; Campopiano, Julia C.; Massis, Liliana M.; Lage, Silvia L.; Cassado, Alexandra A.; Leme-Souza, Rafael; Cunha, Larissa D.; Russo, Momtchilo; Zamboni, Dario S.; Amarante-Mendes, Gustavo P.; Bortoluci, Karina R.

    2010-01-01

    Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1β and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS. PMID:20702413

  3. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  4. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    International Nuclear Information System (INIS)

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-01-01

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression

  5. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    Science.gov (United States)

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

  6. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

    Directory of Open Access Journals (Sweden)

    Cathy W C Bi

    Full Text Available Fo Shou San (FSS is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong and Angelicae Sinensis Radix (ASR; Danggui in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs, and it also stimulated the production of nitric oxide (NO as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+ levels in HUVECs, and the Ca(2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+ and a reduction of ROS.

  7. Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme.

    Science.gov (United States)

    Ghosh, D K; Abu-Soud, H M; Stuehr, D J

    1996-02-06

    The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemeprotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contains heme and H4biopterin, and binds L-arginine and a reductase domain fragment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the reduction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemistry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabilize the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spectral analysis of the dissociation reaction showed that it caused structural changes within the oxygenase domain and exposed the distal side of the heme to solvent, enabling it to bind dithiothreitol as a sixth ligand. Importantly, the oxygenase domain monomers could reassociate into a dimeric form even in the absence of the reductase domain. The reaction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain.

  8. Astaxanthin improves cognitive deficits from oxidative stress, nitric oxide synthase and inflammation through upregulation of PI3K/Akt in diabetes rat.

    Science.gov (United States)

    Xu, Lianbao; Zhu, Juan; Yin, Weibing; Ding, Xinsheng

    2015-01-01

    Diabetes-induced cognitive deficit (DICD) is a prevalent disease with substantial morbidity and mortality and as a global health problem with serious economic burdens. Astaxanthin (AST) has a good prospect in production of nutritional, medical, and particularly functional health drug. The present study was aimed to study the effect of AST on DICD in diabetes mellitus (DM) rat through suppression of oxidative stress, nitric oxide synthase (NOS) pathway, inflammatory reaction and upregulation of PI3K/Akt. In the study, Morris water maze teat was used to detect the cognitive function of DM rat. Afterwards, we measured the body weight and blood glucose levels of DM rats. Then, oxidative stress, the activities of eNOS and iNOS, and inflammatory factors were analyzed using a commercial kit in cerebral cortex and hippocampus. Finally, the caspase-3/9 and phosphoinositide 3-kinase (PI3K)/Akt expressions were also checkout with Real Time PCR and immunoblotting, respectively. In this experiment, AST could availably enhance the body weight and reduce blood glucose levels of DM rats. Moreover, AST could observably perfect cognitive function of DM rat. Next, the activities of oxidative stress, nitric oxide synthase and inflammation were distinctly diminution in DM rat, after the treatment of AST. Furthermore, our present results demonstrated that AST had the protective effect on the brain cell of DM rat, decreased the caspase-3/9 expression and promoted the expression of PI3K/Akt in cerebral cortex and hippocampus.

  9. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

    Science.gov (United States)

    Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

    2013-11-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Observation of an unusual electronically distorted semiquinone radical of PCB metabolites in the active site of prostaglandin H synthase-2.

    Science.gov (United States)

    Wangpradit, Orarat; Moman, Edelmiro; Nolan, Kevin B; Buettner, Garry R; Robertson, Larry W; Luthe, Gregor

    2010-12-01

    The activation of the metabolites of airborne polychlorinated biphenyls (PCBs) into highly reactive radicals is of fundamental importance. We found that human recombinant prostaglandin H synthase-2 (hPGHS-2) biotransforms dihydroxy-PCBs, such as 4-chlorobiphenyl-2',5'-hydroquinone (4-CB-2',5'-H(2)Q), into semiquinone radicals via one-electron oxidation. Using electron paramagnetic resonance (EPR) spectroscopy, we observed the formation of the symmetric quartet spectrum (1:3:3:1 by area) of 4-chlorobiphenyl-2',5'-semiquinone radical (4-CB-2',5'-SQ()(-)) from 4-CB-2',5'-H(2)Q. This spectrum changed to an asymmetric spectrum with time: the change can be explained as the overlap of two different semiquinone radical species. Hindered rotation of the 4-CB-2',5'-SQ()(-) appears not to be a major factor for the change in lineshape because increasing the viscosity of the medium with glycerol produced no significant change in lineshape. Introduction of a fluorine, which increases the steric hindrance for rotation of the dihydroxy-PCB studied, also produced no significant changes. An in silico molecular docking model of 4-CB-2',5'-H(2)Q in the peroxidase site of hPGHS-2 together with ab initio quantum mechanical studies indicate that the close proximity of a negatively charged carboxylic acid in the peroxidase active site may be responsible for the observed perturbation in the spectrum. This study provides new insights into the formation of semiquinones from PCB metabolites and underscores the potential role of PGHS-2 in the metabolic activation of PCBs. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Comparative study of enzyme activity and heme reactivity in Drosophila melanogaster and Homo sapiens cystathionine β-synthases.

    Science.gov (United States)

    Su, Yang; Majtan, Tomas; Freeman, Katherine M; Linck, Rachel; Ponter, Sarah; Kraus, Jan P; Burstyn, Judith N

    2013-01-29

    Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly Drosophila melanogaster (DmCBS) and the CO/NO gas binding reactions of DmCBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), DmCBS is intrinsically highly active and is not regulated by AdoMet. The DmCBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The DmCBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe(II) heme in DmCBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and DmCBS Fe(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme.

  12. Lithium Enhances Axonal Regeneration in Peripheral Nerve by Inhibiting Glycogen Synthase Kinase 3β Activation

    Directory of Open Access Journals (Sweden)

    Huanxing Su

    2014-01-01

    Full Text Available Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS.

  13. Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

    OpenAIRE

    Gregus, Ann M.; Dumlao, Darren S.; Wei, Spencer C.; Norris, Paul C.; Catella, Laura C.; Meyerstein, Flore G.; Buczynski, Matthew W.; Steinauer, Joanne J.; Fitzsimmons, Bethany L.; Yaksh, Tony L.; Dennis, Edward A.

    2013-01-01

    Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presenc...

  14. A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

    International Nuclear Information System (INIS)

    Grum, Daniel; Boom, Johannes van den; Neumann, Daniel; Matena, Anja; Link, Nina M.; Mueller, Jonathan W.

    2010-01-01

    3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

  15. Cooperative functioning between phenylalanine ammonia lyase and isochorishmate synthase activities contributes to salicylic acid biosynthesis in soybean

    Science.gov (United States)

    Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL), or the isochorishmate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We...

  16. The time-dependent effect of provinolsTM on brain NO synthase activity in L-NAME-induced hypertension

    Czech Academy of Sciences Publication Activity Database

    Jendeková, L.; Kojšová, S.; Andriantsitohaina, R.; Pecháňová, Olga

    2006-01-01

    Roč. 55, č. S1 (2006), S31-S37 ISSN 0862-8408 Grant - others:VEGA(SK) 2/6148/26; VEGA(SK) 1/342906 Institutional research plan: CEZ:AV0Z50110509 Keywords : red wine polyphenols * oxidative damage * nitric oxide synthase Subject RIV: ED - Physiology Impact factor: 2.093, year: 2006

  17. The expression and activity of nitric oxide synthase in brainstem and hypothalamic nuclei of adult SHR: Endothelial vs. neuronal isoforms

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Zicha, Josef; Kuneš, Jaroslav

    2006-01-01

    Roč. 24, č. S4 (2006), S345-S346 ISSN 0263-6352. [European Meeting on Hypertension /16./. 12.06.2006-15.06.2006, Madrid] R&D Projects: GA MZd(CZ) NR7786 Keywords : nitric oxide synthase * brain * hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  18. Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes.

    Science.gov (United States)

    Nassi, Alberto; Malorgio, Francesca; Tedesco, Serena; Cignarella, Andrea; Gaion, Rosa Maria

    2016-02-16

    Adenosine has been shown to induce nitric oxide (NO) production via inducible NO synthase (iNOS) activation in vascular smooth muscle cells (VSMCs). Although this is interpreted as a beneficial vasodilating pathway in vaso-occlusive disorders, iNOS is also involved in diabetic vascular dysfunction. Because the turnover of and the potential to modulate iNOS by adenosine in experimental diabetes have not been explored, we hypothesized that both the adenosine system and control of iNOS function are impaired in VSMCs from streptozotocin-diabetic rats. Male Sprague-Dawley rats were injected with streptozotocin once to induce diabetes. Aortic VSMCs from diabetic and nondiabetic rats were isolated, cultured and exposed to lipopolysaccharide (LPS) plus a cytokine mix for 24 h in the presence or absence of (1) exogenous adenosine and related compounds, and/or (2) pharmacological agents affecting adenosine turnover. iNOS functional expression was determined by immunoblotting and NO metabolite assays. Concentrations of adenosine, related compounds and metabolites thereof were assayed by HPLC. Vasomotor responses to adenosine were determined in endothelium-deprived aortic rings. Treatment with adenosine-degrading enzymes or receptor antagonists increased iNOS formation in activated VSMCs from nondiabetic and diabetic rats. Following treatment with the adenosine transport inhibitor NBTI, iNOS levels increased in nondiabetic but decreased in diabetic VSMCs. The amount of secreted NO metabolites was uncoupled from iNOS levels in diabetic VSMCs. Addition of high concentrations of adenosine and its precursors or analogues enhanced iNOS formation solely in diabetic VSMCs. Exogenous adenosine and AMP were completely removed from the culture medium and converted into metabolites. A tendency towards elevated inosine generation was observed in diabetic VSMCs, which were also less sensitive to CD73 inhibition, but inosine supplementation did not affect iNOS levels. Pharmacological

  19. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...

  20. Physical Activity Improves Quality of Life

    Science.gov (United States)

    ... Healthy Workplace Food and Beverage Toolkit Physical activity improves quality of life Updated:Mar 2,2015 Do ... becomes a part of their routine. Physical activity improves physical wellness. Reduced Risk Factors Too much sitting ...

  1. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  2. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  3. Soluble Starch Synthase III-1 in Amylopectin Metabolism of Banana Fruit: Characterization, Expression, Enzyme Activity, and Functional Analyses

    OpenAIRE

    Miao, Hongxia; Sun, Peiguang; Liu, Qing; Jia, Caihong; Liu, Juhua; Hu, Wei; Jin, Zhiqiang; Xu, Biyu

    2017-01-01

    Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline duri...

  4. A Systems Chemical Biology Study of Malate Synthase and Isocitrate Lyase Inhibition in Mycobacterium tuberculosis During Active and NRP Growth

    Science.gov (United States)

    May, Elebeoba E.; Leitão, Andrei; Tropsha, Alexander; Oprea, Tudor I.

    2013-01-01

    The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistent under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system. To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption. Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence. PMID:24121675

  5. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...

  6. The Effect of Aging and Exogenous Testosterone Replacement on Nitric Oxide Concentration and Activity of Nitric Oxide Synthase in the Rat Corpus Cavernosum

    OpenAIRE

    Shiono, Manabu

    2001-01-01

    The effects of testosterone replacement on the nitric oxide (NO) concentration and activity of NO synthase (NOS) in the penis were investigated. Male Wistar rats (n = 39) were divided into 5 groups: 14-week-old males, 13-month-old males, 15-month-old males, 15-month-old males treated with low-dose testosterone replacement and 15-month-old males treated with high-dose testosterone replacement. The testosterone concentration in serum and the NO concentration in penile tissue were measured, and ...

  7. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

    OpenAIRE

    Herasimenka, Yury; Kotasinska, Marta; Walter, Stefan; Schrempf, Hildgund

    2010-01-01

    A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin...

  8. Studies of gene expression and activity of hexokinase, phosphofructokinase and glycogen synthase in human skeletal muscle in states of altered insulin-stimulated glucose metabolism

    DEFF Research Database (Denmark)

    Vestergaard, H

    1999-01-01

    of the review is to discuss our present knowledge of the activities and gene expression of hexokinase II (HKII), phosphofructokinase (PFK) and glycogen synthase (GS) in human skeletal muscle in states of altered insulin-stimulated glucose metabolism. My own experimental studies have comprised patients...... been reported to increase the basal concentration of muscle GS mRNA in NIDDM patients to a level similar to that seen in control subjects although insulin-stimulated glucose disposal rates remain reduced in NIDDM patients. In the insulin resistant states examined so far, basal and insulin...

  9. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and...

  10. Role of 5′AMP-activated protein kinase in glycogen synthase activity and glucose utilization: insights from patients with McArdle's disease

    Science.gov (United States)

    Nielsen, Jakob N; Wojtaszewski, Jørgen F P; Haller, Ronald G; Hardie, D Grahame; Kemp, Bruce E; Richter, Erik A; Vissing, John

    2002-01-01

    It has been suggested that 5′AMP-activated protein kinase (AMPK) is involved in the regulation of glucose and glycogen metabolism in skeletal muscle. We used patients with chronic high muscle glycogen stores and deficient glycogenolysis (McArdle's disease) as a model to address this issue. Six McArdle patients were compared with control subjects during exercise. Muscle α2AMPK activity increased in McArdle patients (from 1.3 ± 0.2 to 1.9 ± 0.2 pmol min−1 mg−1, P = 0.05) but not in control subjects (from 1.0 ± 0.1 to 1.3 ± 0.3 pmol min−1 mg−1). Exercise-induced phosphorylation of the in vivo AMPK substrate acetyl CoA carboxylase (ACCβ; Ser221) was higher (P < 0.01) in McArdle patients than in control subjects (18 ± 3 vs. 10 ± 1 arbitrary units). Exercise-induced whole-body glucose utilization was also higher in McArdle patients than in control subjects (P < 0.05). No correlation between individual AMPK or ACCβ values and glucose utilization was observed. Glycogen synthase (GS) activity was decreased in McArdle patients from 11 ± 1.3 to 5 ± 1.2 % (P < 0.05) and increased in control subjects from 19 ± 1.6 to 23 ± 2.3 % (P < 0.05) in response to exercise. This was not associated with activity changes of GS kinase 3 or protein phosphatase 1, but the changes in GS activity could be due to changes in activity of AMPK or protein kinase A (PKA) as a negative correlation between either ACCβ phosphorylation (Ser221) or plasma adrenaline and GS activity was observed. These findings suggest that GS activity is increased by glycogen breakdown and decreased by AMPK and possibly PKA activation and that the resultant GS activity depends on the relative strengths of the various stimuli. Furthermore, AMPK may be involved in the regulation of glucose utilization during exercise in humans, although the lack of correlation between individual AMPK activity or ACCβ phosphorylation (Ser221) values and individual glucose utilization during exercise implies that AMPK

  11. The Nicotianamine Synthase Gene Is a Useful Candidate for Improving the Nutritional Qualities and Fe-Deficiency Tolerance of Various Crops

    Directory of Open Access Journals (Sweden)

    Tomoko Nozoye

    2018-03-01

    Full Text Available With the global population predicted to grow by at least 25% by the year 2050, the sustainable production of nutritious foods will be necessary for human health and the environment. Iron (Fe is an essential nutrient for both plants and humans. Fe is poorly soluble, especially at high pH levels, at which it is difficult for living organisms to accumulate sufficient Fe. In plants, Fe deficiency leads to low yield and poor nutritional quality, as it significantly affects chlorophyll synthesis. Fe deficiency is a worldwide agricultural problem that is especially serious in soils with a high pH, such as calcareous soils, which comprise approximately 30% of cultivated soils worldwide. Genetic improvements in crops that can tolerate Fe deficiency will be required to meet the demands for crop production and could ultimately contribute to the amelioration of global warming. Nicotianamine (NA is an Fe chelator in plants that is involved in metal translocation in the plant body. In mammals, NA inhibits angiotensin I-converting enzyme, which plays a key role in blood pressure control. It was recently shown that the enhancement of NA production using nicotianamine synthase is useful for increasing not only NA but also Fe and Zn levels in crops such as rice, soybean, and sweet potato. Additionally, these plants showed Fe-deficiency tolerance in calcareous soil. These results suggested that NAS overexpression simultaneously improves food quality and increases plant production. This review summarizes progress in generating crops overexpressing NAS.

  12. Exercise training provides cardioprotection by activating and coupling endothelial nitric oxide synthase via a β3-adrenergic receptor-AMP-activated protein kinase signaling pathway

    Directory of Open Access Journals (Sweden)

    Larry A Barr

    2017-01-01

    Full Text Available Exercise training confers sustainable protection against ischemia/reperfusion injury. However, the mechanism by which this process occurs is not fully understood. Previously, it was shown that β3-adrenergic receptors (β3-ARs play a critical role in regulating the activation of endothelial nitric oxide synthase (eNOS in response to exercise and play a critical role in exercise-mediated cardioprotection. Intriguingly, a deficiency in β3-ARs led to increased myocardial injury following exercise training. The purpose of the current study was to determine mechanisms by which β3-ARs are linked to eNOS activation and to determine the mechanism responsible for the exacerbated ischemia/reperfusion injury displayed by β3-AR deficient (β3-AR KO mice after exercise training. Wild-type (n = 37 and β3-AR KO ( n = 40 mice were subjected to voluntary wheel running for 4 weeks. Western blot analysis revealed that neither protein kinase B nor protein kinase A linked β3-ARs to eNOS following exercise training. However, analysis revealed a role for AMP-activated protein kinase (AMPK. Specifically, exercise training increased the phosphorylation of AMPK in the hearts of wild-type mice, but failed to do so in the hearts of β3-AR KO mice. Additional studies revealed that exercise training rendered eNOS less coupled and increased NOS-dependent superoxide levels in β3-AR KO mice. Finally, supplementing β3-AR KO mice with the eNOS coupler, tetrahydrobiopterin, during the final week of exercise training reduced myocardial infarction. These findings provide important information that exercise training protects the heart in the setting of myocardial ischemia/reperfusion injury by activating and coupling eNOS via the stimulation of a β3-AR-AMPK signaling pathway.

  13. Pharmacologic inhibition of sphingomyelin synthase (SMS) activity reduces apolipoprotein-B secretion from hepatocytes and attenuates endotoxin-mediated macrophage inflammation.

    Science.gov (United States)

    Lou, Bin; Dong, Jibin; Li, Yali; Ding, Tingbo; Bi, Tingting; Li, Yue; Deng, Xiaodong; Ye, Deyong; Jiang, Xian-Cheng

    2014-01-01

    Sphingomyelin synthase (SMS) plays an important role in plasma atherogenic lipoprotein metabolism, inflammation, and the development of atherosclerosis. To understand whether the impaired apoB secretion and inflammation response is a direct result from lack of SMS activity, in this study, we prepared a series of compounds that inhibit SMS activity. Further, we characterized Dy105, the most potent inhibitor. We found that Dy105 treatment significantly reduces SM levels in SM-rich microdomain on cell membranes. Moreover, we found that SMS inhibition reduces apoB secretion in a human hepatoma cell line and reduces the activation of NFκB and p38, a MAP kinase, in bone marrow derived macrophages. These studies provided further evidence that SMS activity regulates atherogenic lipoprotein metabolism and inflammatory responses. Pharmacologic inhibition of SMS may be a new therapy for atherosclerosis by reducing apoB secretion, and reducing inflammation.

  14. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    OpenAIRE

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macropha...

  15. IMPROVING CAUSE DETECTION SYSTEMS WITH ACTIVE LEARNING

    Data.gov (United States)

    National Aeronautics and Space Administration — IMPROVING CAUSE DETECTION SYSTEMS WITH ACTIVE LEARNING ISAAC PERSING AND VINCENT NG Abstract. Active learning has been successfully applied to many natural language...

  16. TCA cycle activity in Staphylococcus aureus is essential for iron-regulated synthesis of staphyloferrin A, but not staphyloferrin B: the benefit of a second citrate synthase.

    Science.gov (United States)

    Sheldon, Jessica R; Marolda, Cristina L; Heinrichs, David E

    2014-05-01

    Staphylococcus aureus elaborates two citrate-containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron-restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron-regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron-restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB-deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron-restricted media, and is significantly more impaired for virulence than either of the single-deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron-restricted growth, without need of TCA cycle activity. © 2014 John Wiley & Sons Ltd.

  17. Microsomal prostaglandin E synthase type 2 (mPGES2) is a glutathione-dependent heme protein, and dithiothreitol dissociates the bound heme to produce active prostaglandin E2 synthase in vitro.

    Science.gov (United States)

    Takusagawa, Fusao

    2013-04-05

    An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.

  18. Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility

    Directory of Open Access Journals (Sweden)

    Florencia Díaz-Viraqué

    2018-03-01

    Full Text Available The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host–parasite interaction.

  19. Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy.

    Science.gov (United States)

    Suborov, Evgeny V; Smetkin, Alexey A; Kondratiev, Timofey V; Valkov, Andrey Y; Kuzkov, Vsevolod V; Kirov, Mikhail Y; Bjertnaes, Lars J

    2012-06-21

    Mechanical ventilation with high tidal volumes may cause ventilator-induced lung injury (VILI) and enhanced generation of nitric oxide (NO). We demonstrated in sheep that pneumonectomy followed by injurious ventilation promotes pulmonary edema. We wished both to test the hypothesis that neuronal NOS (nNOS), which is distributed in airway epithelial and neuronal tissues, could be involved in the pathogenesis of VILI and we also aimed at investigating the influence of an inhibitor of nNOS on the course of VILI after pneumonectomy. Anesthetized sheep underwent right pneumonectomy, mechanical ventilation with tidal volumes (VT) of 6 mL/kg and FiO2 0.5, and were subsequently randomized to a protectively ventilated group (PROTV; n = 8) keeping VT and FiO2 unchanged, respiratory rate (RR) 25 inflations/min and PEEP 4 cm H2O for the following 8 hrs; an injuriously ventilated group with VT of 12 mL/kg, zero end-expiratory pressure, and FiO2 and RR unchanged (INJV; n = 8) and a group, which additionally received the inhibitor of nNOS, 7-nitroindazole (NI) 1.0 mg/kg/h intravenously from 2 hours after the commencement of injurious ventilation (INJV + NI; n = 8). We assessed respiratory, hemodynamic and volumetric variables, including both the extravascular lung water index (EVLWI) and the pulmonary vascular permeability index (PVPI). We measured plasma nitrite/nitrate (NOx) levels and examined lung biopsies for lung injury score (LIS). Both the injuriously ventilated groups demonstrated a 2-3-fold rise in EVLWI and PVPI, with no significant effects of NI. In the INJV group, gas exchange deteriorated in parallel with emerging respiratory acidosis, but administration of NI antagonized the derangement of oxygenation and the respiratory acidosis significantly. NOx displayed no significant changes and NI exerted no significant effect on LIS in the INJV group. Inhibition of nNOS improved gas exchange, but did not reduce lung water extravasation following

  20. Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy

    Directory of Open Access Journals (Sweden)

    Suborov Evgeny V

    2012-06-01

    Full Text Available Abstract Background Mechanical ventilation with high tidal volumes may cause ventilator-induced lung injury (VILI and enhanced generation of nitric oxide (NO. We demonstrated in sheep that pneumonectomy followed by injurious ventilation promotes pulmonary edema. We wished both to test the hypothesis that neuronal NOS (nNOS, which is distributed in airway epithelial and neuronal tissues, could be involved in the pathogenesis of VILI and we also aimed at investigating the influence of an inhibitor of nNOS on the course of VILI after pneumonectomy. Methods Anesthetized sheep underwent right pneumonectomy, mechanical ventilation with tidal volumes (VT of 6 mL/kg and FiO2 0.5, and were subsequently randomized to a protectively ventilated group (PROTV; n = 8 keeping VT and FiO2 unchanged, respiratory rate (RR 25 inflations/min and PEEP 4 cm H2O for the following 8 hrs; an injuriously ventilated group with VT of 12 mL/kg, zero end-expiratory pressure, and FiO2 and RR unchanged (INJV; n = 8 and a group, which additionally received the inhibitor of nNOS, 7-nitroindazole (NI 1.0 mg/kg/h intravenously from 2 hours after the commencement of injurious ventilation (INJV + NI; n = 8. We assessed respiratory, hemodynamic and volumetric variables, including both the extravascular lung water index (EVLWI and the pulmonary vascular permeability index (PVPI. We measured plasma nitrite/nitrate (NOx levels and examined lung biopsies for lung injury score (LIS. Results Both the injuriously ventilated groups demonstrated a 2–3-fold rise in EVLWI and PVPI, with no significant effects of NI. In the INJV group, gas exchange deteriorated in parallel with emerging respiratory acidosis, but administration of NI antagonized the derangement of oxygenation and the respiratory acidosis significantly. NOx displayed no significant changes and NI exerted no significant effect on LIS in the INJV group. Conclusion Inhibition of nNOS improved gas exchange

  1. Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Dong Ju Son

    2014-08-01

    Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

  2. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  3. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    pathway for improving oil quality and increasing oil content of peanut through biotechnology-based approaches. Fatty acid biosynthesis is catalysed by two types of fatty acid synthase (FAS). Type I FAS, as found in vertebrates, yeast and some bacteria, contains all the active sites on one or two multidomain polypeptides.

  4. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    Science.gov (United States)

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  5. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  6. Activating clinical trials: a process improvement approach.

    Science.gov (United States)

    Martinez, Diego A; Tsalatsanis, Athanasios; Yalcin, Ali; Zayas-Castro, José L; Djulbegovic, Benjamin

    2016-02-24

    The administrative process associated with clinical trial activation has been criticized as costly, complex, and time-consuming. Prior research has concentrated on identifying administrative barriers and proposing various solutions to reduce activation time, and consequently associated costs. Here, we expand on previous research by incorporating social network analysis and discrete-event simulation to support process improvement decision-making. We searched for all operational data associated with the administrative process of activating industry-sponsored clinical trials at the Office of Clinical Research of the University of South Florida in Tampa, Florida. We limited the search to those trials initiated and activated between July 2011 and June 2012. We described the process using value stream mapping, studied the interactions of the various process participants using social network analysis, and modeled potential process modifications using discrete-event simulation. The administrative process comprised 5 sub-processes, 30 activities, 11 decision points, 5 loops, and 8 participants. The mean activation time was 76.6 days. Rate-limiting sub-processes were those of contract and budget development. Key participants during contract and budget development were the Office of Clinical Research, sponsors, and the principal investigator. Simulation results indicate that slight increments on the number of trials, arriving to the Office of Clinical Research, would increase activation time by 11 %. Also, incrementing the efficiency of contract and budget development would reduce the activation time by 28 %. Finally, better synchronization between contract and budget development would reduce time spent on batching documentation; however, no improvements would be attained in total activation time. The presented process improvement analytic framework not only identifies administrative barriers, but also helps to devise and evaluate potential improvement scenarios. The strength

  7. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  8. Methylmercury alters the activities of Hsp90 client proteins, prostaglandin E synthase/p23 (PGES/23 and nNOS.

    Directory of Open Access Journals (Sweden)

    Samuel Caito

    Full Text Available Methylmercury (MeHg is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS and Hsp90-prostaglandin E synthase/p23 (PGES/p23 were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2 synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2- and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity.

  9. Controlling Active Site Loop Dynamics in the (β/α8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

    Directory of Open Access Journals (Sweden)

    Kathleen F. O’Rourke

    2016-08-01

    Full Text Available The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N- and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new μs–ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

  10. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

    Science.gov (United States)

    García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel

    2017-12-01

    3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca 2+ ] c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca 2+ , was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca 2+ ] c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    International Nuclear Information System (INIS)

    Hopperton, Kathryn E.; Duncan, Robin E.; Bazinet, Richard P.; Archer, Michael C.

    2014-01-01

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from 14 C-labeled acetate to those supplied exogenously as 14 C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare utilization of

  12. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Hopperton, Kathryn E., E-mail: kathryn.hopperton@mail.utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Duncan, Robin E., E-mail: robin.duncan@uwaterloo.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Bazinet, Richard P., E-mail: richard.bazinet@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Archer, Michael C., E-mail: m.archer@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada)

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  13. Elevated CO2-induced production of nitric oxide (NO) by NO synthase differentially affects nitrate reductase activity in Arabidopsis plants under different nitrate supplies.

    Science.gov (United States)

    Du, Shaoting; Zhang, Ranran; Zhang, Peng; Liu, Huijun; Yan, Minggang; Chen, Ni; Xie, Huaqiang; Ke, Shouwei

    2016-02-01

    CO2 elevation often alters the plant's nitrate reductase (NR) activity, the first enzyme acting in the nitrate assimilation pathway. However, the mechanism underlying this process remains unknown. The association between elevated CO2-induced alterations of NR activity and nitric oxide (NO) was examined in Col-0 Arabidopsis fed with 0.2-10 mM nitrate, using NO donors, NO scavenger, and NO synthase (NOS) inhibitor. The noa1 mutant, in which most NOS activity was lost, and the NR activity-null mutant nia1 nia2 were also used to examine the above association. In response to CO2 elevation, NR activity increased in low-nitrate Col-0 plants but was inhibited in high-nitrate Col-0 plants. NO scavenger and NOS inhibitor could eliminate these two responses, whereas the application of NO donors mimicked these distinct responses in ambient CO2-grown Col-0 plants. Furthermore, in both low- and high-nitrate conditions, elevated CO2 increased NOS activity and NO levels in Col-0 and nia1 nia2 plants but had little effect on NO level and NR activity in noa1 plants. Considering all of these findings, this study concluded that, in response to CO2 elevation, either the NR activity induction in low-nitrate plants or the NR activity inhibition in high-nitrate plants is regulated by NOS-generated NO. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  15. Software Engineering Improvement Activities/Plan

    Science.gov (United States)

    2003-01-01

    bd Systems personnel accomplished the technical responsibilities for this reporting period, as planned. A close working relationship was maintained with personnel of the MSFC Avionics Department Software Group (ED14). Work accomplishments included development, evaluation, and enhancement of a software cost model, performing literature search and evaluation of software tools available for code analysis and requirements analysis, and participating in other relevant software engineering activities. Monthly reports were submitted. This support was provided to the Flight Software Group/ED 1 4 in accomplishing the software engineering improvement engineering activities of the Marshall Space Flight Center (MSFC) Software Engineering Improvement Plan.

  16. Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model

    Directory of Open Access Journals (Sweden)

    Iizuka K

    2018-03-01

    Full Text Available Kenzo Iizuka, Cheng Jin, Kokoro Eshima, Mei Hua Hong, Kiyoshi Eshima, Masakazu Fukushima Division of Research and Development, Delta-Fly Pharma Inc., Tokushima, Japan Introduction: Peritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis. Methods: We developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS and a cationic liposome (DFP-10825 and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors. Results: DFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect. Conclusion: Collectively, the ip delivery of

  17. Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

    Science.gov (United States)

    Fattorini, L; Veloccia, A; Della Rovere, F; D'Angeli, S; Falasca, G; Altamura, M M

    2017-07-11

    Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. Altogether, results showed that IBA induced

  18. Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

    2010-02-15

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  19. The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

    Science.gov (United States)

    Karmohapatra, Soumendra K; Chakraborty, Kushal; Kahn, Nighat N; Sinha, Asru K

    2007-11-01

    Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture. (c) 2007 Wiley-Liss, Inc.

  20. Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

    Science.gov (United States)

    Dziegelewska, Maja; Holtze, Susanne; Vole, Christiane; Wachter, Ulrich; Menzel, Uwe; Morhart, Michaela; Groth, Marco; Szafranski, Karol; Sahm, Arne; Sponholz, Christoph; Dammann, Philip; Huse, Klaus; Hildebrandt, Thomas; Platzer, Matthias

    2016-08-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Low sulfide levels and a high degree of cystathionine β-synthase (CBS activation by S-adenosylmethionine (SAM in the long-lived naked mole-rat

    Directory of Open Access Journals (Sweden)

    Maja Dziegelewska

    2016-08-01

    Full Text Available Hydrogen sulfide (H2S is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS, an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

  2. Saturated free fatty acid sodium palmitate-induced lipoapoptosis by targeting glycogen synthase kinase-3β activation in human liver cells.

    Science.gov (United States)

    Cao, Jie; Feng, Xiao-Xia; Yao, Long; Ning, Bo; Yang, Zhao-Xia; Fang, Dian-Liang; Shen, Wei

    2014-02-01

    Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms. Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48 h with or without lithium chloride, a glycogen synthase kinase-3β (GSK-3β) inhibitor, or GSK-3β shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression. The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3β protein, which was indicated by dephosphorylation of GSK-3β at Ser-9. However, inhibition of GSK-3β activity with lithium chloride treatment or knockdown of GSK-3β expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3β expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3β inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response. The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3β activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3β inhibition may be a potential therapeutic target to control NAFLD.

  3. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  4. Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.

    Directory of Open Access Journals (Sweden)

    Hongzhen Wang

    Full Text Available In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS, epi-cedrol (ECS and β-farnesene (FS synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS, a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

  5. Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.

    Science.gov (United States)

    Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E

    2013-01-01

    In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

  6. Crystal structure of mouse thymidylate synthase in tertiary complex with dUMP and raltitrexed reveals N-terminus architecture and two different active site conformations.

    Science.gov (United States)

    Dowierciał, Anna; Wilk, Piotr; Rypniewski, Wojciech; Rode, Wojciech; Jarmuła, Adam

    2014-01-01

    The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.

  7. Phylogenetic diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff), detection of polyketide synthase gene and their antagonistic activity analysis.

    Science.gov (United States)

    Wang, Ya; Gao, Bo Liang; Li, Xi Xi; Zhang, Zhi Bin; Yan, Ri Ming; Yang, Hui Lin; Zhu, Du

    2015-11-01

    The biodiversity of plant endophytic fungi is enormous, numerous competent endophytic fungi are capable of providing different forms of fitness benefits to host plants and also could produce a wide array of bioactive natural products, which make them a largely unexplored source of novel compounds with potential bioactivity. In this study, we provided a first insights into revealing the diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff.) from China using rDNA-ITS phylogenetic analysis. Here, the potential of fungi in producing bioactive natural products was estimated based on the beta-ketosynthase detected in the polyketide synthase (PKS) gene cluster and on the bioassay of antagonistic activity against two rice phytopathogens Thanatephorus cucumeris and Xanthomonas oryzae. A total of 229 endophytic fungal strains were validated in 19 genera. Among the 24 representative strains, 13 strains displayedantagonistic activity against the phytopathogens. Furthermore, PKS genes were detected in 9 strains, indicating their potential for synthesising PKS compounds. Our study confirms the phylogenetic diversity of endophytic fungi in O. rufipogon G. and highlights that endophytic fungi are not only promising resources of biocontrol agents against phytopathogens of rice plants, but also of bioactive natural products and defensive secondary metabolites. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  8. A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

    Science.gov (United States)

    Bharti, Vishwa Deepak; Gupta, Satya P; Kumar, Harish

    2014-02-01

    QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1chi(v) (Kier's first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

  9. Chiral hydroxylation at the mononuclear nonheme Fe(II center of 4-(S hydroxymandelate synthase--a structure-activity relationship analysis.

    Directory of Open Access Journals (Sweden)

    Cristiana M L Di Giuro

    Full Text Available (S-Hydroxymandelate synthase (Hms is a nonheme Fe(II dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.

  10. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels.

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.J.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraaij-Emmerzaal, D. van; Heijer, M. den; Trijbels, J.M.F.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine beta-synthase (CBS) deficiency has been excluded as a major

  11. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraay-Emmerzaal, van D.; Heijer, den M.; Trijbels, F.J.M.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine -synthase (CBS) deficiency has been excluded as a major

  12. A Copal-8-ol Diphosphate Synthase from the Angiosperm Cistus creticus subsp. creticus Is a Putative Key Enzyme for the Formation of Pharmacologically Active, Oxygen-Containing Labdane-Type Diterpenes1[OA

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K.

    2010-01-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%–70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding. PMID:20595348

  13. Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells.

    Science.gov (United States)

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Raposeiras-Roubín, Sergio; González-Peteiro, Mercedes; González-Juanatey, José R; Álvarez, Ezequiel

    2015-01-01

    Non-enzymatic glycated proteins could mediate diabetes vascular complications, but the molecular mechanisms are unknown. Our objective was to find new targets involved in the glycated human serum albumin (gHSA)-enhanced extracellular reactive oxygen species (ROS) production in human endothelial cells. Some nuclear factors and phosphorylation cascades were analysed. gHSA activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which up-regulated NOX4 and P22PHOX and enhanced ROS production. Pharmacological inhibition of NF-κB reversed gHSA-enhanced NOX4 expression and decreased gHSA-induced ROS production in extra- and intracellular spaces. The inhibition of activator protein-1 (AP-1) induced a rise in NOX4 and P22PHOX subunit expression and a down-regulation of endothelial nitric oxide synthase (eNOS). AP-1 inhibition also enhanced extracellular ROS production in the presence of serum albumin, but not with gHSA. These results were explained by the eNOS uncoupling induced by gHSA, also demonstrated in this study. Phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase 1/2 did not show to be involved in gHSA-induced ROS production. All together, the results suggested that gHSA-enhanced ROS production in endothelium is mediated by: 1) NF-κB activation and subsequence up-regulation of NADPH oxidase, 2) eNOS uncoupling. AP-1, although is not directly affected by gHSA, is another target for regulating NADPH oxidase and eNOS expression in endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes.

    Science.gov (United States)

    Shiao, Young-Ji; Lin, Yun-Lian; Sun, Ya-Hui; Chi, Chih-Wen; Chen, Chieh-Fu; Wang, Chuen-Neu

    2005-01-01

    The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated. Falcarindiol abrogated the LPS/IFN-gamma-mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration-dependent manner. The inhibitory effect of falcarindiol on iNOS induction was attributable to decrease in the protein content and the mRNA level of iNOS. Treatment with 50 microM of falcarindiol for 30 min decreased LPS/IFN-gamma-induced nuclear factor-kappaB (NF-kappaB) activation by 32%. Treatment with 50 microM of falcarindiol for 60 min diminished the LPS/IFN-gamma-mediated activation of IkappaB kinase-alpha (IKK-alpha) and IKK-beta by 28.2 and 29.7%, respectively. Falcarindiol modulated the nuclear translocation of signal transducer and activator of transcription 1 (Stat1) in a time-dependent manner. Falcarindiol (50 microM) decreased the tyrosine phosphorylation of janus kinase 1 (JAK1) by 84.8% at 5 min. Falcarindiol also abrogated the tyrosine phoshorylation of JAK2 by 82.3% at 10 min.The present study demonstrates that falcarindiol attenuated the activation of IKK and JAK contributing to the blockade of activation of NF-kappaB and Stat1, thereby leading to the suppression of iNOS expression.

  15. Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

    Science.gov (United States)

    Gregus, Ann M; Dumlao, Darren S; Wei, Spencer C; Norris, Paul C; Catella, Laura C; Meyerstein, Flore G; Buczynski, Matthew W; Steinauer, Joanne J; Fitzsimmons, Bethany L; Yaksh, Tony L; Dennis, Edward A

    2013-05-01

    Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

  16. Improving activation cross section data with TALYS

    Science.gov (United States)

    Dzysiuk, Nataliia; Koning, Arjan

    2017-09-01

    Needs for accurate (n,x) activation cross sections for fusion technology have been considered with reference to the current status of the TENDL library. The current work is focused on improving activation cross section data for nuclear reactions relevant mainly for fusion and astrophysical needs. The fits have been performed with the TALYS-1.8 code by means of nuclear model parameter variation, mostly for the optical model and level densities, followed by comparison to recent experimental data taken from EXFOR and other evaluated nuclear databases. The updated cross section data are going to be adopted into the new version of TENDL. The improvements have been performed both for differential as well as integral data sets.

  17. Signal-on CoA-dependent electrochemical biosensor for highly sensitive and label-free detection of Citrate synthase activity.

    Science.gov (United States)

    Wang, Qin; Chen, Hongjun; Li, Yong; Wang, Huixia; Nie, Zhou; Hu, Yufang; Yao, Shouzhou

    2016-12-01

    We report here a label-free and sensitive electrochemical method for probing Citrate synthase (CS) activity based on detailed investigations into the nucleic acid-mimicking coordination polymer (CP) formed from the coenzyme A (CoA)-Ag(I) repeat units. Our biosensing approach provides an especial and significant detection mechanism: CS can catalyze the essential condensation reaction between acetyl-coenzyme A (Ac-CoA) and oxaloacetate (OAA) to form citrate and CoA; then, in the presence of Ag(I), CoA-Ag(I) CP can be in situ formed because of the strong complexation ability of thiol groups of CoA toward Ag(I). The generated CoA-Ag(I) CP attaches to graphene-modified glassy carbon electrode surface by multiple adenine bases deriving from CoA and acting as the side groups along the polymeric backbone, which displays efficient H 2 O 2 -electrocatalyzing activity. More importantly, by using the formed polymer as signal output, the process is implemented to quantitatively analyze the activity of CS. Under the optimal conditions, CS with a detection limit as low as 0.00165U/µL could be sensitively probed with a wide linear range from 0.0033 to 0.264U/µL. Furthermore, with the character of label-free detection, high sensitivity and excellent selectivity, this strategy offers a convenient and specific method for CS activity detection and relevant inhibitors screening, which holds a promising potential in the practical application of CS-based biochemical research, disease diagnosis and drug discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Electrochemical, Spectroscopic, and Density Functional Theory Characterization of Redox Activity in Nickel-Substituted Azurin: A Model for Acetyl-CoA Synthase.

    Science.gov (United States)

    Manesis, Anastasia C; Shafaat, Hannah S

    2015-08-17

    Nickel-containing enzymes are key players in global hydrogen, carbon dioxide, and methane cycles. Many of these enzymes rely on Ni(I) oxidation states in critical catalytic intermediates. However, due to the highly reactive nature of these species, their isolation within metalloenzymes has often proved elusive. In this report, we describe and characterize a model biological Ni(I) species that has been generated within the electron transfer protein, azurin. Replacement of the native copper cofactor with nickel is shown to preserve the redox activity of the protein. The Ni(II/I) couple is observed at -590 mV versus NHE, with an interfacial electron transfer rate of 70 s(-1). Chemical reduction of Ni(II)Az generates a stable species with strong absorption features at 350 nm and a highly anisotropic, axial EPR signal with principal g-values of 2.56 and 2.10. Density functional theory calculations provide insight into the electronic and geometric structure of the Ni(I) species, suggesting a trigonal planar coordination environment. The predicted spectroscopic features of this low-coordinate nickel site are in good agreement with the experimental data. Molecular orbital analysis suggests potential for both metal-centered and ligand-centered reactivity, highlighting the covalency of the metal-thiolate bond. Characterization of a stable Ni(I) species within a model protein has implications for understanding the mechanisms of complex enzymes, including acetyl coenzyme A synthase, and developing scaffolds for unique reactivity.

  19. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  20. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    Science.gov (United States)

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  1. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    Science.gov (United States)

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  3. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C. [Athens Univ. (Greece). School of Medicine

    1996-06-28

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 {+-} 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK).

  4. Changes in Phytochemical Synthesis, Chalcone Synthase Activity and Pharmaceutical Qualities of Sabah Snake Grass (Clinacanthus nutans L. in Relation to Plant Age

    Directory of Open Access Journals (Sweden)

    Ali Ghasemzadeh

    2014-10-01

    Full Text Available In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old. The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74 was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW] and total phenolic (TP (18.21 mg/g DW were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW and gallic acid (5.96 mg/g DW were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50 values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP assay showed a higher activity in 6-month-old buds (488 μM of Fe(II/g than in 1-year-old buds (453 μM of Fe(II/g, in contrast to the DPPH result. Significant correlations (p < 0.05 were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity

  5. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  6. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  7. Identification of protein phosphatase involvement in the AT-receptor induced activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Peluso, A Augusto; Bertelsen, Jesper Bork; Andersen, Kenneth

    2018-01-01

    -antagonist), L-NAME (10µM; eNOS inhibitor), MK-2206 (100nM; Akt-inhibitor) sodium fluoride (1nM; serine/threonine-phosphatase inhibitor) or sodium orthovanadate (10nM; tyrosine-phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence. The phosphorylation status of activating (e...

  8. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    Science.gov (United States)

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

  9. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  10. Caffeine restores myocardial cytochrome oxidase activity and improves cardiac function during sepsis.

    Science.gov (United States)

    Verma, Richa; Huang, Zhishan; Deutschman, Clifford S; Levy, Richard J

    2009-04-01

    Impaired mitochondrial function is a potential cause of sepsis-associated myocardial depression. Cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain, is inhibited in the septic heart. Caffeine increases CcOX activity by increasing cyclic adenosine monophosphate and protein kinase A activity. We hypothesized that caffeine will restore myocardial CcOX activity, increase cardiac function, and improve survival during sepsis. Prospective randomized controlled study. University hospital-based laboratory. One hundred twenty Sprague-Dawley male rats. Sprague-Dawley male rats underwent cecal ligation and puncture (CLP) or sham operation. At 24 and 48 hours, rats underwent intraperitoneal injection of either caffeine (7.5 mg/kg, the equivalent of 1-1.5 cups of coffee) or equal volume of saline. One hour following the 48-hour injection, steady-state CcOX kinetic activity was measured in isolated mitochondria and normalized to citrate synthase activity. Cardiac function was assessed using an isolated rat heart preparation and survival was tracked to 96 hours. CLP significantly decreased myocardial CcOX activity, oxygen consumption, left ventricular pressure, and pressure developed during isovolumic contraction (+dP/dt) and relaxation (-dP/dt). Caffeine restored CcOX activity and increased left ventricular pressure and +/-dP/dt toward sham values following CLP. Survival significantly improved following CLP in caffeine-injected animals compared with saline injection. Caffeine may be a novel therapy to treat sepsis-associated myocardial depression.

  11. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfishCyanea capillata.

    Science.gov (United States)

    Wang, Beilei; Liu, Dan; Wang, Chao; Wang, Qianqian; Zhang, Hui; Liu, Guoyan; Tao, Xia; Zhang, Liming

    2017-01-01

    Our previous study demonstrated that tentacle extract (TE) from the jellyfish Cyanea capillata ( C. capillata ) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca 2+ signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which was significantly attenuated by either IP 3 receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca 2+ -free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca 2+ ] i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca 2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca 2+ ] i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP 3 R-sensitive and Ca 2+ -dependent pathways.

  12. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfish Cyanea capillata

    Directory of Open Access Journals (Sweden)

    Beilei Wang

    2017-04-01

    Full Text Available Our previous study demonstrated that tentacle extract (TE from the jellyfish Cyanea capillata (C. capillata could cause a weak relaxation response mediated by nitric oxide (NO using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca2+ signaling pathways in human umbilical vein endothelial cells (HUVECs. Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca2+ concentration ([Ca2+]i, which was significantly attenuated by either IP3 receptor blocker (Heparin or PKC inhibitor (PKC 412. In contrast, extracellular Ca2+-free, L-type calcium channel blocker (Nifedipine, or PKA inhibitor (H89 had no influence on the [Ca2+]i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca2+]i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathways.

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

    Science.gov (United States)

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

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. Discovery and characterization of a novel lachrymatory factor synthase in Petiveria alliacea and its influence on alliinase-mediated formation of biologically active organosulfur compounds.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman

    2009-11-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

  16. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  17. Inhibition of Glycogen Synthase Kinase-3ß Enhances Cognitive Recovery after Stroke: The Role of TAK1

    Science.gov (United States)

    Venna, Venugopal Reddy; Benashski, Sharon E.; Chauhan, Anjali; McCullough, Louise D.

    2015-01-01

    Memory deficits are common among stroke survivors. Identifying neuroprotective agents that can prevent memory impairment or improve memory recovery is a vital area of research. Glycogen synthase kinase-3ß (GSK-3ß) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3ß is active only when…

  18. Regional age-related changes in neuronal nitric oxide synthase (nNOS, messenger RNA levels and activity in SAMP8 brain

    Directory of Open Access Journals (Sweden)

    Guidon Gérard

    2006-12-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8 mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS. To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx- levels, in three brain areas (n = 7 animals in each group. Calibrated reverse transcriptase (RT and real-time polymerase chain reaction (PCR and biochemical procedures were used. Results We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Conclusion Concomitant variations occurring in NO levels

  19. 4G/5G variant of plasminogen activator inhibitor-1 gene and severe pregnancy-induced hypertension: subgroup analyses of variants of angiotensinogen and endothelial nitric oxide synthase.

    Science.gov (United States)

    Kobashi, Gen; Ohta, Kaori; Yamada, Hideto; Hata, Akira; Minakami, Hisanori; Sakuragi, Noriaki; Tamashiro, Hiko; Fujimoto, Seiichiro

    2009-01-01

    Pregnancy-induced hypertension (PIH) is a common cause of perinatal mortality. It is believed to result from the interaction of several factors, including those related to the blood coagulation system. We performed genotyping and subgroup analyses to determine if the 4G/5G genotypes of the plasminogen activator inhibitor-1 gene (PAI-1) play a role in the pathogenesis of PIH, and to evaluate possible interactions of the PAI-1 polymorphisms with those of the angiotensinogen gene (AGT) and the endothelial nitric oxide synthase gene (NOS3). An association study of PAI-1 polymorphism, and subgroup analyses of common variants of AGT and NOS3, among 128 patients with PIH and 376 healthy pregnant controls. No significant differences were found between the cases and controls in the frequencies of allele 4G or the 4G/4G genotype. In subgroup analyses, after adjustment for multiple comparison, a significant association with the AGT TT genotype was found among women with the PAI-1 4G/4G genotype, and an association with the NOS3 GA+AA genotype was found among women with the 5G/5G or 4G/5G genotypes. Our findings suggest that there are at least 2 pathways in the pathogenesis of severe PIH. However, with respect to early prediction and prevention of severe PIH, although the PAI-1 4G/4G genotype alone was not a risk factor for severe PIH, the fact that PAI-1 genotypes are associated with varying risks for severe PIH suggests that PAI-1 genotyping of pregnant women, in combination with other tests, may be useful in the development of individualized measures that may prevent severe PIH.

  20. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  1. Endotoxin and interferon-γ inhibit translation in skeletal muscle cells by stimulating nitric oxide synthase activity

    Science.gov (United States)

    Frost, Robert A.; Nystrom, Gerald J.; Lang, Charles H.

    2012-01-01

    The purpose of the present study was to test the hypothesis that endogenous nitric oxide (NO) negatively affects translation in skeletal muscle cells after exposure to a combination of endotoxin (LPS) and interferon (IFN)γ. Individually LPS and IFNγ did not alter protein synthesis but in combination they inhibited protein synthesis by 80% in C2C12 myotubes. The combination of LPS and IFNγ dramatically down regulated the auto-phosphorylation of the mammalian target of rapamycin (mTOR) and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased whereas phosphorylation of elongation factor-2 (eEF-2) and raptor was enhanced consistent with defects in both translation initiation and elongation. Reduced S6 phosphorylation occurred 8–18 h after LPS/IFNγ and coincided with a prolonged upregulation of NOS2 mRNA and protein. NOS2 protein expression and the LPS/IFNγ –induced fall in phosphorylated S6 were prevented by the proteasome inhibitor MG132. The general NOS inhibitor L-NAME and the specific NOS2 inhibitor 1400W also prevented the LPS/IFNγ-induced decrease in protein synthesis and restored translational signaling. LPS/IFNγ down regulated the phosphorylation of multiple Akt substrates including the proline rich Akt substrate-40 (PRAS40) while enhancing the phosphorylation of raptor on an AMPK regulated site. The negative effects of LPS/IFNγ were blunted by the AMPK inhibitor compound C. The data suggest that in combination LPS and IFNγ induce a prolonged expression of NOS2 and excessive production of NO that reciprocally alters Akt and AMPK activity and consequently down regulates translation via reduced mTOR signaling. PMID:19295495

  2. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

    Directory of Open Access Journals (Sweden)

    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

  3. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-06

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  4. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  5. Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors.

    Science.gov (United States)

    Warrilow, Andrew G S; Hawkesford, Malcolm J

    2002-03-01

    The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.

  6. The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity

    DEFF Research Database (Denmark)

    Gaster, Michael; Petersen, Ingrid; Højlund, Kurt

    2002-01-01

    The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase...

  7. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  8. Physical Activity to Improve Erectile Function

    DEFF Research Database (Denmark)

    Gerbild, Helle Nygaard; Larsen, Camilla Marie; Areskoug Josefsson, Kristina

    2018-01-01

    , and metabolic syndrome. Physical activity (PA) has proved to be a protective factor against erectile problems, and it has been shown to improve erectile function for men affected by vascular ED. This systematic review estimated the levels of PA needed to decrease ED for men with physical inactivity, obesity......Introduction The leading cause of erectile dysfunction (ED) is arterial dysfunction, with cardiovascular disease as the most common comorbidity. Therefore, ED is typically linked to a web of closely interrelated cardiovascular risk factors such as physical inactivity, obesity, hypertension......, hypertension, metabolic syndrome, and/or manifest cardiovascular diseases. Aim To provide recommendations of levels of PA needed to decrease ED for men with physical inactivity, obesity, hypertension, metabolic syndrome, and/or cardiovascular diseases. Methods In accord with the Preferred Reporting Items...

  9. Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity.

    Science.gov (United States)

    Borman, Pınar; Taşbaş, Özgur; Karabulut, Halil; Tukun, Ajlan; Yorgancıoğlu, Rezan

    2015-01-01

    The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and number of patients with MTX-related adverse affects, were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity, were determined. The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The frequency of TS2R and TS3R polymorphisms were found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45μmol/L vs 10.7μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. In conclusion, homocysteine levels might effect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX

  10. USING FUN ACTIVITIES TO IMPROVE LISTENING SKILL

    Directory of Open Access Journals (Sweden)

    Hanna Andyani

    2015-12-01

    Full Text Available Based on the researcher’s experience in teaching English at MTsN Mojokerto, there are three problems dealing with the teaching of listening especially for the third year students: 1 most of the students’ scores on listening test are still under the minimum passing criterion (KKM, which is 60; 2 most students are not very enthusiastic in listening activities; 3 it is difficult for students to understand native speech in a tape recorder. Based on the problems, the main purpose of the study is to improve the ninth grade students’ listening skill using Fun Activity in the form of Games at MTsN Mojokerto. The design of this study was Classroom Action Research. The instruments were the listening tests, observation checklist and questionnaires. With the implementation of the games, the criteria of success were successfully achieved in Cycle 2. 74% of the total number of the students could get the scores more than 60 and 90% have positive responses on the implementation of games. Keywords: tic tac toe game, running dictation game, whispering game, listening skill

  11. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  12. Improvement of Tissue Survival of Skin Flaps by 5α-Reductase Inhibitors: Possible Involvement of Nitric Oxide and Inducible Nitric Oxide Synthase

    Science.gov (United States)

    Karimi, Ali Asghar; Ajami, Marjan; Asadi, Yasin; Aboutaleb, Nahid; Gorjipour, Fazel; Malekloo, Roya; Pazoki-Toroudi, Hamidreza

    2015-01-01

    Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5α-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats were randomly allocated into six groups: 1, normal saline topical application; 2, azelaic acid (100 mg/flap); 3, finasteride (1 mg/flap); 4, injection of L-NG-nitroarginine methyl ester (L-NAME) (i.p., 20 mg/kg); 5, L-NAME (20 mg/kg, i.p.) + azelaic acid (100 mg/flap, topical); 6, L-NAME (20 mg/kg, i.p.) + finasteride (1 mg/flap, topical). Tissue survival, level of nitric oxide, and iNOS expression in groups were measured. Results: Our data revealed that azelaic acid and finasteride significantly increased the expression of iNOS protein and nitric oxide (NO) levels in graft tissue (P azelaic acid- and finasteride-mediated survival of the skin flaps. PMID:25864816

  13. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  14. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  15. Physical activity improves cognition: possible explanations.

    Science.gov (United States)

    Koščak Tivadar, Blanka

    2017-08-01

    Good cognitive abilities (CA) enable autonomy, improve social inclusion and act preventively. Regular physical activity (PA) reduces the risk of developing Alzheimer's disease (AD) and, at the same time, it reduces the decline of CA and stimulates neurogenesis. So PA in connection with cognitive training, nutrition and social interaction has a positive effect on general CA and the central nervous system, the central executor, memory and attention, and reduces the likelihood of developing dementia. Our objective was to examine which sort and intensity of PA is preferred. We did a review, restricted only to human studies, of transparent scientific articles and sample surveys carried out and published in the period between 2001 and 2016 based on the keywords: age, aging, physical activity, physical abilities, cognitive abilities, memory and Alzheimer's disease. According to results CA and PA interact, as an increasing PA of only 10% reduces the risk of dementia and AD significantly. However, there is a question of appropriate intensity of exercise. Low-intensity aerobic exercise has a positive effect on the visual spatial perception and attention, whereas moderate PA has a positive impact on general CA, working memory and attention, verbal memory and attention and vice versa. While the majority of experts recommends vigorous or moderate exercise, many of them warn that higher intensity requires more attention to PA and less to cognitive processes, particularly in terms of reducing reactions, selective attention and flexibility to tasks. There is also a further question what PA should be like. Although some experts believe that the best combination is aerobic PA and exercises against resistance, it is not entirely clear whether the improvement in CA is a result of cardiac vascular fitness. On the other hand, for most elderly it is more suitable to perform an alternative form (not anaerobic) of PA due to comorbidity and actual fragility. We can conclude that PA has a

  16. 77 FR 31615 - Improving Mail Management Policies, Procedures, and Activities

    Science.gov (United States)

    2012-05-29

    ... ADMINISTRATION Improving Mail Management Policies, Procedures, and Activities AGENCY: Office of Governmentwide Policy, General Services Administration (GSA). ACTION: Notice of FMR Bulletin G-03. SUMMARY: The General... guidance to Executive Branch agencies for improving mail management policies, procedures, and activities...

  17. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  18. 42 CFR 423.162 - Quality improvement organization activities.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Quality improvement organization activities. 423... and Quality Improvement Requirements § 423.162 Quality improvement organization activities. (a) General rule. Quality improvement organizations (QIOs) are required to offer providers, practitioners, and...

  19. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Successfully Improving Physical Activity Behavior After Rehabilitation

    NARCIS (Netherlands)

    van der Ploeg, Hidde P.; Streppel, Kitty R.M.; van der Beek, Allard J.; Woude, Luc H.V.; Vollenbroek-Hutten, Miriam Marie Rosé; van Harten, Willem H.; van Mechelen, Willem

    2007-01-01

    Purpose To determine the effects of the physical activity promotion programs Rehabilitation & Sports (R&S) and Active after Rehabilitation (AaR) on sport and daily physical activity 1 year after in- or outpatient rehabilitation. Design Subjects in intervention rehabilitation centers were randomized

  1. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    Science.gov (United States)

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

  2. Nitric oxide synthase in ferret brain: localization and characterization.

    Science.gov (United States)

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  3. Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation

    Science.gov (United States)

    Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj

    2016-01-01

    Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation. PMID:27725707

  4. Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase.

    OpenAIRE

    Franch, J; Aslesen, R; Jensen, J

    1999-01-01

    We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen s...

  5. Human platelet nitric oxide synthase activity: an optimized method Atividade da óxido nítrico sintase em plaquetas humanas: um método otimizado

    Directory of Open Access Journals (Sweden)

    Elisa Mitiko Kawamato

    2002-09-01

    Full Text Available We investigated the kinetic analysis of human platelet Nitric Oxide Synthase (NOS activity by the rate of conversion of [³H] arginine to [³H]-citrulline in unstimulated fresh platelets. NOS activity was present in the membrane fraction and cytosol, and was Ca2+- and calmodulin dependent which is a characteristic of endothelial NOS. NOS activity was also dependent of NADPH since the omission of this cofactor induced an important decrease (85,2% in the enzyme activity. The kinetic varied with protein and arginine concentration but optimum concentrations were found up to 60 minutes, and up to 80 µg of protein at 120 nM of arginine and 0.5 µCi of ³H-arginine. NOS activity in the absence of FAD (flavin adenine dinucleotide, FMN (flavin mononucleotide and BH4 (tetrahydrobiopterin was only 2.8% of the activity measured in the presence of these three cofactors. The enzyme activity was completely inhibited by L-NAME (1 mM (98.1 % and EGTA (5 mM (98.8 %. Trifluoperazine (TFP caused 73.2% inhibition of the enzyme activity at 200 µM and 83.8 % at 500 µM. Under basal conditions, NOS Km for L-arginine was 0.84 ± 0.08 µM and mean Vmax values were 0.122 ± 0.025 pmol.mg-1.min-1. Mean human NOS platelet activity was 0.020 ± 0.010 pmol.mg-1.min-1. Results indicate that the eNOS in human platelet can be evaluated by conversion of [³H]-arginine to [³H]citrulline in an optimized method, which provide reproducible and accurate results with good sensitivity to clinical experiments involving neurological and psychiatric diseases.A análise cinética da atividade da óxido nítrico sintase (NOS plaquetária foi avaliada pela conversão de [³H]-arginina em [³H]-citrulina em plaquetas humanas frescas não estimuladas. A atividade da NOS foi detectada na fração citosólica e na membrana, além de ser dependente de Ca2+-calmodulina, que é uma característica da NOS endotelial (eNOS. A omissão de NADPH levou à diminuição da atividade da NOS dependente da

  6. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    OpenAIRE

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates S...

  7. Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes

    OpenAIRE

    Shiao, Young-Ji; Lin, Yun-Lian; Sun, Ya-Hui; Chi, Chih-Wen; Chen, Chieh-Fu; Wang, Chuen-Neu

    2004-01-01

    The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon-γ (LPS/IFN-γ) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated.Falcarindiol abrogated the LPS/IFN-γ-mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration-dependent manner.The inhibitory effect of falcarindio...

  8. The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pindjáková, Jana; Zajícová, Alena; Krulová, Magdalena; Železná, Blanka; Matoušek, Petr; Svoboda, Petr

    2005-01-01

    Roč. 12, č. 3 (2005), s. 227-234 ISSN 0908-665X R&D Projects: GA MZd(CZ) NR7816; GA ČR(CZ) GP310/02/D162; GA ČR(CZ) GD310/03/H147; GA MŠk(CZ) ME 300; GA AV ČR KSK5020115 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50110509 Keywords : inducible nitric oxide synthase production * nitric oxide * suppressive molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.114, year: 2005

  9. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

  10. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

    Science.gov (United States)

    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  11. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  12. Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse Inhibition of brain citrate synthase activity in an animal model of sepsis

    Directory of Open Access Journals (Sweden)

    Giselli Scaini

    2011-06-01

    fisiopatologia desta doença.OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS. Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

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

    Science.gov (United States)

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

    2013-01-01

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

  14. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  15. Research reactor management. Safety improvement activities in HANARO

    International Nuclear Information System (INIS)

    Wu, Jong-Sup; Jung, Hoan-Sung; Hong, Sung Taek; Ahn, Guk-Hoon

    2012-01-01

    Safety activities in HANARO have been continuously conducted to enhance its safe operation. Great effort has been placed on a normalization and improvement of the safety attitude of the regular staff and other employees working at the reactor and other experimental facilities. This paper introduces the activities on safety improvement that were performed over the last few years. (author)

  16. Forms and Methods of Agricultural Sector Innovative Activity Improvement

    Directory of Open Access Journals (Sweden)

    Aisha S. Ablyaeva

    2013-01-01

    Full Text Available The article is focused on basic forms and methods to improve the efficiency of innovative activity in the agricultural sector of Ukraine. It was determined that the development of agriculture in Ukraine is affected by a number of factors that must be considered to design innovative models of entrepreneurship development and ways to improve the efficiency of innovative entrepreneurship activity.

  17. Current activities on safety improvement at Ukrainian NPPs

    International Nuclear Information System (INIS)

    Stovbun, V.V.

    2000-01-01

    This report describes general development status of the national programs on safety improvement of the Ukrainian NPPs, basic approaches adopted for planning and implementation of safety improvement works, and state of implementation of principal technical activities aimed at safety improvement of Ukrainian NPPs. (author)

  18. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  19. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c....... solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization....

  20. [Experimental models of diabetes mellitus of the 1st and 2nd types in rats: regulation of activity of glycogen synthase by peptides of the insulin superfamily and by epidermal growth factor in skeletal muscles].

    Science.gov (United States)

    Kuznetsova, L A; Chistiakova, O V

    2012-01-01

    The regulatory effect of peptides of the insulin hyperfamily--insulin, insulin-like growth factor (IGF-1), and relaxin, as well as of epidermal growth factor (EGF) on activity of glycogen synthase (GS) in rat skeletal muscles was studied in norm and in experimental diabetes mellitus of the 1st and 2nd types (DM1, DM2). In norm, peptides in vitro stimulated maximally the GS activity at a concentration of 10-8 M. The row of efficiency of the peptide action was as follows: insulin > IGF-1 > relaxin. In DM1 the basal GS activity did not change, while effect of insulin in vitro was decreased more sharply as compared with action of IGF-1 and relaxin at the 30th day of development of diabetes, i. e., the efficiency row was as follows: IGF-1 = relaxin > insulin. Administration of insulin in vivo did not restore sensitivity of the enzyme to the action of hormone in DM1. In DM2, the GS activity (both the total and active form) decreased. while the stimulatory effect ofpeptides and EGF on the enzyme was absent. Insulin introduced in vitro did not lead to restoration of the enzyme reaction. The conclusion has been made that the insulin resistance affects the basal GS activity in rat skeletal muscles as well as the regulation of the enzyme by peptides of the insulin nature and by EGF, which is more obvious in DM2, than in DM1.

  1. Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

    International Nuclear Information System (INIS)

    Sun, Xin; Huang, Luqi; Zhang, Min; Sun, Shenggang; Wu, Yan

    2010-01-01

    Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP + ). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP + as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP + -induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP + from apoptosis via the GSK-3beta signaling pathway.

  2. Are students' impressions of improved learning through active learning methods reflected by improved test scores?

    Science.gov (United States)

    Everly, Marcee C

    2013-02-01

    To report the transformation from lecture to more active learning methods in a maternity nursing course and to evaluate whether student perception of improved learning through active-learning methods is supported by improved test scores. The process of transforming a course into an active-learning model of teaching is described. A voluntary mid-semester survey for student acceptance of the new teaching method was conducted. Course examination results, from both a standardized exam and a cumulative final exam, among students who received lecture in the classroom and students who had active learning activities in the classroom were compared. Active learning activities were very acceptable to students. The majority of students reported learning more from having active-learning activities in the classroom rather than lecture-only and this belief was supported by improved test scores. Students who had active learning activities in the classroom scored significantly higher on a standardized assessment test than students who received lecture only. The findings support the use of student reflection to evaluate the effectiveness of active-learning methods and help validate the use of student reflection of improved learning in other research projects. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Continuous improvement in teams : The (mis)fit between improvement and operational activities of improvement teams

    NARCIS (Netherlands)

    Ros, D.J.

    1999-01-01

    Since the 1970s and 1980s, increasing attention has been paid to the Japanese ways of organising production. One of the subjects often discussed is the importance of continuous incremental improvements. Nowadays, for many organisations, continuous improvement has become an important topic; many

  4. Prevention Research Matters-Communities Working to Improve Physical Activity

    Centers for Disease Control (CDC) Podcasts

    2018-02-15

    We know that children who are physically active every day are less likely to develop chronic diseases as adults, including obesity. Dr. Sandy Slater, a researcher with the University of Illinois, Chicago Prevention Research Center, discusses how a park improvement project in Chicago helped engage communities to improve areas for play and activity.  Created: 2/15/2018 by National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP).   Date Released: 2/15/2018.

  5. Resveratrol as a Bioenhancer to Improve Anti-Inflammatory Activities of Apigenin

    Directory of Open Access Journals (Sweden)

    Jin-Ah Lee

    2015-11-01

    Full Text Available The aim of this study was to improve the anti-inflammatory activities of apigenin through co-treatment with resveratrol as a bioenhancer of apigenin. RAW 264.7 cells pretreated with hepatic metabolites formed by the co-metabolism of apigenin and resveratrol (ARMs in HepG2 cells were stimulated with lipopolysaccharide (LPS. ARMs prominently inhibited (p < 0.05 the production of nitric oxide (NO, prostaglandin E2 (PGE2, interleukin (IL-1β, IL-6 and TNF-α. Otherwise no such activity was observed by hepatic metabolites of apigenin alone (AMs. ARMs also effectively suppressed protein expressions of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2. Co-administration of apigenin (50 mg/kg and resveratrol (25 mg/kg also showed a significant reduction of carrageenan-induced paw edema in mice (61.20% to 23.81%. Co-administration of apigenin and resveratrol led to a 2.39 fold increase in plasma apigenin levels compared to administration of apigenin alone, suggesting that co-administration of resveratrol could increase bioavailability of apigenin. When the action of resveratrol on the main apigenin metabolizing enzymes, UDP-glucuronosyltransferases (UGTs, was investigated, resveratrol mainly inhibited the formation of apigenin glucuronides by UGT1A9 in a non-competitive manner with a Ki value of 7.782 μM. These results suggested that resveratrol helps apigenin to bypass hepatic metabolism and maintain apigenin’s anti-inflammatory activities in the body.

  6. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    Science.gov (United States)

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  7. Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment.

    Science.gov (United States)

    Frangioudakis, Georgia; Diakanastasis, Barbara; Liao, Bing-Qing M; Saville, Jennifer T; Hoffman, Nolan J; Mitchell, Todd W; Schmitz-Peiffer, Carsten

    2013-12-01

    An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention. CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen. Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression. Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.

  8. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

    Science.gov (United States)

    Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

    2008-05-01

    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

  9. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  10. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph

    2017-01-01

    , linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards...

  11. Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1999-01-01

    ) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported...

  12. DHEA and non-alcoholic fat liver disease: increased gene expression of peroxisome proliferation-activated receptor γ (PPARγ and fatty acid synthase (FAS

    Directory of Open Access Journals (Sweden)

    Felipe Natali Almeida

    2014-05-01

    Full Text Available Dehydroespiandrosterone (DHEA is associated with improvements in chronic degenerative diseases, including obesity, insulin resistance, and cardiovascular diseases. Nevertheless, it is observed an increase in its concentration in individuals with liver lipid infiltration, but it is not precise if this condition emerges as a cause or a consequence. In this way, we aimed to identify gene expression alterations in lipid and glucose liver metabolism markers, as well as oxidative stress markers. For this purpose, male Wistar rats, 12-14 months old were treated with subcutaneous injections of DHEA (only dose of 10 mg kg-1; and after 7 days, hepatic gene expression by PCR real time were performed for the following genes:  G6Pase, PEPCK, FAS, PPARγ, malic enzyme, ChREBP, LXR, catalase, GPx, iNOS, NADPH oxidase subunits and PCNA. We observed a tendency of reduction in G6Pase gene expression in treated group (p = 0.08. In addition, it was identified an increase in liver PPARγ and FAS gene expressions, two markers of increased activity of lipogenic pathway. We also observed an increase in iNOS gene expression, a known inductor of systemic and hepatic insulin resistance. In conclusion, our data indicates that the treatment with DHEA can be associated with the development of liver lipid infiltration and hepatic insulin resistance.

  13. Active Listening Improve Your Ability to Listen and Lead

    CERN Document Server

    (CCL), Center for Creative Leadership

    2011-01-01

    Active listening is a person's willingness and ability to hear and understand. At its core, active listening is a state of mind that involves paying full and careful attention to the other person, avoiding premature judgment, reflecting understanding, clarifying information, summarizing, and sharing. By learning and committing to the skills and behaviors of active listening, leaders can become more effective listeners and, over time, improve their ability to lead.

  14. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2009-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to ...

  15. Further analysis of multicentre cystathionine beta synthase deficiency thrombosis data and metabolic pathways suggests potentially better treatment via improved cysteine supplementation, diet, antioxidant supplementation, follow-up and testing for thrombophilic mutations

    Directory of Open Access Journals (Sweden)

    David Vance

    2017-01-01

    Full Text Available Background Homozygous or compound heterozygous Cystathionine-beta-synthase deficiency (CBS-- may result in thrombosis. Treatment has included various combinations of: low-methionine diets, cystine (cystine dimer-enriched amino acid supplementation, vitamin B6, folic acid, vitamin B12 and betaine. Treatment compliance and outcomes even in the most-developed countries are mostly sub-optimal and variable, and the differing theoretical metabolic ramifications due to differing treatments have not been well addressed. The aim of this work was to further analyse the thrombosis events data of Yap et al (2001/2003, and to compare these with the rate of thrombosis in the general population, and to examine the theoretical significance of the metabolic pathways affected by CBS-- and its treatments, and so find any potential improvements in treatments, considering also less-developed areas. Methods Yap et al’s (2001/2003 data of the thrombosis outcomes of five major (CBS---treating centers: in Dublin, Sydney, Nijmegen, Manchester and London; were statistically compared with outcomes predicted by Mudd et al’s (1985 untreated natural history outcomes, and then Dublin versus the others; these rates were then compared with those of general populations; and treatments were examined regarding their theoretical metabolic ramifications. Results There were less thrombosis outcomes (P<.05 in the treated and followed CBS-- patient groups of each of the five centers, even when considered singly, than that expected in the absence of treatment by reference to the natural history data of Mudd et al (1985, but the reduction was less than half that claimed by Yap et al, and the remaining level of thrombosis is roughly 10 times that of the general population. The thromboses outcome (nil of the Dublin group is better than that of the other four groups, but only at P ~ 0.16 with the other four groups combined, or P = 0.14 to 0.23 singly. Treatment regimens differ, including

  16. The influence of corporate structure and quality improvement activities on outcome improvement in residential care homes

    NARCIS (Netherlands)

    Winters, S.; Kool, R. B.; Klazinga, N. S.; Huijsman, R.

    2014-01-01

    To examine the impact of corporate structure and quality improvement (QI) activities on improvements in client-reported and professional indicators between 2007 and 2009. A cross-sectional study using organizational survey and indicator multilevel modelling to test relationships between corporate

  17. Performance in physiology evaluation: possible improvement by active learning strategies.

    Science.gov (United States)

    Montrezor, Luís H

    2016-12-01

    The evaluation process is complex and extremely important in the teaching/learning process. Evaluations are constantly employed in the classroom to assist students in the learning process and to help teachers improve the teaching process. The use of active methodologies encourages students to participate in the learning process, encourages interaction with their peers, and stimulates thinking about physiological mechanisms. This study examined the performance of medical students on physiology over four semesters with and without active engagement methodologies. Four activities were used: a puzzle, a board game, a debate, and a video. The results show that engaging in activities with active methodologies before a physiology cognitive monitoring test significantly improved student performance compared with not performing the activities. We integrate the use of these methodologies with classic lectures, and this integration appears to improve the teaching/learning process in the discipline of physiology and improves the integration of physiology with cardiology and neurology. In addition, students enjoy the activities and perform better on their evaluations when they use them. Copyright © 2016 The American Physiological Society.

  18. Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum.

    Science.gov (United States)

    Wang, Ting-Ting; Zhu, Chen-Ye; Zheng, Shuang; Meng, Cai-Cai; Wang, Tian-Tian; Meng, Dan-Hua; Li, Yi-Jun; Zhu, Hao-Miao; Wang, Feng-Shan; Sheng, Ju-Zheng

    2018-04-03

    Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.

  19. Physical activities to improve the aged people’s health

    Directory of Open Access Journals (Sweden)

    Raydelin Hernández-Porraspita

    2013-08-01

    Full Text Available The physical activities have as main function the establishment of ways, forms and means that facilitate a stable health and a high capacity of physical yield in the practitioners, so that they can satisfactorily carry out their activities in all the spheres of their lives serving as base for their development as active citizens of the society. The imperious necessity to increase daily realization of physicalrecreational activities to improve the state of our adults' health is one of the highpriority tasks of the Revolution, besides the mediator role that professionals of physical culture and sport carry out when favouring the benefit of a healthier elderly.

  20. HMM Adaptation for Improving a Human Activity Recognition System

    Directory of Open Access Journals (Sweden)

    Rubén San-Segundo

    2016-09-01

    Full Text Available When developing a fully automatic system for evaluating motor activities performed by a person, it is necessary to segment and recognize the different activities in order to focus the analysis. This process must be carried out by a Human Activity Recognition (HAR system. This paper proposes a user adaptation technique for improving a HAR system based on Hidden Markov Models (HMMs. This system segments and recognizes six different physical activities (walking, walking upstairs, walking downstairs, sitting, standing and lying down using inertial signals from a smartphone. The system is composed of a feature extractor for obtaining the most relevant characteristics from the inertial signals, a module for training the six HMMs (one per activity, and the last module for segmenting new activity sequences using these models. The user adaptation technique consists of a Maximum A Posteriori (MAP approach that adapts the activity HMMs to the user, using some activity examples from this specific user. The main results on a public dataset have reported a significant relative error rate reduction of more than 30%. In conclusion, adapting a HAR system to the user who is performing the physical activities provides significant improvement in the system’s performance.

  1. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    HP

    Purpose: To explore the antioxidant properties of the methanol extract of Pericarpium Zanthoxyli and its effect on inducible nitric oxide synthase (iNOS), cycleooxygenase-2 (COX-2) and lipopolysaccharides (LPS)-induced cell damage in macrophage cells. Methods: Anti-oxidant activities were tested by measuring free ...

  2. Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms ...

    African Journals Online (AJOL)

    Background: Previous studies indicated an association between endothelial nitric oxide synthase (eNOS) activity and maintenance of pregnancy, but it is rather controversial whether polymorphisms of the gene encoding for eNOS are associated with recurrent spontaneous abortions (RSAs). Aim: The aim was to investigate ...

  3. Predicting the catalytic sites of isopenicillin N synthase (IPNS ...

    African Journals Online (AJOL)

    Isopenicillin N synthase (IPNS) related Non-haem iron-dependent oxygenases and oxidases (NHIDOX) demonstrated a striking structural conservativeness, even with low protein sequence homology. It is evident that these enzymes have an architecturally similar catalytic centre with active ligands lining the reactive pocket.

  4. Mechanical Behaviour of Soil Improved by Alkali Activated Binders

    Directory of Open Access Journals (Sweden)

    Enza Vitale

    2017-11-01

    Full Text Available The use of alkali activated binders to improve engineering properties of clayey soils is a novel solution, and an alternative to the widely diffused improvement based on the use of traditional binders such as lime and cement. In the paper the alkaline activation of two fly ashes, by-products of coal combustion thermoelectric power plants, has been presented. These alkali activated binders have been mixed with a clayey soil for evaluating the improvement of its mechanical behaviour. One-dimensional compression tests on raw and treated samples have been performed with reference to the effects induced by type of binder, binder contents and curing time. The experimental evidences at volume scale of the treated samples have been directly linked to the chemo-physical evolution of the binders, investigated over curing time by means of X Ray Diffraction. Test results showed a high reactivity of the alkali activated binders promoting the formation of new mineralogical phases responsible for the mechanical improvement of treated soil. The efficiency of alkali activated binders soil treatment has been highlighted by comparison with mechanical performance induced by Portland cement.

  5. Improving activities of daily living ability in women with fibromyalgia

    DEFF Research Database (Denmark)

    von Bülow, Cecilie; Amris, Kirstine; Bandak, Elisabeth

    2017-01-01

    OBJECTIVE: To explore and compare the outcomes of adaptation and physical activity programmes regarding activities of daily living (ADL) ability following interdisciplinary rehabilitation in women with fibromyalgia. METHODS: Participants (n = 85) were quasi-randomized to 16-week adaptation (ADAPT......) or physical activity (ACTIVE) programmes following 2-week interdisciplinary rehabilitation. Primary outcomes were ADL motor and ADL process ability, measured with the Assessment of Motor and Process Skills (AMPS) at 4-week follow-up. Data were analysed per protocol. RESULTS: Participants (ADAPT, n = 21......: Although limited by a large drop-out, this exploratory study showed that both adaptation and physical activity programmes following interdisciplinary rehabilitation improved ADL ability in the majority of participants. ADL ability outcomes were independent of group allocation (ADAPT vs ACTIVE), suggesting...

  6. Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

    Science.gov (United States)

    Ramírez, Marco A; Morales, Jorge; Cornejo, Marcelo; Blanco, Elias H; Mancilla-Sierpe, Edgardo; Toledo, Fernando; Beltrán, Ana R; Sobrevia, Luis

    2018-04-01

    l-Arginine is taken up via the cationic amino acid transporters (system y + /CATs) and system y + L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y + /CATs and system y + L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH 4 Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 μmol/L, 10 s, 37 °C) in the absence or presence of 200 μmol/L N-ethylmaleimide (NEM) (system y + /CATs inhibitor) or 2 mmol/L l-leucine (systemy + L substrate) was measured. Protein abundance for eNOS and serine 1177 or threonine 495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y + L but not system y + /CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine 1177 phosphorylation. Thus, system y + L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Early interleukin-6 enhances hepatic ketogenesis in APPSWE/PSEN1dE9 mice via 3-hydroxy-3-methylglutary-CoA synthase 2 signaling activation by p38/nuclear factor κB p65.

    Science.gov (United States)

    Shi, Le; Zhao, Daina; Hou, Chen; Peng, Yunhua; Liu, Jing; Zhang, Shuangxi; Liu, Jiankang; Long, Jiangang

    2017-08-01

    Alzheimer's disease (AD) is considered a multifactorial disease that affects the central nervous system and periphery. A decline in brain glucose metabolism is an early feature of AD and is accompanied by a phenotypic shift from aerobic glycolysis to ketogenesis. The liver is responsible for the generation of the ketone body. However, the mechanism that underlies hepatic ketogenesis in AD remains unclear. Here, we investigated hepatic ketogenesis during the early stage of AD pathogenesis in amyloid precursor protein (APP SWE ) and presenilin (PSEN1dE9) (APP/PS1) mice. We observed that β-hydroxybutyric acid was increased in the brain of the postmortem mild cognitive impairment and AD subjects and in 3-month-old APP/PS1 AD mice. A rise in 3-hydroxy-3-methylglutary-CoA synthase 2 (HMGCS2), a key enzyme for catalyzing β-hydroxybutyric acid production, was observed in early AD mice. We further showed that proinflammatory cytokines were activated in the liver prior to their activation in the brain of 3-month-old APP/PS1 mice. Among the cytokines, interleukin-6 significantly activated HMGCS2 through the binding of nuclear factor κB (NF-κB) p65 to the HMGCS2 promoter. Additionally, interleukin-6 stimulated phosphorylation of p38 mitogen activated protein kinases, an upstream molecule for NF-κB p65 signaling. We have demonstrated that a hepatic inflammatory factor enhances ketogenesis through HMGCS2 signaling activation by p38/NF-κB p65. These results provide a novel peripheral metabolic mechanism for enhanced ketone production and suggest a plausible early AD phenotype to diagnose AD. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. [The regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by insulin superfamily peptides in myometrium of pregnant women and its impairments under different types of diabetes mellitus].

    Science.gov (United States)

    Kuznetsova, L A; Chistiakova, O V

    2009-01-01

    The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10(-9) M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10(-9) M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women

  9. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-03-01

    The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions.

  10. Thyroid hormone improves insulin signaling and reduces the activation of neurodegenerative pathway in the hippocampus of diabetic adult male rats.

    Science.gov (United States)

    Prieto-Almeida, Fernanda; Panveloski-Costa, Ana Carolina; Crunfli, Fernanda; da Silva Teixeira, Silvania; Nunes, Maria Tereza; Torrão, Andréada Silva

    2018-01-01

    Diabetes mellitus (DM) and impairments of glucose metabolism and insulin resistance in the brain have been suggested as a likely etiology of Alzheimer's disease (AD). Studies have shown that thyroid hormones (THs) improve insulin sensitivity in DM rats and act as mediators of the plasticity of the nervous system altering behavior and cognitive function. Based on these findings, this study aimed to evaluate the effects of diabetes and triiodothyronine (T3) treatment upon proteins associated with DM and AD in the central nervous system. Euglycemic and Diabetic (alloxan-induced) male Wistar rats were daily treated with T3 (1.5μg/100g body weight) or vehicle (saline) for a 4-week period and subdivided into the following groups: euglycemic treated with saline (Control=C); diabetic treated with saline (Diabetic=D); euglycemic treated with T3 (T3); diabetic treated with T3 (DT3). The expression of insulin signaling, neurodegenerative and neuron survival markers was evaluated in the hippocampus by immunoblotting, ELISA, and RT-PCR. T3 treatment decreased glycemia, restored the insulin signaling and reduced the activation of glycogen synthase kinase 3 (GSK3) and tau proteins content in the hippocampus of diabetic rats. The present data provide evidence that T3 treatment of diabetic rats is able to improve insulin sensitivity and reduce the activation of the neurodegenerative pathway in the brain, which might provide neuroprotection in this experimental model. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Prioritising Investments in Marketing Activities to Improve Business Performance

    DEFF Research Database (Denmark)

    Martensen, Anne; Mouritsen, Jan

    2014-01-01

    The purpose of this study is to prioritise investments in marketing activities based on their effect on business performance (BP). On the basis of the European Foundation for Quality Management (EFQM) model adapted to a marketing context, four generic marketing activities are structured in two...... dimensions: (1) Small m: marketing strategy and marketing implementation and (2) big M: cross-functional coordination and innovation. Big M and small m interact and influence BP similarly. When considering investing in marketing activities to improve financial performance, the first priority is to recruit...

  12. Improving Student Active Listening Skills across the Curriculum.

    Science.gov (United States)

    Graves, Jennifer; Loaiza, Karyn

    This research report demonstrates strategies aimed to improve active listening skills in students, which proposed to increase students' academic achievement. The targeted population being studied consisted of first and third grade students from growing low to middle income communities located in a major metropolitan area in central Illinois. Data…

  13. Improvement of nutritional quality and antioxidant activities of yeast ...

    African Journals Online (AJOL)

    This study evaluated the potential use and improves the health beneficial properties of the soybean waste manufacturing products by solid-state fermentation of six GRAS different yeast strains, including extractable antioxidant activities and bioavailable nutritional compositions. In comparison with nonfermented okara ...

  14. Working Memory Training: Improving Intelligence--Changing Brain Activity

    Science.gov (United States)

    Jausovec, Norbert; Jausovec, Ksenija

    2012-01-01

    The main objectives of the study were: to investigate whether training on working memory (WM) could improve fluid intelligence, and to investigate the effects WM training had on neuroelectric (electroencephalography--EEG) and hemodynamic (near-infrared spectroscopy--NIRS) patterns of brain activity. In a parallel group experimental design,…

  15. Active Learning Improves Student Performance in a Respiratory Physiology Lab

    Science.gov (United States)

    Wolf, Alex M.; Liachovitzky, Carlos; Abdullahi, Abass S.

    2015-01-01

    This study assessed the effectiveness of the introduction of active learning exercises into the anatomy and physiology curriculum in a community college setting. Specifically, the incorporation of a spirometry-based respiratory physiology lab resulted in improved student performance in two concepts (respiratory volumes and the hallmarks of…

  16. Performance in Physiology Evaluation: Possible Improvement by Active Learning Strategies

    Science.gov (United States)

    Montrezor, Luís H.

    2016-01-01

    The evaluation process is complex and extremely important in the teaching/learning process. Evaluations are constantly employed in the classroom to assist students in the learning process and to help teachers improve the teaching process. The use of active methodologies encourages students to participate in the learning process, encourages…

  17. Improving Listening Skills through the Use of Active Listening Strategies.

    Science.gov (United States)

    Engraffia, Margie; Graff, Nora; Jezuit, Sue; Schall, Leslie

    This report describes a program for improving active and critical listening skills for elementary age students. The targeted population consists of middle school children located near a large midwestern city. The problems of poor listening skills are documented through data revealing the need for attending interventions that have been put in…

  18. Active video gaming to improve balance in the elderly

    NARCIS (Netherlands)

    Lamoth, C.J.; Caljouw, S.R.; Postema, K.

    2011-01-01

    The combination of active video gaming and exercise (exergaming) is suggested to improve elderly people's balance, thereby decreasing fall risk. Exergaming has been shown to increase motivation during exercise therapy, due to the enjoyable and challenging nature, which could support long-term

  19. Synthetic analogs of anoplin show improved antimicrobial activities

    DEFF Research Database (Denmark)

    Munk, Jens; Uggerhøj, Lars Erik; Poulsen, Tanja Juul

    2013-01-01

    We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin-resistant Enterococcus faecium (ATCC...... compounds are more specific than anoplin. Both 2Nal(6) and Cha(6) show improved therapeutic index against all strains tested....

  20. Improved efficiency of budesonide nebulization using surface-active agents

    NARCIS (Netherlands)

    Heijstra, M. P.; Schaefer, N. C.; Duiverman, E. J.; LeSouef, P. N.; Devadason, S. G.

    2006-01-01

    Our aim was to improve the efficiency of nebulised budesonide using surface-active agents. Cationic, anionic, and nonionic detergents were added to commercial budesonide suspension, and the particle size distribution during nebulization was measured using both cascade impaction and laser

  1. Enhanced thermotolerance for ethanol fermentation of Saccharomyces cerevisiae strain by overexpression of the gene coding for trehalose-6-phosphate synthase.

    Science.gov (United States)

    An, Ming-Zhe; Tang, Yue-Qin; Mitsumasu, Kanako; Liu, Ze-Shen; Shigeru, Morimura; Kenji, Kida

    2011-07-01

    The effect of overexpression of the trehalose-6-phosphate (T6P) synthase gene (TPS1) on ethanol fermentation of Saccharomyces cerevisiae has been studied at 30 and 38°C. The activity of T6P synthase and the accumulation of trehalose during ethanol fermentation were significantly improved by overexpression of TPS1, and especially at 38°C. Ethanol produced by transformants with and without TPS1 gene overexpression at 38°C was approx. 60 and 37 g/l, respectively. The fermentation efficiency of transformants with TPS1 gene overexpression at 38°C was similar to that at 30°C. The critical growth temperature was increased from 36 to 42°C by TPS1 gene overexpression. These results indicated that overexpression of the TPS1 gene had a beneficial effect on the fermentation capacity of the title yeast strain at high temperatures.

  2. Working memory training: improving intelligence--changing brain activity.

    Science.gov (United States)

    Jaušovec, Norbert; Jaušovec, Ksenija

    2012-07-01

    The main objectives of the study were: to investigate whether training on working memory (WM) could improve fluid intelligence, and to investigate the effects WM training had on neuroelectric (electroencephalography - EEG) and hemodynamic (near-infrared spectroscopy - NIRS) patterns of brain activity. In a parallel group experimental design, respondents of the working memory group after 30 h of training significantly increased performance on all tests of fluid intelligence. By contrast, respondents of the active control group (participating in a 30-h communication training course) showed no improvements in performance. The influence of WM training on patterns of neuroelectric brain activity was most pronounced in the theta and alpha bands. Theta and lower-1 alpha band synchronization was accompanied by increased lower-2 and upper alpha desynchronization. The hemodynamic patterns of brain activity after the training changed from higher right hemispheric activation to a balanced activity of both frontal areas. The neuroelectric as well as hemodynamic patterns of brain activity suggest that the training influenced WM maintenance functions as well as processes directed by the central executive. The changes in upper alpha band desynchronization could further indicate that processes related to long term memory were also influenced. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Do workplace physical activity interventions improve mental health outcomes?

    Science.gov (United States)

    Chu, A H Y; Koh, D; Moy, F M; Müller-Riemenschneider, F

    2014-06-01

    Mental health is an important issue in the working population. Interventions to improve mental health have included physical activity. To review evidence for the effectiveness of workplace physical activity interventions on mental health outcomes. A literature search was conducted for studies published between 1990 and August 2013. Inclusion criteria were physical activity trials, working populations and mental health outcomes. Study quality was assessed using the Jadad scale. Of 3684 unique articles identified, 17 met all selection criteria, including 13 randomized controlled trials, 2 comparison trials and 2 controlled trials. Studies were grouped into two key intervention areas: physical activity and yoga exercise. Of eight high-quality trials, two provided strong evidence for a reduction in anxiety, one reported moderate evidence for an improvement in depression symptoms and one provided limited evidence on relieving stress. The remaining trials did not provide evidence on improved mental well-being. Workplace physical activity and yoga programmes are associated with a significant reduction in depressive symptoms and anxiety, respectively. Their impact on stress relief is less conclusive. © The Author 2014. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Novel applications of plant polyketide synthases.

    Science.gov (United States)

    Abe, Ikuro

    2012-04-01

    The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Reduced-activation steels: Future development for improved creep strength

    International Nuclear Information System (INIS)

    Klueh, R.L.

    2008-01-01

    Reduced-activation steels for fusion applications were developed in the 1980s to replace the elevated-temperature commercial steels first considered. The new steels were patterned after the commercial steels, with the objective that the new steels have yield stress and ultimate tensile strength and impact toughness in a Charpy test comparable to or better than the steels they replaced. That objective was achieved in reduced-activation steels developed in Japan, Europe, and the United States. Although tensile and impact toughness of the reduced-activation steels exceed those of the commercial steels they were patterned after, their creep-rupture properties are inferior to some commercial steels they replaced. They are even more inferior to commercial steels developed since the 1980s. In this paper, compositional differences between reduced-activation steels and new commercial steels are examined, and compositions are proposed for development of new-and-improved reduced-activation steels

  6. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase

    Directory of Open Access Journals (Sweden)

    Gila Idelman

    2015-08-01

    Full Text Available It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4 signaling. LPS-stimulated iNOS and NADPH oxidase (Nox activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O2− production, respectively. The generation of both nitrate and O2− in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD and bilirubin effectively abolished LPS-mediated O2− production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α, an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.

  7. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase.

    Science.gov (United States)

    Idelman, Gila; Smith, Darcey L H; Zucker, Stephen D

    2015-08-01

    It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide ( [Formula: see text] ) production, respectively. The generation of both nitrate and [Formula: see text] in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated [Formula: see text] production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin. Copyright © 2015. Published by Elsevier B.V.

  8. The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose

    Directory of Open Access Journals (Sweden)

    Madoka eYonekura

    2013-03-01

    Full Text Available Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter–luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light–dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both POsSPS1::LUC and POsSPS11::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the 2 OsSPS genes.

  9. Reconstitution of T Cell Proliferation under Arginine Limitation: Activated Human T Cells Take Up Citrulline via L-Type Amino Acid Transporter 1 and Use It to Regenerate Arginine after Induction of Argininosuccinate Synthase Expression

    Directory of Open Access Journals (Sweden)

    Anke Werner

    2017-07-01

    Full Text Available In the tumor microenvironment, arginine is metabolized by arginase-expressing myeloid cells. This arginine depletion profoundly inhibits T cell functions and is crucially involved in tumor-induced immunosuppression. Reconstitution of adaptive immune functions in the context of arginase-mediated tumor immune escape is a promising therapeutic strategy to boost the immunological antitumor response. Arginine can be recycled in certain mammalian tissues from citrulline via argininosuccinate (ASA in a two-step enzymatic process involving the enzymes argininosuccinate synthase (ASS and argininosuccinate lyase (ASL. Here, we demonstrate that anti-CD3/anti-CD28-activated human primary CD4+ and CD8+ T cells upregulate ASS expression in response to low extracellular arginine concentrations, while ASL is expressed constitutively. ASS expression peaked under moderate arginine restriction (20 µM, but no relevant induction was detectable in the complete absence of extracellular arginine. The upregulated ASS correlated with a reconstitution of T cell proliferation upon supplementation of citrulline, while the suppressed production of IFN-γ was refractory to citrulline substitution. In contrast, ASA reconstituted proliferation and cytokine synthesis even in the complete absence of arginine. By direct quantification of intracellular metabolites we show that activated primary human T cells import citrulline but only metabolize it further to ASA and arginine when ASS is expressed in the context of low amounts of extracellular arginine. We then clarify that citrulline transport is largely mediated by the L-type amino acid transporter 1 (LAT1, induced upon human T cell activation. Upon siRNA-mediated knockdown of LAT1, activated T cells lost the ability to import citrulline. These data underline the potential of citrulline substitution as a promising pharmacological way to treat immunosuppression in settings of arginine deprivation.

  10. IMPROVEMENT OF QUALITY ASSURANCE SYSTEM ACTIVITIES OF HIGHER EDUCATION INSTITUTIONS

    Directory of Open Access Journals (Sweden)

    Z. M. Sultalieva

    2016-01-01

    Full Text Available Abstract. The aim of the article is the improvement of quality assessment system of higher education institutions in the aspect of management. The problems of quality improvement are revealed and classified. The analysis of criteria assessment sets used to define the efficiency of higher education institutions activity is carried out. The components of quality of higher education institutions activity are specified. The structural model of quality assessment system of higher education institutions activity is offered. The analysis of macro environment of a university based on the method of strategic management is carried out, i.e. PEST analysis. As a result of the research a new model of macro criteria model of quality assessment system of higher education institutions, characterizing quality management as an approach to university efficiency is offered, moreover, this system can define the level of its competitiveness in the aspect of quality management. 

  11. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  12. Performance improvements on passive activated charcoal 222Rn samplers

    International Nuclear Information System (INIS)

    Wei Suxia

    1996-01-01

    Improvements have been made on passive activated charcoal 222 Rn samplers with sintered metal filters. Based on the samplers of good adaptability to temperature and humidity developed before, better charcoal was selected to further improve their performance in radon absorption ability and moisture-resistance. And charcoal quantity in samplers was strictly controlled. The integration time constant of the improved samplers was about 4.3 days. As the sampler was combined with gamma spectrometer to measure radon concentration, the calibration factor was 0.518 min -1 ·Bq -1 ·m 3 for samplers of 7 days exposure time, and the minimum detectable concentration 0.28 Bq·m -3 if counting time for both background and sample is 1000 minutes. The improved samplers are suited to accurately determine the indoor and outdoor average radon concentration under conditions of great variation in temperature and humidity

  13. Intensification of Doxorubicin-Related Oxidative Stress in the Heart by Hypothyroidism Is Not Related to the Expression of Cytochrome P450 NADPH-Reductase and Inducible Nitric Oxide Synthase, As Well As Activity of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Jaroslaw Dudka

    2012-01-01

    Full Text Available Cytochrome P450 NADPH-reductase (P450R, inducible synthase (iNOS and xanthine oxidase play an important role in the antracycline-related cardiotoxicity. The expression of P450R and iNOS is regulated by triiodothyronine. The aim of this study was to evaluate the effect of methimazole-induced hypothyreosis on oxidative stress secondary to doxorubicin administration. 48 hours after methimazole giving cessation, rats were exposed to doxorubicin (2.0, 5.0 and 15 mg/kg. Blood and heart were collected 4, 48 and 96 h after the drug administration. Animals exposed exclusively to doxorubicin or untreated ones were also assessed. The hypothyreosis (0.025% of methimazole significantly increased the doxorubicin effect on the cardiac carbonyl group and they may increase the glutathione level. An insignificant effect of methimazole was noticed in case of the cardiac lipid peroxidation product, the amount of DNA oxidative damages, iNOS and xanthine oxidase-enzymes responsible for red-ox activation of doxorubicin. However, the concentration of P450R was affected by a lower dose of methimazole in rats administered with doxorubicin. Since in rats receiving doxorubicin changes in oxidative stress caused by methimazole were not accompanied by elevation of bioreductive enzymes, it may be concluded that these changes in the oxidative stress were not related to the tested enzymes.

  14. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  15. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  16. Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

    Directory of Open Access Journals (Sweden)

    Jennifer M Atkinson

    Full Text Available It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment in contrast to other solid tumor cell lines (IC50 >10 uM as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

  17. Improving aerobic capacity through active videogames: A randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Jorge Luiz de Brito-Gomes

    2015-09-01

    Full Text Available AbstractThe rate of peak workload improvement between different types of Active Video Games (AVG in young sedentary adults was investigated. Aerobic capacity improvement after a 6-week intervention between AVG types was also compared. Twenty participants, after baseline assessments, were randomized into one of three parallel groups: structured AVG (n= 6, unstructured AVG (n= 7 and a control group (n= 7. Participants played their respective AVG 3 times a week for 6-weeks (30 minutes-session. The control group maintained normal activities. Both structured and unstructured AVG improved peak workload after four weeks but only the structured group maintained this improvement through week five and six. Aerobic capacity improved in the unstructured (Pre: 36.0 ± 5.2ml.kg.min-¹,Post: 39.7 ± 4.9ml.kg.min-¹, p = .038 and structured AVG (Pre: 39.0 ± 5.9ml.kg.min-¹,Post: 47.8 ± 4.3ml.kg.min-¹, p = .006 groups. Structured AVG provide greater health benefits to aerobic capacity and peak workload in young sedentary but otherwise healthy males relative to unstructured AVG.

  18. Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

    OpenAIRE

    Mafu, Sibongile; Karunanithi, Prema Sambandaswami; Palazzo, Teresa Ann; Harrod, Bronwyn Lee; Rodriguez, Selina Marakana; Mollhoff, Iris Natalie; O’Brien, Terrence Edward; Tong, Shen; Fiehn, Oliver; Tantillo, Dean J.; Bohlmann, Jörg; Zerbe, Philipp

    2017-01-01

    Diterpenes play important roles in plant biology and serve as industrial bioproducts and therapeutics, including the anticancer drug Taxol. Enzymes of the diterpene synthase family produce the many core structural scaffolds that form the foundation of the large diversity of biologically active diterpenes. This paper describes the identification and the mechanism of a distinct diterpene synthase, pseudolaratriene synthase, from the golden larch tree, Pseudolarix amabilis. The enzyme catalyzes ...

  19. Physical activity to improve strength and balance in old people

    Directory of Open Access Journals (Sweden)

    Luz Marina Chalapud Narváez

    2017-05-01

    Full Text Available Introduction: Old age encompasses a fairly broad age in a person’s life, and physical activity becomes the strategy to improve the elderly’s life quality. Objective: To determine the effectiveness of a physical activity program in improving strength to the lower limbs and balance for the elderly. Materials and methods: Semi-experimental longitudinal study with a sample of 57 elderly people to whom an initial and final assessment of balance and strength was performed, as well as a 4-week physical activity program with 2 training sessions each, comprising posture, proprioception, balance and muscle strength exercises. The program used for data analysis was SPSS v.24.0 for Windows. An univariate analysis was performed applying central trend measurements (mean, median, mode, standard deviation, range and a statistical analysis with non-parametric testing for two related Wilcoxon samples. Results: The following tests showed statistical significance: Functional extension (p=0,000, open eyes tandem (p=0,20, closed eyes tandem (p=0,002, closed eyes unipodal (p=0,012 and sitting/standing (p=0,000. Conclusion: This study determined that physical activity is effective to improve balance and muscle strength for lower limbs and a suitable tool to preserve senior’s functionality and Independence.

  20. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison...

  1. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K

    2001-01-01

    that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol....... Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction...... basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were...

  2. Studies of gene expression and activity of hexokinase, phosphofructokinase and glycogen synthase in human skeletal muscle in states of altered insulin-stimulated glucose metabolism

    DEFF Research Database (Denmark)

    Vestergaard, H

    1999-01-01

    When whole body insulin-stimulated glucose disposal rate is measured in man applying the euglycaemic, hyperinsulinaemic clamp technique it has been shown that approximately 75% of glucose is taken up by skeletal muscle. After the initial transport step, glucose is rapidly phosphorylated to glucose...... due to an increased glycogen synthesis rate in muscle, which is paralleled by an increased total GS activity, increased GS mRNA levels and enhanced insulin-stimulated activation of GS. These changes are probably due to local contraction-dependent mechanisms. Likewise, one-legged exercise training has...

  3. BACK and DRAW activities for improving writing skills

    Directory of Open Access Journals (Sweden)

    Muhammad Lukman Syafii

    2017-09-01

    Full Text Available This present paper addresses the issue of writing as an important aspect of EFL instruction. Writing as one of the four basic language skills plays a pivotal role in the context of English teaching as a foreign language in Indonesia. Considering these problems, it is important to modify the writing process in such a way to cope the writing. The process of writing the writer means is BACK and DRAW activities. What the writer termed as BACK and DRAW is a set of writing activities involving students’ active participation in the process, their intense learning experience in producing a required text, as well as their interactive work with peers and the teacher. BACK and DRAW stands for Brainstorming, Attaching, Correcting, Keeping, Developing, Revising, Arranging, and Wow! Or Wonderful! Or Well done!. These activities are good ways to improve the writing Ability.

  4. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

  5. Perceiving active listening activates the reward system and improves the impression of relevant experiences

    Science.gov (United States)

    Kawamichi, Hiroaki; Yoshihara, Kazufumi; Sasaki, Akihiro T.; Sugawara, Sho K.; Tanabe, Hiroki C.; Shinohara, Ryoji; Sugisawa, Yuka; Tokutake, Kentaro; Mochizuki, Yukiko; Anme, Tokie; Sadato, Norihiro

    2015-01-01

    Although active listening is an influential behavior, which can affect the social responses of others, the neural correlates underlying its perception have remained unclear. Sensing active listening in social interactions is accompanied by an improvement in the recollected impressions of relevant experiences and is thought to arouse positive feelings. We therefore hypothesized that the recognition of active listening activates the reward system, and that the emotional appraisal of experiences that had been subject to active listening would be improved. To test these hypotheses, we conducted functional magnetic resonance imaging (fMRI) on participants viewing assessments of their own personal experiences made by evaluators with or without active listening attitude. Subjects rated evaluators who showed active listening more positively. Furthermore, they rated episodes more positively when they were evaluated by individuals showing active listening. Neural activation in the ventral striatum was enhanced by perceiving active listening, suggesting that this was processed as rewarding. It also activated the right anterior insula, representing positive emotional reappraisal processes. Furthermore, the mentalizing network was activated when participants were being evaluated, irrespective of active listening behavior. Therefore, perceiving active listening appeared to result in positive emotional appraisal and to invoke mental state attribution to the active listener. PMID:25188354

  6. Activation of Nrf2/HO-1 Pathway by Glycogen Synthase Kinase-3β Inhibition Attenuates Renal Ischemia/Reperfusion Injury in Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Xiaohua Shen

    2017-06-01

    Full Text Available Background/Aims: Diabetes mellitus can exacerbate renal ischemia-reperfusion (I/R injury (RI/RI. The aim of the present study was to evaluate the protective effect of GSK-3β inhibition (TDZD-8 on I/R-induced renal injury through the Nrf2/HO-1 pathway in a streptozocin (STZ-induced diabetic rat model. Methods: STZ-induced diabetic rats preconditioned with TDZD-8 and ZnPP were subjected to renal I/R. The extent of renal morphologic lesions. Renal function was assessed from blood urea nitrogen (BUN and serum creatinine (Scr, as determined utlizing commercial kits. Oxidative stress and inflammatory activity in the kidney tissue was estimated from levels of malondialdehyde (MDA, interleukin-10 (IL-10, tumor necrosis factor-α (TNF-α, and nitric oxide (NO, as well as the activities of superoxide dismutase (SOD and glutathione (GSH using qRT-PCR and ELISA. The expressions of Nrf2, HO-1, Bcl-2 and NF-κB in the renal tissue were measured by qRT-PCR and western blotting. Results: I/R-induced renal inflammation was reduced significantly by TDZD-8 pretreatment. Preconditioning with TDZD-8 suppressed NF-κB expression and enhanced Bcl-2 expression in the renal tissue. The upregulated level of malondialdehyde (MDA, and reduced activities of superoxide dismutase (SOD and glutathione (GSH in I/R-shocked rats were markedly restored by TDZD-8 pretreatment. Furthermore, pretreatment with TDZD-8 enhanced activation of the Nrf2/HO-1 pathway in the renal tissue of diabetic RI/RI rats. Conclusion: These findings suggest that preconditioning with TDZD-8 may protect the kidney from I/R-induced damage via the activation of the Nrf2/HO-1 pathway in STZ-induced diabetic rats. Further detailed studies are needed to further clarify the underlying mechanisms.

  7. Training to Support Standardization and Improvement of Configuration Management Activities

    Energy Technology Data Exchange (ETDEWEB)

    Dauber, V.; Scheffer, N.; Schoenfelder, C.

    2015-07-01

    In recent years AREVA has conducted several measures to enhance the effectiveness and efficiency of configuration management-related activities within a nuclear power plant (NPP) new build as well as modernization projects, thereby further strengthening its commitment to achieving the highest level of safety in nuclear facilities. This paper describes a new training course that shall support the standardization and improvement of configuration management activities. Recommendations will be given that should be considered when designing and developing comparable training programs to support change processes within engineering units. (Author)

  8. Expression of Cyclooxygenase-2, Nitric Oxide Synthase 2 and Heme Oxygenase-1 mRNA Induced byBis-Eugenol in RAW264.7 Cells and their Antioxidant Activity Determined Using the Induction Period Method.

    Science.gov (United States)

    Murakami, Yukio; Kawata, Akifumi; Fujisawa, Seiichiro

    2017-01-01

    To clarify the mechanisms responsible for the anti-inflammatory/proinflammatory activities of eugenol-related compounds, we investigated the cytotoxicity and up-regulatory/down-refgulatory effects of the biphenols curcumin, bis-eugenol, magnolol and honokiol, and the monophenols eugenol and isoeugenol, on major regulators of cyclooxygenase-2 (Cox-2), nitric oxide synthase 2 (Nos2) and heme oxygenase-1 (HO-1) mRNA in RAW264.7 cells. mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and the theoretical parameters were calculated using the DFT/B3LYP/6-31* method. Also, the antioxidant activity of eugenol-related compounds in combination with 2-mercapto-1-methylimidazole (MMI, as a model for glutathione (GSH)) was investigated using the induction period method for polymerization of methyl methacrylate initiated by benzoyl peroxide (BPO). The cytotoxicity of eugenol-related compounds showed a linear relationship with their softness (σ) and electrophilicity (ω). At a concentration of 50 μM, biphenols except for bis-eugenol elicited the expression of mRNA for both Cox-2 and Nos2, but monophenols did not. In contrast, bis-eugenol elicited Cox-2 gene expression, but down-regulated Nos2 gene expression. bis-Eugenol alone induced the expression of HO-1 mRNA, and when combined with MMI it showed a potent antagonistic effect on BPO-induced antioxidant activity. The ability of methoxyphenols to inhibit LPS-stimulated Cox-2 gene expression declined in the order curcumin > isoeugenol > bis-eugenol > eugenol, and the rank of ability was related to their ω value. Most eugenol-related compounds had proinflammatory activity at high concentrations. However, they had also anti-inflammatory activity at lower concentrations. Eugenol-related compounds may exert antioxidant and anti-inflammatory activity in LPS-stimulated RAW264.7 cells possibly by inhibiting the activation of nuclear factor-kappa B (Nf-ĸB), whereas bis

  9. Active robotic training improves locomotor function in a stroke survivor

    Directory of Open Access Journals (Sweden)

    Krishnan Chandramouli

    2012-08-01

    Full Text Available Abstract Background Clinical outcomes after robotic training are often not superior to conventional therapy. One key factor responsible for this is the use of control strategies that provide substantial guidance. This strategy not only leads to a reduction in volitional physical effort, but also interferes with motor relearning. Methods We tested the feasibility of a novel training approach (active robotic training using a powered gait orthosis (Lokomat in mitigating post-stroke gait impairments of a 52-year-old male stroke survivor. This gait training paradigm combined patient-cooperative robot-aided walking with a target-tracking task. The training lasted for 4-weeks (12 visits, 3 × per week. The subject’s neuromotor performance and recovery were evaluated using biomechanical, neuromuscular and clinical measures recorded at various time-points (pre-training, post-training, and 6-weeks after training. Results Active robotic training resulted in considerable increase in target-tracking accuracy and reduction in the kinematic variability of ankle trajectory during robot-aided treadmill walking. These improvements also transferred to overground walking as characterized by larger propulsive forces and more symmetric ground reaction forces (GRFs. Training also resulted in improvements in muscle coordination, which resembled patterns observed in healthy controls. These changes were accompanied by a reduction in motor cortical excitability (MCE of the vastus medialis, medial hamstrings, and gluteus medius muscles during treadmill walking. Importantly, active robotic training resulted in substantial improvements in several standard clinical and functional parameters. These improvements persisted during the follow-up evaluation at 6 weeks. Conclusions The results indicate that active robotic training appears to be a promising way of facilitating gait and physical function in moderately impaired stroke survivors.

  10. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  11. A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases.

    Science.gov (United States)

    Ye, Zhixia; Bair, Morgan; Desai, Hemant; Williams, Gavin J

    2011-11-01

    Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies. This journal is © The Royal Society of Chemistry 2011

  12. Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase.

    Science.gov (United States)

    Smith, A M

    1990-11-01

    The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between "waxy" proteins and starch-granule-bound starch synthases generally.

  13. Rubisco activity and regulation as targets for crop improvement.

    Science.gov (United States)

    Parry, Martin A J; Andralojc, P John; Scales, Joanna C; Salvucci, Michael E; Carmo-Silva, A Elizabete; Alonso, Hernan; Whitney, Spencer M

    2013-01-01

    Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.

  14. An improved active contour model for glacial lake extraction

    Science.gov (United States)

    Zhao, H.; Chen, F.; Zhang, M.

    2017-12-01

    Active contour model is a widely used method in visual tracking and image segmentation. Under the driven of objective function, the initial curve defined in active contour model will evolve to a stable condition - a desired result in given image. As a typical region-based active contour model, C-V model has a good effect on weak boundaries detection and anti noise ability which shows great potential in glacial lake extraction. Glacial lake is a sensitive indicator for reflecting global climate change, therefore accurate delineate glacial lake boundaries is essential to evaluate hydrologic environment and living environment. However, the current method in glacial lake extraction mainly contains water index method and recognition classification method are diffcult to directly applied in large scale glacial lake extraction due to the diversity of glacial lakes and masses impacted factors in the image, such as image noise, shadows, snow and ice, etc. Regarding the abovementioned advantanges of C-V model and diffcults in glacial lake extraction, we introduce the signed pressure force function to improve the C-V model for adapting to processing of glacial lake extraction. To inspect the effect of glacial lake extraction results, three typical glacial lake development sites were selected, include Altai mountains, Centre Himalayas, South-eastern Tibet, and Landsat8 OLI imagery was conducted as experiment data source, Google earth imagery as reference data for varifying the results. The experiment consequence suggests that improved active contour model we proposed can effectively discriminate the glacial lakes from complex backgound with a higher Kappa Coefficient - 0.895, especially in some small glacial lakes which belongs to weak information in the image. Our finding provide a new approach to improved accuracy under the condition of large proportion of small glacial lakes and the possibility for automated glacial lake mapping in large-scale area.

  15. Improvement of active filter for HIMAC power sources

    Energy Technology Data Exchange (ETDEWEB)

    Kumada, Masayuki [National Inst. of Radiological Sciences, Chiba (Japan); Kubo, Hiroshi; Furuzeki, Shoichiro; Kanazawa, Toru

    1996-03-01

    For the power sources of the synchrotron electromagnets for the heavy particle beam cancer therapy apparatus HIMAC in National Institute of Radiological Science, in order to stabilize the taken-out beam, the ripple property as low as below 1 x 10{sup -6} is required. As for this electromagnet power sources, various devices were applied to lower ripples, and the required specifications have been satisfied. Also the beam spill is stable, but slight variation has been observed, therefore, by improving the performance of the active filter, the ripples were improved. The specifications of the electromagnet power sources and the whole constitution of the power source system are shown. In the HIMAC power sources, the means for having realized the low ripple performance so far are explained. Those are the absorption of the ineffective power generated from the power sources, the control of the ripples of common made due to the transducer thyristor, and the sure compensation of ripples by the control circuit for the power sources. By adding the band pass filters to the active filter, its characteristics were improved. As the result, 1200 Hz ripple component was reduced by 41 db, thus the sufficient effect was obtained. Hereafter, by the high sensitivity measurement of the current of electromagnets and the evaluation of magnetic fields, the validity will be evaluated. (K.I.)

  16. Engaging clinical nurses in quality and performance improvement activities.

    Science.gov (United States)

    Albanese, Madeline P; Evans, Dietra A; Schantz, Cathy A; Bowen, Margaret; Disbot, Maureen; Moffa, Joseph S; Piesieski, Patricia; Polomano, Rosemary C

    2010-01-01

    Nursing performance measures are an integral part of quality initiatives in acute care; however, organizations face numerous challenges in developing infrastructures to support quality improvement processes and timely dissemination of outcomes data. At the Hospital of the University of Pennsylvania, a Magnet-designated organization, extensive work has been conducted to incorporate nursing-related outcomes in the organization's quality plan and to integrate roles for clinical nurses into the Department of Nursing and organization's core performance-based programs. Content and strategies that promote active involvement of nurses and prepare them to be competent and confident stakeholders in quality initiatives are presented. Engaging clinical nurses in the work of quality and performance improvement is essential to achieving excellence in clinical care. It is important to have structures and processes in place to bring meaningful data to the bedside; however, it is equally important to incorporate outcomes into practice. When nurses are educated about performance and quality measures, are engaged in identifying outcomes and collecting meaningful data, are active participants in disseminating quality reports, and are able to recognize the value of these activities, data become one with practice.

  17. Improving Activity of Salt-Lyophilized Enzymes in Organic Media

    Science.gov (United States)

    Borole, Abhijeet P.; Davison, Brian H.

    Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.

  18. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  19. Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid.

    Science.gov (United States)

    Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Stec-Martyna, Emilia; Pawlowska, Zofia; Watala, Cezary

    2016-06-25

    Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1

  20. Physician Perceptions of Performance Feedback in a Quality Improvement Activity.

    Science.gov (United States)

    Eden, Aimee R; Hansen, Elizabeth; Hagen, Michael D; Peterson, Lars E

    2017-10-01

    Physician performance and peer comparison feedback can affect physician care quality and patient outcomes. This study aimed to understand family physician perspectives of the value of performance feedback in quality improvement (QI) activities. This study analyzed American Board of Family Medicine open-ended survey data collected between 2004 and 2014 from physicians who completed a QI module that provided pre- and post-QI project individual performance data and peer comparisons. Physicians made 3480 comments in response to a question about this performance feedback, which were generally positive in nature (86%). Main themes that emerged were importance of accurate feedback data, enhanced detail in the content of feedback, and ability to customize peer comparison groups to compare performance to peers with similar patient populations or practice characteristics. Meaningful and tailored performance feedback may be an important tool for physicians to improve their care quality and should be considered an integral part of QI project design.

  1. Improving the palatability of activated charcoal in pediatric patients.

    Science.gov (United States)

    Cheng, Adam; Ratnapalan, Savithiri

    2007-06-01

    To compare the taste preference and ease of swallowing of activated charcoal among healthy teenagers when mixed separately with 3 different additives: chocolate milk, Coca-Cola, and water. Healthy volunteers between 14 to 19 years of age were selected for the study. Five grams of activated charcoal (25 mL of 0.2 g/mL of Charcodote [Pharma Science, Montreal, Canada]) was mixed with 25 mL of chocolate milk, Coca-Cola, or water individually to make up 50 mL. The volunteers drank the 3 cups of the charcoal-additive mixture separately and then rated taste and ease of swallowing on a 10-cm visual analogue scale. The subjects then indicated their preferred charcoal-additive mixture if he/she had to drink 9 more portions of charcoal (this would estimate the dose of charcoal for a 50-kg child). A total of 44 subjects were recruited (25 boys and 19 girls). The mean scores for taste preference for chocolate milk, Coca-Cola, and water mixtures of charcoal were 5.5, 6.3, and 2.0, respectively, on a 10-cm visual analogue scale. Thus, subjects preferred the taste of charcoal mixed with chocolate milk or Coca-Cola over charcoal mixed with water (P = 0.0003 for both comparisons). The subjects did not show a statistically significant difference for ease of swallowing between the 3 charcoal-additive mixtures. Overall, 48% preferred the chocolate milk mixture, 45% preferred the Coca-Cola mixture, and 7% preferred charcoal mixed with water. Healthy teenaged subjects identified that activated charcoal (Charcodote) mixed with chocolate milk or Coca-Cola (in a 1:1 ratio) improved taste but had no significant effect on improving ease of swallowing. Overall, the addition of chocolate milk or coke improves the palatability of charcoal and is favored over charcoal mixed with water alone.

  2. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  3. ASMPKS: an analysis system for modular polyketide synthases

    Directory of Open Access Journals (Sweden)

    Kong Eun-Bae

    2007-09-01

    Full Text Available Abstract Background Polyketides are secondary metabolites of microorganisms with diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. Polyketides are synthesized by serialized reactions of a set of enzymes called polyketide synthase(PKSs, which coordinate the elongation of carbon skeletons by the stepwise condensation of short carbon precursors. Due to their importance as drugs, the volume of data on polyketides is rapidly increasing and creating a need for computational analysis methods for efficient polyketide research. Moreover, the increasing use of genetic engineering to research new kinds of polyketides requires genome wide analysis. Results We describe a system named ASMPKS (Analysis System for Modular Polyketide Synthesis for computational analysis of PKSs against genome sequences. It also provides overall management of information on modular PKS, including polyketide database construction, new PKS assembly, and chain visualization. ASMPKS operates on a web interface to construct the database and to analyze PKSs, allowing polyketide researchers to add their data to this database and to use it easily. In addition, the ASMPKS can predict functional modules for a protein sequence submitted by users, estimate the chemical composition of a polyketide synthesized from the modules, and display the carbon chain structure on the web interface. Conclusion ASMPKS has powerful computation features to aid modular PKS research. As various factors, such as starter units and post-processing, are related to polyketide biosynthesis, ASMPKS will be improved through further development for study of the factors.

  4. Improved thermal neutron activation sensor for detection of bulk explosives

    Science.gov (United States)

    McFee, John E.; Faust, Anthony A.; Andrews, H. Robert; Clifford, Edward T. H.; Mosquera, Cristian M.

    2012-06-01

    Defence R&D Canada - Suffield and Bubble Technology Industries have been developing thermal neutron activation (TNA) sensors for detection of buried bulk explosives since 1994. First generation sensors, employing an isotopic source and NaI(Tl) gamma ray detectors, were deployed by Canadian Forces in 2002 as confirmation sensors on the ILDS teleoperated, vehicle-mounted, multi-sensor anti-tank landmine detection systems. The first generation TNA could detect anti-tank mines buried 10 cm or less in no more than a minute, but deeper mines and those significantly displaced horizontally required considerably longer times. Mines as deep as 30 cm could be detected with long counting times (1000 s). The second generation TNA detector is being developed with a number of improvements aimed at increasing sensitivity and facilitating ease of operation. Among these are an electronic neutron generator to increase sensitivity for deeper and horizontally displaced explosives; LaBr3(Ce) scintillators, to improve time response and energy resolution; improved thermal and electronic stability; improved sensor head geometry to minimize spatial response nonuniformity; and more robust data processing. This improved sensitivity can translate to either decreased counting times, decreased minimum detectable explosive quantities, increased maximum sensor-to-target displacement, or a trade off among all three. Experiments to characterize the performance of the latest generation TNA in detecting buried landmines and IEDs hidden in culverts were conducted during 2011. This paper describes the second generation system. The experimental setup and methodology are detailed and preliminary comparisons between the performance of first and second generation systems are presented.

  5. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    Science.gov (United States)

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  6. Topical Rosiglitazone Treatment Improves Ulcerative Colitis by Restoring Peroxisome Proliferator-Activated Receptor-gamma Activity

    DEFF Research Database (Denmark)

    Pedersen, G.; Brynskov, Jørn

    2010-01-01

    for 14 days. RESULTS: PPAR gamma expression was fourfold reduced in epithelial cells from inflamed compared with uninflamed mucosa and controls. Adipophillin levels were decreased in parallel. Rosiglitazone induced a concentration-dependent increase in adipophillin levels and restored PPAR gamma activity...... in epithelial cells from inflamed mucosa in vitro. Rosiglitazone enema treatment was well tolerated and reduced the Mayo ulcerative colitis score from 8.9 to 4.3 (P ... gamma activation in vivo. CONCLUSIONS: Roziglitasone enemas improve impaired PPAR gamma activity in inflamed colonic epithelium and have beneficial clinical effect in patients with active distal ulcerative colitis. These findings raise interest in further studies of PPAR gamma ligands that exhibit...

  7. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  8. Nanostructured delivery systems with improved leishmanicidal activity: a critical review.

    Science.gov (United States)

    Bruni, Natascia; Stella, Barbara; Giraudo, Leonardo; Della Pepa, Carlo; Gastaldi, Daniela; Dosio, Franco

    2017-01-01

    Leishmaniasis is a vector-borne zoonotic disease caused by protozoan parasites of the genus Leishmania , which are responsible for numerous clinical manifestations, such as cutaneous, visceral, and mucocutaneous leishmaniasis, depending on the site of infection for particular species. These complexities threaten 350 million people in 98 countries worldwide. Amastigotes living within macrophage phagolysosomes are the principal target of antileishmanial treatment, but these are not an easy target as drugs must overcome major structural barriers. Furthermore, limitations on current therapy are related to efficacy, toxicity, and cost, as well as the length of treatment, which can increase parasitic resistance. Nanotechnology has emerged as an attractive alternative as conventional drugs delivered by nanosized carriers have improved bioavailability and reduced toxicity, together with other characteristics that help to relieve the burden of this disease. The significance of using colloidal carriers loaded with active agents derives from the physiological uptake route of intravenous administered nanosystems (the phagocyte system). Nanosystems are thus able to promote a high drug concentration in intracellular mononuclear phagocyte system (MPS)-infected cells. Moreover, the versatility of nanometric drug delivery systems for the deliberate transport of a range of molecules plays a pivotal role in the design of therapeutic strategies against leishmaniasis. This review discusses studies on nanocarriers that have greatly contributed to improving the efficacy of antileishmaniasis drugs, presenting a critical review and some suggestions for improving drug delivery.

  9. ASMPKS: an analysis system for modular polyketide synthases

    OpenAIRE

    Kong Eun-Bae; Tae Hongseok; Park Kiejung

    2007-01-01

    Abstract Background Polyketides are secondary metabolites of microorganisms with diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. Polyketides are synthesized by serialized reactions of a set of enzymes called polyketide synthase(PKS)s, which coordinate the elongation of carbon skeletons by the stepwise condensation of short carbon precursors. Due to their importance as drugs, the volume of data on polyketides is rapi...

  10. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine

  11. A new salicylate synthase AmS is identified for siderophores biosynthesis in Amycolatopsis methanolica 239(T).

    Science.gov (United States)

    Xie, Feng; Dai, Shengwang; Shen, Jinzhao; Ren, Biao; Huang, Pei; Wang, Qiushui; Liu, Xueting; Zhang, Buchang; Dai, Huanqin; Zhang, Lixin

    2015-07-01

    Siderophores are important for the growth of bacteria or the applications in treatment of iron overload-associated diseases due to the iron-chelating property. Salicylate synthase played a key role in the biosynthesis of some NRPS-derived siderophores by the providing of an iron coordination moiety as the initial building block. A new salicylate synthase, namely AmS, was identified in the biosynthesis pathway of siderophore amychelin in Amycolatopsis methanolica 239(T), since it shunt chorismate, an integrant precursor, from primary to secondary metabolite flow. The amino acid sequence alignment and phylogenetic analysis showed that AmS grouped into a new cluster. In vitro assays of AmS revealed its wide temperature tolerance ranged from 0 to 40 °C and narrow pH tolerant ranged from 7.0 to 9.0. AmS was resistant to organic solvents and non-ionic detergents. Moreover, AmS converted chorismate to salicylate with K m of 129.05 μM, k cat of 2.20 min(-1) at optimal conditions, indicating its low substrate specificity and comparable velocity to reported counterparts (Irp9 and MbtI). These properties of AmS may improve the iron-seizing ability of A. methanolica to compete with its neighbors growing in natural environments. Most importantly, serine and cysteine residues were found to be important for the catalytic activity of AmS. This study presented AmS as a new cluster of salicylate synthase and the reaction mechanism and potential applications of salicylate synthase were highlighted as well.

  12. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  13. Process benchmarking for improvement of environmental restoration activities

    Energy Technology Data Exchange (ETDEWEB)

    Celorie, J.A. [CH2M Hill, Corvallis, OR (United States); Selman, J.R.; Larson, N.B. [Department of Energy, Germantown, MD (United States)

    1995-12-31

    A process benchmarking study was initiated by the Office of Environmental Management (EM) of the US Department of Energy (DOE) to analyze and improve the department`s environmental assessment and environmental restoration (ER) processes. The purpose of this study was to identify specific differences in the processes and implementation procedures used at comparable remediation sites to determine best practices which had the greatest potential to minimize the cost and time required to conduct remedial investigation/ feasibility study (RI/FS) activities. Technical criteria were identified and used to select four DOE, two Department of Defense (DOD), and two Environmental Protection Agency (EPA) restoration sites that exhibited comparable characteristics and regulatory environments. By comparing the process elements and activities executed at the different sites for similar endpoints, best practices were identified for streamlining process elements and minimizing non-value-added activities. Critical measures that influenced process performance were identified and characterized for the sites. This benchmarking study focused on two processes and the internal/external review of documents and the development of the initial evaluation and data collection plan (IEDCP)--since these had a great potential for savings, a high impact on other processes, and a high probability for implementation.

  14. Discovery and Characterization of a Novel Lachrymatory Factor Synthase in Petiveria alliacea and Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds1[W][OA

    Science.gov (United States)

    Musah, Rabi A.; He, Quan; Kubec, Roman

    2009-01-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two α-subunits (68.8 kD each), one γ-subunit (22.5 kD), and one δ-subunit (11.9 kD). The two α-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules. PMID:19692535

  15. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    Directory of Open Access Journals (Sweden)

    Mirian Perez Maluf

    2009-01-01

    Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

  16. Improved Isotherm Data for Adsorption of Methane on Activated Carbons

    KAUST Repository

    Loh, Wai Soong

    2010-08-12

    This article presents the adsorption isotherms of methane onto two different types of activated carbons, namely, Maxsorb III and ACF (A-20) at temperatures from (5 to 75) °C and pressures up to 2.5 MPa. The volumetric technique has been employed to measure the adsorption isotherms. The experimental results presented herein demonstrate the improved accuracy of the uptake values compared with previous measurement techniques for similar adsorbate-adsorbent combinations. The results are analyzed with various adsorption isotherm models. The heat of adsorption, which is concentration and temperature dependent, has been calculated from the measured isotherm data. Henry\\'s law coefficients for these adsorbent-methane pairs are also evaluated at various temperatures. © 2010 American Chemical Society.

  17. Improved efficiency of budesonide nebulization using surface-active agents.

    Science.gov (United States)

    Bouwman, A M; Heijstra, M P; Schaefer, N C; Duiverman, E J; Lesouëf, P N; Devadason, S G

    2006-01-01

    Our aim was to improve the efficiency of nebulised budesonide using surface-active agents. Cationic, anionic, and nonionic detergents were added to commercial budesonide suspension, and the particle size distribution during nebulization was measured using both cascade impaction and laser diffraction. Our results showed that the emitted dose was increased after addition of cationic (p < 0.001) and nonionic detergents (p < 0.01) compared with the commercial formulation alone. The respirable fraction was increased for all detergent formulations (p < 0.001) compared with the commercial formulation. We concluded that cationic and nonionic detergent increased the total output of budesonide from the Sidestream. All detergent formulations increased the respirable fraction of nebulized budesonide.

  18. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Science.gov (United States)

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

    Science.gov (United States)

    Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

    2016-11-01

    Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

  20. Improved Active Harmonic Current Elimination Based on Voltage Detection.

    Directory of Open Access Journals (Sweden)

    Tianyuan Tan

    Full Text Available With the increasing penetration of power electronic equipment in modern residential distribution systems, harmonics mitigation through the distributed generation (DG interfacing converters has received significant attention. Among recently proposed methods, the so-called active resonance damper (ARD and harmonic voltage compensator (HVC based on voltage detection can effectively reduce the harmonic distortions in selected areas of distribution systems. However, it is found out that when traditional ARD algorithm is used to eliminate harmonic current injected by non-linear loads, its performance is constrained by stability problems and can at most eliminate half of the load harmonic currents. Thus, inspired by the duality between ARD and HVC, this paper presents a novel improved resistive active power filter (R-APF algorithm based on integral-decoupling control. The design guideline for its parameters is then investigated through carefully analyzing the closed-loop poles' trajectory. Computer studies demonstrate that the proposed algorithm can effectively mitigate the load harmonic currents and its performance is much better than traditional ARD based on proportional control.

  1. Improved Active Harmonic Current Elimination Based on Voltage Detection.

    Science.gov (United States)

    Tan, Tianyuan; Dong, Shuan; Huang, Yingwei; Liu, Jian; Le, Jian; Liu, Kaipei

    2016-01-01

    With the increasing penetration of power electronic equipment in modern residential distribution systems, harmonics mitigation through the distributed generation (DG) interfacing converters has received significant attention. Among recently proposed methods, the so-called active resonance damper (ARD) and harmonic voltage compensator (HVC) based on voltage detection can effectively reduce the harmonic distortions in selected areas of distribution systems. However, it is found out that when traditional ARD algorithm is used to eliminate harmonic current injected by non-linear loads, its performance is constrained by stability problems and can at most eliminate half of the load harmonic currents. Thus, inspired by the duality between ARD and HVC, this paper presents a novel improved resistive active power filter (R-APF) algorithm based on integral-decoupling control. The design guideline for its parameters is then investigated through carefully analyzing the closed-loop poles' trajectory. Computer studies demonstrate that the proposed algorithm can effectively mitigate the load harmonic currents and its performance is much better than traditional ARD based on proportional control.

  2. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    Science.gov (United States)

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.

  3. Attitudes to Improving Speaking Skills by Guided Individual Activities

    Directory of Open Access Journals (Sweden)

    Galina Kavaliauskienė

    2014-06-01

    Full Text Available Students’ perceptions of difficulties in speaking on professional issues are in the focus of the present article. It is generally assumed that the skill of speaking a foreign language is very difficult to master, while speaking on professional topics involves such difficulties as the usage of specific vocabulary and ability to deal with listeners’ oncoming arguments. The aims of the current research are to investigate learners’ attitudes to the level of difficulty in speaking activi - ties on a subject matter at university and apply an innovative approach to improving their speaking skills. The methodology applied was focused on guided individual learning (GIL, with gradually increasing amount of spontaneity in public talks on the subject matter, starting with prepared short talks on an ESP issue leading to group discussions; moving on to Power Point presentations, involving spontaneous deviations from the subject and followed by question time; further, adding some complex subject matter, such as a discussion on a problematic professional subject suggested by learning materials; and, eventually, speaking impromptu on an issue, with a high level of control of one’s speaking skills. The research method of the learners’ attitudes employed the survey on learner attitudes to four different speaking activities in the classroom, which included short talks, Power Point Presentations, discussions and speaking impromptu. The questionnaire was administered to students of two different specializations by the end of the semester. The respondents were students who studied Psychology and Social Work at the Faculty of Social Policy, at Mykolas Romeris University in Vilnius, Lithuania. The respondents were asked to indicate the degree of difficulty they had with the various speaking activities on the Likert’s scale ranging from “very difficult” (1 to “very easy” (5. The results indicated that perceptions of difficulties to developing speaking

  4. Linalool and linalool nerolidol synthases in roses, several genes for little scent.

    Science.gov (United States)

    Magnard, Jean-Louis; Bony, Aurélie Rius; Bettini, Fabienne; Campanaro, Ausilia; Blerot, Bernard; Baudino, Sylvie; Jullien, Frédéric

    2018-03-11

    Roses are widely appreciated for the appearance of their flowers and for their fragrance. This latter character results from the combination of different odorant molecules among which monoterpenes are often prevalent constituents. In this study, we report the cloning and characterization of three rose monoterpene synthases. In vitro functional characterization of these enzymes showed that one is a (-)-(3R)-linalool synthase whereas the others have a dual (+)-(3S)-linalool nerolidol synthase activity. However, given that the characterized rose cultivars were only able to produce the (-)-(3R)-linalool stereoisomer, the linalool nerolidol synthases are probably not active in planta. Furthermore, these three enzymes were also characterized by a weak expression level as assessed by RT-qPCR and by the low abundance of the corresponding sequences in an EST library. This characteristic is likely to explain why linalool is generally a minor constituent in rose flowers' scents. On this basis, we propose that in roses the monoterpene biosynthesis effort is focused on the production of acyclic monoterpenes derived from geraniol through the recently characterized Nudix biosynthesis pathway, at the expense of conventional monoterpene biosynthesis via terpene synthases such as linalool or linalool nerolidol synthases. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

    Jiang, He-Zhong; Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing; Fan, Hui-Jin; Ma, Xiao-Feng

    2012-01-01

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC 50 value of 10.3 μmol L -1 . (author)

  6. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  7. Characterization of a 1-aminocyclopropane-1-carboxylate synthase gene from loblolly pine (Pinus taeda L.).

    Science.gov (United States)

    Barnes, J R; Lorenz, W W; Dean, J F D

    2008-04-30

    1-Aminocyclopropane-1-carboxylate (ACC) synthase catalyzes what is typically the rate-limiting step in the biosynthesis of ethylene, a gaseous plant growth regulator that plays numerous roles in the growth and development of higher plants. Although ACC synthase genes have been characterized from a wide variety of angiosperm plant species, no ACC synthase genes have been described previously for gymnosperms. Evidence suggests that ethylene helps to regulate wood formation in trees, and may also signal for the metabolic shifts that lead to compression wood formation on the undersides of branches and leaning stems in gymnosperm trees. Since compression wood is an inferior feedstock for the manufacturing of most wood products, a better understanding of the factors influencing its formation could lead to substantial economic benefits. This study describes the isolation and characterization of a putative ACC synthase gene, PtaACS1, from loblolly pine (Pinus taeda L.), an important commercial forest tree species. Also described is an apparent splice variant of PtaACS1 (PtaACS1s) that is missing 138 bp from the 5' end of the transcript, including bases that encode a conserved amino acid residue considered critical for ACC synthase activity. The two sequences share interesting homologies with a group of plant aminotransferases, in addition to ACC synthases, but structural models and the conservation of critical catalytic amino acid residues strongly support PtaACS1 as encoding an active ACC synthase. The two transcripts were differentially expressed in various tissues of loblolly pine, as well as in response to perturbations of pine seedling stems. Transcript levels of this ACC synthase gene increased rapidly in response to bending stress but returned to near starting levels within 30 min. It remains unclear to what extent bending-induced expression of this gene product plays a role in compression wood formation.

  8. Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization

    International Nuclear Information System (INIS)

    Anstrom, David M.; Colip, Leslie; Moshofsky, Brian; Hatcher, Eric; Remington, S. James

    2005-01-01

    Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement. Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success

  9. Can schoolyard improvements increase physical activity for the least active students, or just provide better opportunities for the most active?

    DEFF Research Database (Denmark)

    Breum, Lars; Toftager, M.; Troelsen, J.

    2014-01-01

    Introduction School physical activity interventions can improve the physical environment and organizational environment of schools to increase the opportunities for physical activity (PA) in recess. The purpose of this study was to evaluate if improvements to the outdoor physical environment...... at the school had equal impact on all students regardless of their PA at baseline [1]. Method The SPACE-study used a cluster randomized controlled study design with a 2-year follow-up, and enrolled 1348 students aged 11–13 years from 14 schools in Denmark. A web-based questionnaire was used to obtain knowledge...

  10. [Improvement of the activity of activated sludge by low intensity ultrasound].

    Science.gov (United States)

    Liu, Hong; Yan, Yi-Xin; Wang, Wen-Yan; Yu, Yong-Yong

    2005-07-01

    Aerobic activated sludge from domestic wastewater treatment plant was used as an experimental material. Oxygen Uptake Rate(OUR) was detected as the index to indicate the changes of sludge activity induced by ultrasound(US) at 35kHz in 0 - 40 min with the US intensities of 0 - 1 .2 W/cm2. The best results were achieved at a US intensity of 0.3 W/cm2 and irradiation time of 10 min. Improper US intensity and irradiation time lead to a decrease of sludge activity. Therefore, the stimulating effects of US on the sludge activity could be enhanced at an optimized US intensity and irradiation time. In addition, changes of sludge activities (within 48 hours) after US irradiation at 0.3 W/cm2 in 10 min were examined. The activity of sludge took 8 hours after irradiation to reach peak level, and the activity was as double as that of the initial level after irradiation. In 24 hours after irradiation, the enhancement effects induced by ultrasound disappeared. Therefore, to improve the treatment efficiency of biological reactor, ultrasound with intensity of 0.3W/cm2 could be employed to irradiate part of the sludge in the biological treatment system for 10 minutes every 8 hours. It also presented the hypothetical explanation of the mechanism of biological activity enhancement stimulated by ultrasound.

  11. Improving management of student clinical placements: insights from activity theory.

    Science.gov (United States)

    O'Keefe, Maree; Wade, Victoria; McAllister, Sue; Stupans, Ieva; Burgess, Teresa

    2016-08-24

    An approach to improve management of student clinical placements, the Building Teams for Quality Learning project, was trialed in three different health services. In a previous paper the authors explored in some detail the factors associated with considerable success of this approach at one of these services. In this paper, the authors extend this work with further analysis to determine if the more limited outcomes observed with participants at the other two services could be explained by application of activity theory and in particular the expansive learning cycle. Staff at three health services participated in the Building Teams for Quality Learning project: a dental clinic, a community aged care facility and a rural hospital. At each site a team of seven multi-disciplinary staff completed the project over 9 to 12 months (total 21 participants). Evaluation data were collected through interviews, focus groups and direct observation of staff and students. Following initial thematic analysis, further analysis was conducted to compare the processes and outcomes at each participating health service drawing on activity theory and the expansive learning cycle. Fifty-one interview transcripts, 33 h of workplace observation and 31 sets of workshop field notes (from 36 h of workshops) were generated. All participants were individually supportive of, and committed to, high quality student learning experiences. As was observed with staff at the dental clinic, a number of potentially effective strategies were discussed at the aged care facility and the rural hospital workshops. However, participants in these two health services could not develop a successful implementation plan. The expansive learning cycle element of modeling and testing new solutions was not achieved and participants were unable, collectively to reassess and reinterpret the object of their activities. The application of activity theory and the expansive learning cycle assisted a deeper understanding of

  12. Improving cardiac rehabilitation attendance and completion through quality improvement activities and a motivational program.

    Science.gov (United States)

    Pack, Quinn R; Johnson, Lezlie L; Barr, Laurie M; Daniels, Stephanie R; Wolter, Anne D; Squires, Ray W; Perez-Terzic, Carmen M; Thomas, Randal J

    2013-01-01

    Recent studies have demonstrated that patients who attend more cardiac rehabilitation (CR) sessions have lower subsequent mortality rates than those who attend fewer sessions. We analyzed the impact of several phased-in policy and process changes implemented to increase patient participation in CR. In March 2010, our CR program changed from a policy of individualizing the recommended number of CR sessions per patient to a policy that recommended all 36 CR sessions. In October 2010, we introduced a 7-minute video describing the benefits of CR. In August 2011, we introduced a motivational program that rewarded patients after every sixth CR session. The number of CR sessions attended was determined through review of billing records. Enrollment and completion were defined as attending ≥1 session and ≥30 sessions, respectively. We identified 1103 patients sequentially enrolled in CR between May 2009 and January 2012. Overall, the median number of sessions per patient improved from 12 to 20 (P attendance by a median of 3 sessions per patient (P = .04), but this effect was limited to local CR participants. Financial analysis suggested that for every $100 spent on motivational rewards, patients attended an additional 6.6 (95% CI, -1 to 14) sessions of CR. Quality improvement activities significantly increased CR participation. Wide implementation of such programs may favorably impact patient participation in CR and potentially decrease the rate of subsequent cardiac events.

  13. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

  14. Radiolabeling of a wound-inducible pyridoxal phosphate utilizing protein from tomato: evidence for its identification as ACC synthase

    International Nuclear Information System (INIS)

    Privalle, L.S.; Graham, J.S.; Caughey, P.A.

    1986-01-01

    Aminocyclopropane 1-carboxylic acid (ACC) synthase, a pyridoxal phosphate utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate limiting step in the biosynthesis of the plant hormone, ethylene. Ethylene, besides being involved in normal plant growth processes, is also produced in response to stress, e.g. wounding, pathogen infection, etc. The authors report the partial purification (400 fold) of ACC synthase from wounded pink tomato pericarp by classical techniques including ammonium sulfate precipitation, ion exchange and phenyl sepharose chromatography. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme and the low levels present in plant tissue. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase enriched fraction was achieved. Evidence that this radiolabeled protein is ACC synthase will be presented. Amino acid sequence determination of putative ACC synthase-derived peptides is underway

  15. Improved Fission Neutron Data Base for Active Interrogation of Actinides

    Energy Technology Data Exchange (ETDEWEB)

    Pozzi, Sara; Czirr, J. Bart; Haight, Robert; Kovash, Michael; Tsvetkov, Pavel

    2013-11-06

    This project will develop an innovative neutron detection system for active interrogation measurements. Many active interrogation methods to detect fissionable material are based on the detection of neutrons from fission induced by fast neutrons or high-energy gamma rays. The energy spectrum of the fission neutrons provides data to identify the fissionable isotopes and materials such as shielding between the fissionable material and the detector. The proposed path for the project is as follows. First, the team will develop new neutron detection systems and algorithms by Monte Carlo simulations and bench-top experiments. Next, They will characterize and calibrate detection systems both with monoenergetic and white neutron sources. Finally, high-fidelity measurements of neutron emission from fissions induced by fast neutrons will be performed. Several existing fission chambers containing U-235, Pu-239, U-238, or Th-232 will be used to measure the neutron-induced fission neutron emission spectra. The challenge for making confident measurements is the detection of neutrons in the energy ranges of 0.01 – 1 MeV and above 8 MeV, regions where the basic data on the neutron energy spectrum emitted from fission is least well known. In addition, improvements in the specificity of neutron detectors are required throughout the complete energy range: they must be able to clearly distinguish neutrons from other radiations, in particular gamma rays and cosmic rays. The team believes that all of these challenges can be addressed successfully with emerging technologies under development by this collaboration. In particular, the collaboration will address the area of fission neutron emission spectra for isotopes of interest in the advanced fuel cycle initiative (AFCI).

  16. Highly active vitrification plant remote handling operational experience and improvements

    International Nuclear Information System (INIS)

    Milgate, I.

    1996-01-01

    All the main process plant and equipment at the Sellafield Waste Vitrification Plant (WVP) is enclosed in heavily shielded concrete walled cells. There is a large quantity of relatively complex plant and equipment which must be remotely operated, maintained or replaced in-cell in a severe environment. The WVP has five in-cell polar cranes which are of modular construction to aid replacement of failed components. Each can be withdrawn into a shielded cell extension for decontamination and hands-on maintenance. The cells have a total of 80 through wall tube positions to receive Master Slave Manipulators (MSMs). The MSMs are used where possible for ''pick and place'' purposes but are often called upon to position substantial pieces of mechanical equipment and thus are subject to heavy loading and high failure rates. An inward flow of air is maintained in the active cells. The discharged air passes through a filter cell where remote damper operation filter changing and maintenance is carried out by means of a PAR3000 manipulator. A Nuclear Engineered Advanced Teleoperated Robot (Neater) swabs the vitrified product container to ensure cleanliness before storage. There is a significant arising of solid radioactive waste from replaced in-cell items which undergoes sorting and size reduction in a breakdown cell equipped with a large reciprocating saw and a hydraulic shear. Improvements to the remote handling facilities made in the light of operational experience are described. (UK)

  17. Using RF Smart Points for the Improvement of Metrological Activities

    Directory of Open Access Journals (Sweden)

    Claudio de Capua

    2007-03-01

    Full Text Available This work describes the realization of a “radio-frequency identification system” for the improvement of the activities of a metrological laboratory. Some radio-frequency modules, called by the authors RF Smart Points (“radio-frequency smart points”, have been designed to store into their memories all data which are necessary for the instruments tracking (the type of instruments, their identification numbers or serial numbers, the manufacturer, the date when they have been admitted to the installed base of the laboratory, their working state, the elapsed time from the last calibration procedure. The insertion of the data and the inquiry of the instruments are executed by the technical staff of the laboratory through a PDA (Personal Digital Assistant or a PC, which manage the radio-frequency communication by using the RS 232 interface for sending messages to a RF Transceiver. The executable software for managing the communication between the Smart Points and the “PDA/PC-Controllers” is realized in LabVIEW graphical programming environment.

  18. The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  19. Type III polyketide synthases in microorganisms.

    Science.gov (United States)

    Katsuyama, Yohei; Ohnishi, Yasuo

    2012-01-01

    Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Activation of central angiotensin type 2 receptors by compound 21 improves arterial baroreflex sensitivity in rats with heart failure.

    Science.gov (United States)

    Gao, Juan; Zucker, Irving H; Gao, Lie

    2014-10-01

    In a previous study we demonstrated that central administration of compound 21 (C21), a nonpeptide AT2R agonist, inhibited sympathetic tone in normal rats. In this study, we hypothesized that C21 exerts a similar effect in rats with coronary ligation-induced heart failure (HF). C21 was intracerebroventricularly infused for 7 days by osmotic mini pump. Blood pressure (BP) and heart rate (HR) were recorded by radiotelemetry in the conscious state to measure spontaneous arterial baroreflex sensitivity. Urine was collected for measurement of norepinephrine excretion. On the last day of C21 treatment, renal sympathetic nerve activity, BP, and HR were directly recorded under anesthesia, and the induced arterial baroreflex sensitivity was evaluated. Protein expressions of neuronal nitric oxide synthase (nNOS) and angiotensin II type 1 receptor (AT1R) in the subfornical organ, paraventricular nucleus, rostral ventrolateral medulla, and nucleus tractus solitarius were determined by Western blot analysis. C21-treated HF rats displayed significantly less norepinephrine excretion (2,385.6 ± 121.1 vs. 3,677.3 ± 147.6 ng/24 hours; P baroreflex sensitivity and induced arterial baroreflex sensitivity. Bolus intracerebroventricular injection of angiotensin II-evoked pressor and sympatho-excitatory responses were attenuated in the C21-treated HF rats, which displayed upregulated nNOS and downregulated AT1R expression in the subfornical organ, paraventricular nucleus, and rostral ventrolateral medulla. Activation of central angiotensin II type 2 receptor AT2R by C21 suppresses sympathetic outflow in rats with HF by improving baroreflex sensitivity and may provide important benefit in the HF syndrome. © American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  3. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  4. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

    Science.gov (United States)

    Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

    2014-02-01

    Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison...

  6. Isolation, characterization, and mechanistic studies of (-)-alpha-gurjunene synthase from Solidago canadensis.

    Science.gov (United States)

    Schmidt, C O; Bouwmeester, H J; Bülow, N; König, W A

    1999-04-15

    The leaves of the composite Solidago canadensis (goldenrod) were shown to contain (-)-alpha-gurjunene synthase activity. This sesquiterpene is likely to be the precursor for cyclocolorenone, a sesquiterpene ketone present in high amounts in S. canadensis leaves. (-)-alpha-Gurjunene synthase was purified to apparent homogeneity (741-fold) by anion-exchange chromatography (on several matrices), dye ligand chromatography, hydroxylapatite chromatography, and gel filtration. Chromatography on a gel filtration matrix indicated a native molecular mass of 48 kDa, and SDS-PAGE showed the enzyme to be composed of one subunit with a denatured mass of 60 kDa. Its maximum activity was observed at pH 7.8 in the presence of 10 mM Mg2+ and the KM value for the substrate farnesyl diphosphate was 5.5 microM. Over a range of purification steps (-)-alpha-gurjunene and (+)-gamma-gurjunene synthase activities copurified. In addition, the product ratio of the enzyme activity under several different assay conditions was always 91% (-)-alpha-gurjunene and 9% (+)-gamma-gurjunene. This suggests that the formation of these two structurally related products is catalyzed by one enzyme. For further confirmation, we carried out a number of mechanistic studies with (-)-alpha-gurjunene synthase, in which an enzyme preparation was incubated with deuterated substrate analogues. Based on mass spectrometry analysis of the products formed, a cyclization mechanism was postulated which makes it plausible that the synthase catalyzes the formation of both sesquiterpenes. Copyright 1999 Academic Press.

  7. Increased β-cyanoalanine nitrilase activity improves cyanide tolerance and assimilation in Arabidopsis.

    Science.gov (United States)

    O'Leary, Brendan; Preston, Gail M; Sweetlove, Lee J

    2014-01-01

    Plants naturally produce cyanide (CN) which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens β-cyanoalanine nitrilase pinA were compared with wild-type and a β-cyanoalanine nitrilase knockout line (ΔAtnit4) for growth in the presence of exogenous CN. After incubation with CN, +PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in β-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [¹³C]KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered β-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.

  8. Underlying Mechanisms of Improving Physical Activity Behavior after Rehabilitation

    NARCIS (Netherlands)

    van der Ploeg, Hidde P.; Streppel, Kitty R.M.; van der Beek, Allard J.; Woude, Luc H.V.; van Harten, Willem H.; Vollenbroek-Hutten, Miriam Marie Rosé; van Mechelen, Willem

    2008-01-01

    Background: Regular physical activity is beneficial for the health and functioning of people with a disability. Effective components of successful physical activity promotion interventions should be identified and disseminated. Purpose: To study the underlying mechanisms of the combined sport

  9. Ack kinase regulates CTP synthase filaments during Drosophila oogenesis.

    Science.gov (United States)

    Strochlic, Todd I; Stavrides, Kevin P; Thomas, Sam V; Nicolas, Emmanuelle; O'Reilly, Alana M; Peterson, Jeffrey R

    2014-11-01

    The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis. © 2014 The Authors.

  10. Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

    Science.gov (United States)

    Radwan, Alzahraa; Kleinwächter, Maik; Selmar, Dirk

    2017-09-01

    In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Physical activity stimulation program for children with cerebral palsy did not improve physical activity: a randomised trial

    NARCIS (Netherlands)

    van Wely, L.; Balemans, A.C.J.; Becher, J.G.; Dallmeijer, A.J.

    2014-01-01

    Question: In children with cerebral palsy, does a 6-month physical activity stimulation program improve physical activity, mobility capacity, fitness, fatigue and attitude towards sports more than usual paediatric physiotherapy? Design: Multicentre randomised controlled trial with concealed

  12. The improved physical activity index for measuring physical activity in EPIC Germany.

    Directory of Open Access Journals (Sweden)

    Angelika Wientzek

    Full Text Available In the European Investigation into Cancer and Nutrition study (EPIC, physical activity (PA has been indexed as a cross-tabulation between PA at work and recreational activity. As the proportion of non-working participants increases, other categorization strategies are needed. Therefore, our aim was to develop a valid PA index for this population, which will also be able to express PA continuously. In the German EPIC centers Potsdam and Heidelberg, a clustered sample of 3,766 participants was re-invited to the study center. 1,615 participants agreed to participate and 1,344 participants were finally included in this study. PA was measured by questionnaires on defined activities and a 7-day combined heart rate and acceleration sensor. In a training sample of 433 participants, the Improved Physical Activity Index (IPAI was developed. Its performance was evaluated in a validation sample of 911 participants and compared with the Cambridge Index and the Total PA Index. The IPAI consists of items covering five areas including PA at work, sport, cycling, television viewing, and computer use. The correlations of the IPAI with accelerometer counts in the training and validation sample ranged r = 0.40-0.43 and with physical activity energy expenditure (PAEE r = 0.33-0.40 and were higher than for the Cambridge Index and the Total PA Index previously applied in EPIC. In non-working participants the IPAI showed higher correlations than the Cambridge Index and the Total PA Index, with r = 0.34 for accelerometer counts and r = 0.29 for PAEE. In conclusion, we developed a valid physical activity index which is able to express PA continuously as well as to categorize participants according to their PA level. In populations with increasing rates of non-working people the performance of the IPAI is better than the established indices used in EPIC.

  13. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Using waste of Tofu production improved antagonistic activities of a ...

    African Journals Online (AJOL)

    Mrs. Hoa

    2012-10-04

    Oct 4, 2012 ... screened for their antagonistic activity against 10 races of Xoo causing rice bacterial blight disease. Three actinomycete strains ... antagonistic activity of VN10-A-44 against the Xoo pathogen and to make use of tofu waste for large- ..... vitamins and some essential amino acids which are very important for ...

  15. Improved Antitumoral Activity of Extracts Derived from Cultured ...

    African Journals Online (AJOL)

    After 10, 20 and 30 days, alga samples were collected and their organic extracts prepared. Lipid and phenol contents were evaluated by standardized methods. Antiproliferative activity was assayed in four cancer cell lines (Hep-2, HeLa, SiHa, and KB) while cytotoxic activity was evaluated on a normal cell line (MDCK).

  16. Improving active Mealy machine learning for protocol conformance testing

    NARCIS (Netherlands)

    Aarts, F.; Kuppens, H.; Tretmans, J.; Vaandrager, F.; Verwer, S.

    2014-01-01

    Using a well-known industrial case study from the verification literature, the bounded retransmission protocol, we show how active learning can be used to establish the correctness of protocol implementation I relative to a given reference implementation R. Using active learning, we learn a model M

  17. Can dance exergaming contribute to improving physical activity ...

    African Journals Online (AJOL)

    Physically active video games that involve dancing are being marketed as possible forms of group physical activity (PA) in schools. However, the efficacy of a group video game dance intervention for reducing sedentary behaviour is unknown. The purpose of the present study was to compare before-school group video ...

  18. Improved Oxidase Mimetic Activity by Praseodymium Incorporation into Ceria Nanocubes.

    Science.gov (United States)

    Jiang, Lei; Fernandez-Garcia, Susana; Tinoco, Miguel; Yan, Zhaoxia; Xue, Qi; Blanco, Ginesa; Calvino, Jose J; Hungria, Ana B; Chen, Xiaowei

    2017-06-07

    Ceria nanocubes (NC) modified with increasing concentrations of praseodymium (5, 10, 15, and 20 mol %) have been successfully synthesized by a hydrothermal method. The as-synthesized Pr-modified ceria nanocubes exhibit an enhanced oxidase-like activity on the organic dye TMB within a wide range of concentrations and durations. The oxidase activity increases with increasing Pr amounts in Pr-modified ceria nanocubes within the investigated concentration range. Meanwhile, these Pr-modified ceria nanocubes also show higher reducibility than pure ceria nanocubes. The kinetics of their oxidase mimetic activity is fitted with the Michaelis-Menten equation. A mechanism has been proposed on how the Pr incorporation could affect the energy level of the bands in ceria and hence facilitate the TMB oxidation reaction. The presence of Pr 3+ species on the surface also contributes to the increasing activity of the Pr-modified ceria nanocubes present higher oxidase activity than pure ceria nanocubes.

  19. Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

    2012-01-01

    The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

  20. Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Liang, Y-L; Zhao, S-J; Xu, L-X; Zhang, X-Y

    2012-11-01

    Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. A dammarenediol-producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT-PCR. Fermentation was carried out in a 5-L GRJY-bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250μgl(-1). However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. This study showed that the high-value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene-derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  1. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  2. Active Learning to Improve Fifth Grade Mathematics Achievement in Banten

    Directory of Open Access Journals (Sweden)

    Andri Suherman

    2011-12-01

    Full Text Available Teaching for active learning is a pedagogical technique that has been actively promoted in Indonesian education through government reform efforts and international development assistance projects for decades. Recently, elementary schools in Banten province received training in active learning instructional strategies from the USAID-funded project, Decentralized Basic Education 2. Post-training evaluations conducted by lecturers from the University of Sultan Ageng Tirtayasa (UNTIRTA: Universitas Sultan Ageng Tirtayasa suggested that teachers were successfully employing active learning strategies in some subjects, but not mathematics. In order to understand the difficulties teachers were having in teaching for active learning in mathematics, and to assist them in using active learning strategies, a team of lecturers from UNTIRTA designed and carried out an action research project to train teachers in an elementary school in the city of Cilegon to use a technique called Magic Fingers in teaching Grade 5 multiplication. During the course of the project the research team discovered that teachers were having problems transferring knowledge gained from training in one context and subject to other school subjects and contexts. Key Words: Mathematics, Teaching for Active Learning, Indonesia, Banten

  3. Multiple sclerosis outpatient future groups: improving the quality of participant interaction and ideation tools within service improvement activities.

    Science.gov (United States)

    Thomson, Alison; Rivas, Carol; Giovannoni, Gavin

    2015-04-23

    Improving the patient experience is a key focus within the National Health Service. This has led us to consider how health services are experienced, from both staff and patient perspectives. Novel service improvement activities bring staff and patients together to use design-led methods to improve how health services are delivered. The Multiple Sclerosis Outpatient Future Group study aimed to explore how analogies and props can be used to facilitate rich interactions between staff and patients within these activities. This paper will consider how these interactions supported participants to share experiences, generate ideas and suggest service improvements. Qualitative explorative study using 'future groups,' a reinterpretation of the recognised focus groups method directed towards exploring future alternatives through employing analogies and physical props to engage participants to speculate about future service interactions and health experiences. Participants were people with multiple sclerosis (PwMS) and outpatient staff: staff nurses, nursing assistants, junior sisters and reception staff. Use of future groups, analogies and physical props enabled PwMS and outpatient staff to invest their own ideas and feelings in the service improvement activity and envisage alternative health care scenarios. The combination of participants in the groups with their diverse perspectives and knowledge of the service led to a collaborative approach in which staff highlighted potential practical problems and patients ensured ideas were holistic. Service improvements were prototyped and tested in the outpatient clinic. Design-led methods such as future groups using analogies and physical props can be used to facilitate interactions between staff and patients in service improvement activities, leading to the generation of meaningful ideas. It is hoped that improving the quality of ideation tools within design-led methods can contribute to developing successful service interventions

  4. Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice.

    Science.gov (United States)

    Gauba, Esha; Guo, Lan; Du, Heng

    2017-01-01

    Brain aging is the known strongest risk factor for Alzheimer's disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD.

  5. Improving health through policies that promote active travel

    DEFF Research Database (Denmark)

    de Nazelle, Audrey; Nieuwenhuijsen, Mark J; Antó, Josep M

    2011-01-01

    Substantial policy changes to control obesity, limit chronic disease, and reduce air pollution emissions, including greenhouse gasses, have been recommended. Transportation and planning policies that promote active travel by walking and cycling can contribute to these goals, potentially yielding ...

  6. Angelica Dahurica ethanolic extract improves impaired wound healing by activating angiogenesis in diabetes.

    Directory of Open Access Journals (Sweden)

    Xiao-Na Zhang

    Full Text Available Abnormal angiogenesis plays an important role in impaired wound healing and development of chronic wounds in diabetes mellitus. Angelica dahurica radix is a common traditional Chinese medicine with wide spectrum medicinal effects. In this study, we analyzed the potential roles of Angelica dahurica ethanolic extract (ADEE in correcting impaired angiogenesis and delayed wound healing in diabetes by using streptozotocin-induced diabetic rats. ADEE treatment accelerated diabetic wound healing through inducing angiogenesis and granulation tissue formation. The angiogenic property of ADEE was subsequently verified ex vivo using aortic ring assays. Furthermore, we investigated the in vitro angiogenic activity of ADEE and its underlying mechanisms using human umbilical vein endothelial cells. ADEE treatment induced HUVECs proliferation, migration, and tube formation, which are typical phenomena of angiogenesis, in dose-dependent manners. These effects were associated with activation of angiogenic signal modulators, including extracellular signal-regulated kinase 1/2 (ERK1/2, Akt, endothelial nitric oxide synthase (eNOS as well as increased NO production, and independent of affecting VEGF expression. ADEE-induced angiogenic events were inhibited by the MEK inhibitor PD98059, the PI3K inhibitor Wortmannin, and the eNOS inhibitor L-NAME. Our findings highlight an angiogenic role of ADEE and its ability to protect against impaired wound healing, which may be developed as a promising therapy for impaired angiogenesis and delayed wound healing in diabetes.

  7. Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics.

    Science.gov (United States)

    Schroeder, Marie A; Ali, Mohammad A; Hulikova, Alzbeta; Supuran, Claudiu T; Clarke, Kieran; Vaughan-Jones, Richard D; Tyler, Damian J; Swietach, Pawel

    2013-03-05

    CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic

  8. Facility optimization to improve activation rate distributions during IVNAA

    International Nuclear Information System (INIS)

    Ebrahimi Khankook, Atiyeh; Rafat Motavalli, Laleh; Miri Hakimabad, Hashem

    2013-01-01

    Currently, determination of body composition is the most useful method for distinguishing between certain diseases. The prompt-gamma in vivo neutron activation analysis (IVNAA) facility for non-destructive elemental analysis of the human body is the gold standard method for this type of analysis. In order to obtain accurate measurements using the IVNAA system, the activation probability in the body must be uniform. This can be difficult to achieve, as body shape and body composition affect the rate of activation. The aim of this study was to determine the optimum pre-moderator, in terms of material for attaining uniform activation probability with a CV value of about 10% and changing the collimator role to increase activation rate within the body. Such uniformity was obtained with a high thickness of paraffin pre-moderator, however, because of increasing secondary photon flux received by the detectors it was not an appropriate choice. Our final calculations indicated that using two paraffin slabs with a thickness of 3 cm as a pre-moderator, in the presence of 2 cm Bi on the collimator, achieves a satisfactory distribution of activation rate in the body. (author)

  9. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

    Gonzalez Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J.; Faraldos, Juan A.; Allemann, Rudolf Konrad

    2014-01-01

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and

  10. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  11. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  12. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  13. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  14. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  15. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  16. Improved Control Strategy for Active Bouncers used in Klystron Modulators

    CERN Document Server

    Aguglia, D; Benedetti, M; Garcia Retegui, R; Maestri, S; Nisbet, D

    2012-01-01

    This paper introduces a closed-loop control system for klystron modulators. The system is based on the discharge of a capacitor into a step-up voltage transformer and an active bouncer implemented with a multiphase buck converter. In order to obtain a constant Klystron voltage at the at-top, the active bouncer must compensate both the capacitor discharge and the pulse transformer characteristic. The proposed control includes an inner voltage regulation loop that controls the active bouncer output voltage and an outer one that controls the klystron voltage. The primary side current and main capacitor voltage are included in the regulation loops to simplify the controllers. Simulations demonstrate that the strategy adopted allows to obtain a precision better than 0:1% on a 110 kV klystron. Experimental tests have shown that the multiphase converter is able to track a high dynamics reference even under variable output voltage conditions.

  17. Synthesis of N-(Methoxycarbonylthienylmethylthioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Michael D. Threadgill

    2010-04-01

    Full Text Available Two isomeric N-(methoxycarbonylthienylmethylthioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS.

  18. On the road to improved scheduling - fitting activities to capacity

    DEFF Research Database (Denmark)

    Lindhard, Søren Munch; Wandahl, Søren

    2012-01-01

    Last Planner System has through the sounding process increased the reliability of the schedule. The sound activities are moved to a buffer and afterwards selected to the Weekly Work Plans to match capacity. Therefore, in order to maximise productivity it is essential to ensure that the sounding p...

  19. Improved Antitumoral Activity of Extracts Derived from Cultured ...

    African Journals Online (AJOL)

    Erah

    with the total polyphenol content suggesting a causal link related to extract content of polyphenols and phenolic acids [8], while other authors have reported a variety of fatty acids and derivatives with ..... the effectiveness of anticancer therapy; some anticancer drugs show their anticancer activity by inducing apoptosis of ...

  20. Improvement of nutritional quality and antioxidant activities of yeast ...

    African Journals Online (AJOL)

    Jane

    2011-06-27

    Jun 27, 2011 ... the nutritional quality and antioxidant activities of the substrate were enhanced by solid yeast treatment fermentation. Thus, scope exists for ..... may have definite advantages in a diet. It can act as a suitable replacement ..... varying degrees, while giving a good performance in its scavenging of free radicals.

  1. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis.

    Science.gov (United States)

    Hegardt, F G

    1999-03-15

    Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal-neonatal and suckling-weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues.

  2. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  3. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation.

    Science.gov (United States)

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle

    2014-02-15

    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.

  4. Improving adherence physical activity, quality of life and social support for activities in people with intellectual disabilities, through multimodal intervention (education + activity)

    OpenAIRE

    Perez Cruzado, David; Cuesta-Vargas, Antonio; Merchan Baeza, Jose Antonio; Roldan Jimenez, Cristina

    2014-01-01

    Background People with intellectual disabilities (ID) have a poor level of physical activity as they experience many barriers towards doing it (Bodde & Seo, 2009). Physical activity provides many benefits so there are studies to improve adherence to physical activity (Stanish & Frey, 2008). In our study we carried out a multimodal intervention (educational advice + physical activity) to improve adherence to physical activity in people with ID. Method. The sample consisted of 40 peop...

  5. Improvement in dry active waste segregation and processing

    International Nuclear Information System (INIS)

    Hillmer, T.P.; Anderson, K.D.; Dahlen, D.E.

    1989-01-01

    At the Palo Verde Nuclear Generating Station (PVNGS) the majority of dry active waste (DAW) volume reduction activities are performed in the site's new DAW processing and storage facility. This facility houses an interim storage area for a five year volume of compacted DAW, a shredder/compactor, and a DAW segregation area. The DAW segregation program locates and separates non-radioactive and reusable materials from DAW generated at the three unit PVNGS site. This program has saved more than 24,000 cubic feet of burial space and has reclaimed more than $1,000,000 worth of materials. Palo Verde has made numerous changes to the DAW segregation program since its inception. To ensure that the DAW segregation program remained cost effective and in compliance with applicable regulatory guidance, segregation techniques were revised and new equipment was evaluated and procured. This paper details that effort and summarizes the operational data that has been collected

  6. On the road to improved scheduling - fitting activities to capacity

    DEFF Research Database (Denmark)

    Lindhard, Søren Munch; Wandahl, Søren

    2012-01-01

    Last Planner System has through the sounding process increased the reliability of the schedule. The sound activities are moved to a buffer and afterwards selected to the Weekly Work Plans to match capacity. Therefore, in order to maximise productivity it is essential to ensure that the sounding...... solutions to the problem are suggested and discussed. It is proposed to simplify the production by decreasing the number of trades and tasks completed at site. This can be achieved by increasing...

  7. Religion and health: does religious activity improve health outcomes?

    Science.gov (United States)

    Mulligan, Thomas; Gunaratnam, Magdalena; Martin, Caren McHenry

    2005-12-01

    A majority of Americans believe in God and say that religion plays a major role in their lives. Several studies have found a potential link between religious activity and longevity, overall health status, and the ability to recover from an adverse health event. The high level of interest among patients and health care providers for incorporating religion into health care suggests the need for further investigation into the role of religion in health outcomes.

  8. Active Experiencing Training Improves Episodic Memory Recall in Older Adults

    Directory of Open Access Journals (Sweden)

    Sarah E. Banducci

    2017-05-01

    Full Text Available Active experiencing (AE is an intervention aimed at attenuating cognitive declines with mindfulness training via an immersive acting program, and has produced promising results in older adults with limited formal education. Yet, the cognitive mechanism(s of intervention benefits and generalizability of gains across cognitive domains in the course of healthy aging is unclear. We addressed these issues in an intervention trial of older adults (N = 179; mean age = 69.46 years at enrollment; mean education = 16.80 years assigned to an AE condition (n = 86 or an active control group (i.e., theatre history; n = 93 for 4 weeks. A cognitive battery was administered before and after intervention, and again at a 4-month follow-up. Group differences in change in cognition were tested in latent change score models (LCSM. In the total sample, several cognitive abilities demonstrated significant repeated-testing gains. AE produced greater gains relative to the active control only in episodic recall, with gains still evident up to 4 months after intervention. Intervention conditions were similar in the magnitude of gains in working memory, executive function and processing speed. Episodic memory is vulnerable to declines in aging and related neurodegenerative disease, and AE may be an alternative or supplement to traditional cognitive interventions with older adults.

  9. Variability in individual activity bursts improves ant foraging success.

    Science.gov (United States)

    Campos, Daniel; Bartumeus, Frederic; Méndez, Vicenç; Andrade, José S; Espadaler, Xavier

    2016-12-01

    Using experimental and computational methods, we study the role of behavioural variability in activity bursts (or temporal activity patterns) for individual and collective regulation of foraging in A. senilis ants. First, foraging experiments were carried out under special conditions (low densities of ants and food and absence of external cues or stimuli) where individual-based strategies are most prevalent. By using marked individuals and recording all foraging trajectories, we were then able to precisely quantify behavioural variability among individuals. Our main conclusions are that (i) variability of ant trajectories (turning angles, speed, etc.) is low compared with variability of temporal activity profiles, and (ii) this variability seems to be driven by plasticity of individual behaviour through time, rather than the presence of fixed behavioural stereotypes or specialists within the group. The statistical measures obtained from these experimental foraging patterns are then used to build a general agent-based model (ABM) which includes the most relevant properties of ant foraging under natural conditions, including recruitment through pheromone communication. Using the ABM, we are able to provide computational evidence that the characteristics of individual variability observed in our experiments can provide a functional advantage (in terms of foraging success) to the group; thus, we propose the biological basis underpinning our observations. Altogether, our study reveals the potential utility of experiments under simplified (laboratory) conditions for understanding information-gathering in biological systems. © 2016 The Author(s).

  10. Transcriptional regulation of mitochondrial HMG-CoA synthase in the control of ketogenesis.

    Science.gov (United States)

    Hegardt, F G

    1998-10-01

    Mitochondrial and cytosolic HMG-CoA synthases are encoded by two different genes. Control of ketogenesis is exerted by transcriptional regulation of mitochondrial HMG-CoA synthase. Fasting, cAMP, and fatty acids increase its transcriptional rate, while refeeding and insulin repress it. Fatty acids increase transcription through peroxisomal proliferator regulatory element (PPRE), to which peroxisome proliferator activated receptor (PPAR) can bind. Other transcription factors such as chicken ovalbumin upstream promoter transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF-4) compete for the PPRE site, modulating the response of PPAR.

  11. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

    Science.gov (United States)

    Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

    2009-02-01

    Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

  12. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

    Science.gov (United States)

    Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-02-01

    Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. An improved affine projection algorithm for active noise cancellation

    Science.gov (United States)

    Zhang, Congyan; Wang, Mingjiang; Han, Yufei; Sun, Yunzhuo

    2017-08-01

    Affine projection algorithm is a signal reuse algorithm, and it has a good convergence rate compared to other traditional adaptive filtering algorithm. There are two factors that affect the performance of the algorithm, which are step factor and the projection length. In the paper, we propose a new variable step size affine projection algorithm (VSS-APA). It dynamically changes the step size according to certain rules, so that it can get smaller steady-state error and faster convergence speed. Simulation results can prove that its performance is superior to the traditional affine projection algorithm and in the active noise control (ANC) applications, the new algorithm can get very good results.

  14. Application Of Active Screen Method For Ion Nitriding Efficiency Improvement

    Directory of Open Access Journals (Sweden)

    Ogórek M.

    2015-06-01

    Full Text Available Paper presents the research of austenitic steel AISI 304 after ion nitriding at 400°C and at t =4h, for the two different variants of samples distribution in the working plasma reactive chamber tube. In order to assess the effectiveness of ion nitriding variants emission spectroscopy – GDOES, surface hardness tests, microstructure research (LM of nitrided layers were made. It has been found that the use of active screens increases the surface layer thickness and depth of nitrogen diffusion into austenitic steel 304.

  15. Highly active vitrification plant remote handling operational experience and improvements

    International Nuclear Information System (INIS)

    Milgate, I.

    1996-01-01

    Operational experience and technological innovation in the area of remote handling is described for the Sellafield Waste Vitrification Plant (WVP). This plant turns Highly Active Liquid Wastes (HALW) into radioactively immobile, solid forms. The technology needed for remote handling of HALWs, such as ejectors and power fluidics is described as is the mechanical handling needed after the vitrification process. Key features of WVP are described, such as the in-cell cranes, master-slave manipulators and swabbing robots. The severity of the in-cell environment has highlighted the need for innovation in the remote handling equipment and these changes are also described. (UK)

  16. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    International Nuclear Information System (INIS)

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-01-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study

  17. Improving Decision-Making Activities for Meningitis and Malaria

    Science.gov (United States)

    Ceccato, P.; Trzaska, S.; Perez, C.; Kalashnikova, O. V.; del Corral, J.; Cousin, R.; Blumenthal, M. B.; Connor, S.; Thomson, M. C.

    2012-12-01

    Public health professionals are increasingly concerned about the potential impact that climate variability and change can have on infectious disease. The International Research Institute for Climate and Society (IRI) is developing new products to increase the public health community's capacity to understand, use, and demand the appropriate climate data and climate information to mitigate the public health impacts of climate on infectious disease, in particular Meningitis and Malaria. In this paper we present the new and improved products that have been developed for monitoring dust, temperature, rainfall and vectorial capacity model for monitoring and forecasting risks of Meningitis and Malaria epidemics. We also present how the products have been integrated into a knowledge system (IRI Data Library Map room, SERVIR) to support the use of climate and environmental information in climate-sensitive health decision-making.

  18. Improving Decision-Making Activities for Meningitis and Malaria

    Science.gov (United States)

    Ceccato, Pietro; Trzaska, Sylwia; Garcia-Pando, Carlos Perez; Kalashnikova, Olga; del Corral, John; Cousin, Remi; Blumenthal, M. Benno; Bell, Michael; Connor, Stephen J.; Thomson, Madeleine C.

    2013-01-01

    Public health professionals are increasingly concerned about the potential impact that climate variability and change can have on infectious disease. The International Research Institute for Climate and Society (IRI) is developing new products to increase the public health community's capacity to understand, use and demand the appropriate climate data and climate information to mitigate the public health impacts of climate on infectious disease, in particular meningitis and malaria. In this paper, we present the new and improved products that have been developed for: (i) estimating dust aerosol for forecasting risks of meningitis and (ii) for monitoring temperature and rainfall and integrating them into a vectorial capacity model for forecasting risks of malaria epidemics. We also present how the products have been integrated into a knowledge system (IRI Data Library Map Room, SERVIR) to support the use of climate and environmental information in climate-sensitive health decision-making.

  19. Glycogen synthase kinase-3β in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Jacoby, Anne S; Munkholm, Klaus; Vinberg, Maj

    2016-01-01

    OBJECTIVES: The enzyme glycogen synthase kinase-3β (GSK3β) is involved in the mechanisms of action of lithium and may play a role in relation to affective states in bipolar disorder. The objectives of the present study were to compare the activity of GSK-3β (measured as levels of phosphorylated GSK...

  20. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease

    NARCIS (Netherlands)

    McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S.; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A.; Grabowski, Gregory A.; Aerts, Johannes M. F. G.; Cheng, Seng H.; Copeland, Diane P.; Marshall, John

    2007-01-01

    An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease

  1. Expanding the product portfolio of fungal type I fatty acid synthases

    DEFF Research Database (Denmark)

    Zhu, Zhiwei; Zhou, Yongjin J.; Krivoruchko, Anastasia

    2017-01-01

    Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes...

  2. Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kelley, M.J.; Carman, G.M.

    1987-01-01

    The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism

  3. Nitric oxide synthase expression and apoptotic cell death in brains of AIDS and AIDS dementia patients

    NARCIS (Netherlands)

    Vincent, V. A.; de Groot, C. J.; Lucassen, P. J.; Portegies, P.; Troost, D.; Tilders, F. J.; van Dam, A. M.

    1999-01-01

    To determine the occurrence and cellular localization of inducible nitric oxide synthase (iNOS), NOS activity and its association with cell death in brains of AIDS and AIDS dementia complex (ADC) patients. Post-mortem cerebral cortex tissue of eight AIDS patients, eight ADC patients and eight

  4. THYMIDYLATE SYNTHASE LEVELS IN TUMOR-BIOPSIES FROM PATIENTS WITH COLORECTAL-CANCER

    NARCIS (Netherlands)

    MULDER, NH; TIMMERBOSSCHA, H; MEERSMA, GJ; VERSCHUEREN, RCJ

    1994-01-01

    Catalytic activity and FdUMP binding characteristics of thymidylate synthase (TS) were determined in 22 rumor biopsies of patients to be treated (15) or just treated (7) for colorectal cancer with 5-fluorouracil and leucovorin. In 19 samples both parameters could be determined and were found to

  5. The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs

    NARCIS (Netherlands)

    Veldman, RJ; Mita, A; Cuvillier, O; Garcia, [No Value; Klappe, K; Medin, JA; Campbell, JD; Carpentier, S; Kok, JW; Levade, T

    Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected

  6. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion....

  7. Random mutagenesis of 1-aminocyclopropane-1-carboxylate synthase: a key enzyme in ethylene biosynthesis.

    Science.gov (United States)

    Tarun, A S; Lee, J S; Theologis, A

    1998-08-18

    1-Aminocyclopropane-1-carboxylate synthase (ACC synthase, EC 4.4.1. 14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. To determine the amino acid residues critical for the structure and function of this enzyme, the tomato Le-ACS2 isoenzyme has been subjected to both site-directed and PCR random mutagenesis. Mutant ACC synthases with reduced enzyme activity have been selected by using a genetic screen based on the functional complementation of an Escherichia coli Ile auxotroph that has been engineered to express ACC deaminase from Pseudomonas sp. The DNA sequence of almost 1,000 clones has been determined, and 334 single missense mutations have been selected for analysis. We have identified three classes of mutants based on their activity and expression in E. coli. Class I and II mutants have the same level of protein expression as the wild type, but their enzyme activity is reduced to 0-5% and 5-50%, respectively. Class III mutants have neither activity nor detectable protein expression. The inactive mutations are clustered in regions that are highly conserved among various ACC synthases. This library of mutants will facilitate the elucidation of structure-function relationships of this regulatory enzyme.

  8. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    Science.gov (United States)

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

  9. Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases*

    Science.gov (United States)

    Shaw, Jeffrey J.; Berbasova, Tetyana; Sasaki, Tomoaki; Jefferson-George, Kyra; Spakowicz, Daniel J.; Dunican, Brian F.; Portero, Carolina E.; Narváez-Trujillo, Alexandra; Strobel, Scott A.

    2015-01-01

    Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds. PMID:25648891

  10. Improvement in Activities of Daily Living among Danish Centenarians?

    DEFF Research Database (Denmark)

    Rasmussen, Signe Høi; Thinggaard, Mikael; Højgaard, Majken Boris

    2018-01-01

    at investigating if the later born cohort had better functioning in terms of activities of daily living (ADL). Methods: Identification, methodology and assessment instruments were identical in the 1895-West and 1915-West Birth Cohort Studies: All persons living in the western part of Denmark and turning 100 years......Background: With the continued rise in the proportion of the oldest-old in high income countries, it is of interest to know whether the functional health of today's oldest-olds is better or worse than in previous cohorts. Using two Danish centenarian birth cohorts born twenty years apart we aimed...... old in 1995 and 2015, respectively. Data were collected through structured in-home interviews. Participation rates were 74% (n=106) and 79% (n=238), respectively. Results: The proportion of non-disabled women of the 1915-West cohort was more than twice as high compared to the 1895-West cohort...

  11. Kidney Rehabilitation Technology by Improving Blood Flow and Nerve Activation

    International Nuclear Information System (INIS)

    Mohd Jamil Hashim

    2016-01-01

    The rehabilitation of kidney is impossible from doctors point of view. Kidney failure happens when nephron in kidney fail to filter blood and water. Two major causes of kidney failure. First is the shrinkage of kidney and the second is the blockage of kidney medulla. Kidney shrinkage is because nephron damage due to long term diabetes (Nephrology expert point of view). Whereas blockage of kidney is due to food consume which in turn build up deposit at the blood duct connecting to the medulla. Experiment specimen own body. The rehabilitation methodology is to build up your blood flow system and nerve activation. Result from the study is through analyzing blood components such as creatinine, hemoglobin, urea and potassium. Conclusion, creatinine value has lowered and kidney shrinkage has normalize to its original size. It is hopeful I regain my health 100 % when my GFR reading achieved below 100. (author)

  12. Using robust statistics to improve neutron activation analysis results

    International Nuclear Information System (INIS)

    Zahn, Guilherme S.; Genezini, Frederico A.; Ticianelli, Regina B.; Figueiredo, Ana Maria G.

    2011-01-01

    Neutron activation analysis (NAA) is an analytical technique where an unknown sample is submitted to a neutron flux in a nuclear reactor, and its elemental composition is calculated by measuring the induced activity produced. By using the relative NAA method, one or more well-characterized samples (usually certified reference materials - CRMs) are irradiated together with the unknown ones, and the concentration of each element is then calculated by comparing the areas of the gamma ray peaks related to that element. When two or more CRMs are used as reference, the concentration of each element can be determined by several different ways, either using more than one gamma ray peak for that element (when available), or using the results obtained in the comparison with each CRM. Therefore, determining the best estimate for the concentration of each element in the sample can be a delicate issue. In this work, samples from three CRMs were irradiated together and the elemental concentration in one of them was calculated using the other two as reference. Two sets of peaks were analyzed for each element: a smaller set containing only the literature-recommended gamma-ray peaks and a larger one containing all peaks related to that element that could be quantified in the gamma-ray spectra; the most recommended transition was also used as a benchmark. The resulting data for each element was then reduced using up to five different statistical approaches: the usual (and not robust) unweighted and weighted means, together with three robust means: the Limitation of Relative Statistical Weight, Normalized Residuals and Rajeval. The resulting concentration values were then compared to the certified value for each element, allowing for discussion on both the performance of each statistical tool and on the best choice of peaks for each element. (author)

  13. Strategies to improve effectiveness of physical activity coaching systems: Development of personas for providing tailored feedback

    NARCIS (Netherlands)

    Achterkamp, Reinoud; van Weering, Marit; Evering, R.M.H.; Tabak, Monique; Timmerman, Josien; Hermens, Hermanus J.; Vollenbroek-Hutten, Miriam Marie Rosé

    2017-01-01

    Mobile physical activity interventions can be improved by incorporating behavioural change theories. Relations between self-efficacy, stage of change, and physical activity are investigated, enabling development of feedback strategies that can be used to improve their effectiveness. A total of 325

  14. Interacting Patients: The construction of active patientship in quality improvement initiatives

    NARCIS (Netherlands)

    F.D. Vennik (Femke)

    2016-01-01

    markdownabstractThe promotion of active patient participation in healthcare quality improvement projects is an important policy goal in the Netherlands and other Western countries. Healthcare quality improvement is no longer perceived to be an exclusive professional activity; patients, who may be

  15. Using NLP meta, Milton, metaphor models, for improving the activity of the organization

    Directory of Open Access Journals (Sweden)

    Cornel Marian IOSIF

    2010-12-01

    Full Text Available The objective of this paper is the improving of the three methods from the neuro- linguistic programming – metaphor, Milton model and the meta-model, so by using this in daily activities by an organization to improve the activities witch, are performed and to have a more efficient allocation of the available resources.

  16. TENDENCY OF IMPROVEMENT ANALYSIS OF VENTURE ACTIVITY FOR MANAGEMENT DECISIONS

    Directory of Open Access Journals (Sweden)

    G.Yu. Iakovetс

    2015-03-01

    Full Text Available The questions concerning the definition of current trends and prospects of venture financing new innovative enterprises as one of the most effective and alternative, but with a high degree of risk financing sources of the entity. The features of venture financing that is different from other sources of business financing, as well as income from investments of venture capital can greatly exceed the volume of investments, but at the same time such financing risks are significant, so it all makes it necessary to build an effective system of venture capital investments in the workplace. In the course of the study also revealed problems of analysis and minimization of risks in the performance of venture financing of innovative enterprises. Defining characteristics analysis and risk assessment of venture financing helps to find ways to minimize and systematization, avoidance and prevention of risks in the performance of venture capital. The study also identified the major areas of improvement analysis of venture capital for management decisions.

  17. Improved installation prototype for measurement of low argon-37 activity

    Science.gov (United States)

    Pakhomov, Sergei; Dubasov, Yuri

    2015-04-01

    On-site Inspection (OSI) is a key element of verification of State Parties' compliance with the Comprehensive Nuclear-Test-Ban Treaty (CTBT). An on-site inspection is launched to establish whether or not a nuclear explosion has been carried out. One of the most significant evidence of n underground nuclear explosion (UNE) is detection above background concentrations of argon-37 in near surface air. Argon-37 is formed in large amounts at interaction of neutrons of UNE with the potassium which is a part of the majority of rocks. Its estimated contents for the 100th days after explosion with a energy of 1000 t of TNT near a surface can vary from 1 to 1000 mBq/m3. The background concentrations of argon-37 in subsoil air vary 1 do100 mBq/m3. Traditionally, for argon-37 activity measurement the gas-proportional counters are used. But at Khlopin Radium institute the developments of the new type of highly sensitive and low-background installation capable to provide the required range of measurements of the argon-37 concentration are conducted. The liquid scintillation method of the registration of the low-energetic argon-37 electrons is the basic installation principle and as scintillator, the itself condensed air argon sample is used. Registration of scintillations of liquid argon is made by means of system from 3 PMT which cathodes are cooled near to the temperature of liquid nitrogen together with the measuring chamber in which placed the quartz glass ampule, containing the measured sample of the liquefied argon. For converse the short wavelength photons (λ = 127 nm) of liquid argon scintillations to more long-wave, corresponding to the range of PMT sensitivity, the polymer film with tetra-phenyl-butadiene (TPB) is provided. Even the insignificant impurities of nitrogen, oxygen and others gaseous in the liquid argon samples can to cause the quenching of scintillation, especially their slow components. To account this effect and it influence on change of registration

  18. Mesoporous Metal-Containing Carbon Nitrides for Improved Photocatalytic Activities

    Directory of Open Access Journals (Sweden)

    Jie Luo

    2013-01-01

    Full Text Available Graphitic carbon nitrides (g-C3N4 have attracted increasing interest due to their unusual properties and promising applications in water splitting, heterogeneous catalysis, and organic contaminant degradation. In this study, a new method was developed for the synthesis of mesoporous Fe contained g-C3N4 (m-Fe-C3N4 photocatalyst by using SiO2 nanoparticles as hard template and dicyandiamide as precursor. The physicochemical properties of m-Fe-C3N4 were thoroughly investigated. The XRD and XPS results indicated that Fe was strongly coordinated with the g-C3N4 matrix and that the doping and mesoporous structure partially deteriorated its crystalline structure. The UV-visible absorption spectra revealed that m-Fe-C3N4 with a unique electronic structure displays an increased band gap in combination with a slightly reduced absorbance, implying that mesoporous structure modified the electronic properties of g-Fe-C3N4. The photocatalytic activity of m-Fe-C3N4 for photodegradation of Rhodamine B (RhB was much higher than that of g-Fe-C3N4, clearly demonstrating porous structure positive effect.

  19. Improved grazing activity of dairy heifers in shaded tropical grasslands

    Directory of Open Access Journals (Sweden)

    Andréia Cristina Tavares de Mello

    Full Text Available ABSTRACT: Trees in the production systems can effectively reduce hot weather-induced stress in the Brazilian Midwest. High temperatures cause changes in animals daily routine, and trees into pastures can promote benefits. The aim of this research was to evaluate the behavior of dairy heifers in silvopastoral systems in the state of Mato Grosso, Brazil. A herd of 24 crossbreed heifers (3/4 and 7/8 Holstein/Zebu, 350kg average weight, was evaluated over three seasons. Piatã grass was managed under three shade levels: full-sun, moderate-shade, and intensive-shade provided by 10 to 12m high Eucalyptus trees. Behavior data were collected every 15 minutes from 8:30h to 16h. Shade availability significantly impacted heifer behavior, mainly affecting grazing frequency and time during the hottest hours. Grazing behavior was affected by shade levels during the different seasons. Heifers showed preferred grazing times. Heifers in the intensive-shade system visited shady areas during the hottest hours throughout the seasons. Heifers in the full sun-system avoided grazing during the warmer times, ceasing feeding activities. Our results from the Brazilian Midwest showed that shade availability causes breed heifers to change their daily routine.

  20. Activities of CEZ Inc. and improvement of the environment

    Energy Technology Data Exchange (ETDEWEB)

    Kindl, I.V. [Czech Power Co. (CEZ), Prague (Czechoslovakia)

    1995-12-01

    All highly developed nations round the world have gradually assumed the responsibility for the quality of the environment at their respective territories by creating the preconditions (by setting forth concepts, and the legislative, economic and institutional framework as well as the educational information and resources systems) stimulating both individuals and corporate bodies to take care of the environment. Damaging the environment is punished. The approach has been successful in most of the countries. Companies and individuals have begun to recognize the correlations existing between their production and consumption activities and the environment and their direct responsibility for the environmental conditions, realizing that to remove a source of environmental damage or to minimis the damage caused by it in both necessary and more beneficial than being penalized (up to ban of operation) both economically and in broader social terms for failing to comply with the relevant laws. Since 1990 a number of the so-called {open_quotes}ecological{close_quotes} laws, formerly sorely lacking, have been enacted, such as the Environment Act, the Environmental Effects Assessment Act, and the Wastes Act, and the Clean Air Act has been fundamentally amended so that to meet again the requirements of effective protection of the atmosphere from pollutants. In the area of economic tools, the duty of the waste producer to pay for the waste produced was introduced, the fines for air pollution were raised significantly and the polluted waste water discharge fines were amended to keep pace with the devaluation of the local currency.

  1. Optimization to improve precision in neutron activation analysis

    International Nuclear Information System (INIS)

    Yustina Tri Handayani

    2010-01-01

    The level of precision or accuracy required in analysis should be satisfied the general requirements and customer needs. In presenting the results of the analysis, the level of precision is expressed as uncertainty. Requirement general is Horwitz prediction. Factors affecting the uncertainty in the Neutron Activation Analysis (NAA) include the mass of sample, mass standards, concentration in standard, count of sample, count of standard and counting geometry. Therefore, to achieve the expected level of precision, these parameters need to be optimized. A standard concentration of similar materials is applied as a basis of calculation. In the calculation NIST SRM 2704 is applied for sediment samples. Mass of sample, irradiation time and cooling time can be modified to obtain the expected uncertainty. The prediction results show the level of precision for Al, V, Mg, Mn, K, Na, As, Cr, Co, Fe, and Zn eligible the Horwitz. The predictive the count and standard deviation for Mg-27 and Zn-65 were higher than the actual value occurred due to overlapping of Mg-27 and Mn-54 peaks and Zn-65 and Fe-59 peaks. Precision level of Ca is greater than the Horwitz, since the value of microscopic cross section, the probability of radiation emission of Ca-49 and gamma spectrometer efficiency at 3084 keV is relatively small. Increased precision can only be done by extending the counting time and multiply the number of samples, because of the fixed value. The prediction results are in accordance with experimental results. (author)

  2. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    Science.gov (United States)

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Active immunization against IL-23p19 improves experimental arthritis.

    Science.gov (United States)

    Ratsimandresy, Rojo Anthony; Duvallet, Emilie; Assier, Eric; Semerano, Luca; Delavallée, Laure; Bessis, Natacha; Zagury, Jean-François; Boissier, Marie-Christophe

    2011-11-21

    IL-23 is a pro-inflammatory cytokine essential for the differentiation of Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. IL-23 shares a subunit with IL-12, IL-12/23p40, and comprises a specific subunit, IL-23p19. We previously demonstrated that active immunization against entire TNF-α and against peptides of IL-1β was protective in animal models of rheumatoid arthritis. The aim of this study was to evaluate the effect of peptide-based vaccines targeting the IL-23p19 subunit in collagen-induced arthritis (CIA). Using bioinformatics, the murine IL-23p19 subunit was modeled and two peptides were defined in the receptor interacting domain. Each peptide was coupled to keyhole limpet hemocyanin (KLH) to obtain two vaccines IL23-K1 and IL23-K2. Both vaccines were used for immunizations in incomplete Freund adjuvant (IFA) in groups of DBA/1 mice. Control groups received KLH or PBS at the same dates. CIA was induced by two subcutaneous injections of bovine type II collagen (CIIb), and the development of disease assessed during the next two months. Anti-CIIb and anti-IL-23 antibody levels were assessed by ELISA. Pro- and anti-inflammatory cytokines mRNA were quantified by qRT-PCR in the spleen and the synovium. T-cell populations in the spleen were evaluated by FACS analysis. The clinical scores showed that mice treated with IL23-K1 developed less arthritis than negative controls (panti-IL-23 antibodies than those immunized with IL23-K2 (peffect on IL-17 level. Histological examination showed that IL23-K1 strongly protected against joint destruction and inflammation (pvaccination strategy is protective in CIA. This specific targeting of IL-23 might constitute a promising therapeutic approach to explore in rheumatoid arthritis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Physcomitrella PpORS, basal to plant type III polyketide synthases in phylogenetic trees, is a very long chain 2'-oxoalkylresorcinol synthase.

    Science.gov (United States)

    Kim, Sun Young; Colpitts, Che C; Wiedemann, Gertrud; Jepson, Christina; Rahimi, Mehrieh; Rothwell, Jordan R; McInnes, Adam D; Hasebe, Mitsuyasu; Reski, Ralf; Sterenberg, Brian T; Suh, Dae-Yeon

    2013-01-25

    The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2'-oxoalkylresorcinol. Therefore, PpORS is a 2'-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln(218), Val(277), and Ala(286)) at the putative PpORS active site. Substitution of the Ala(286) to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.

  5. Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathway.

    Science.gov (United States)

    Hourdé, C; Jagerschmidt, C; Clément-Lacroix, P; Vignaud, A; Ammann, P; Butler-Browne, G S; Ferry, A

    2009-04-01

    We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing.

  6. Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

    Directory of Open Access Journals (Sweden)

    Ro Dae-Kyun

    2009-07-01

    Full Text Available Abstract Background Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development. Results Glandular trichomes of sunflower (Helianthus annuus L. were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes

  7. Thiazolopyridines Improve Adipocyte Function by Inhibiting 11 Beta-HSD1 Oxoreductase Activity

    Directory of Open Access Journals (Sweden)

    Thirumurugan Rathinasabapathy

    2017-01-01

    Full Text Available Background. Glucocorticoid excess has been linked to clinical observations associated with the pathophysiology of metabolic syndrome. The intracellular glucocorticoid levels are primarily modulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 enzyme that is highly expressed in key metabolic tissues including fat, liver, and the central nervous system. Methods. In this study we synthesized a set of novel tetrahydrothiazolopyridine derivatives, TR-01–4, that specifically target 11β-HSD1 and studied their ability to interfere with the glucocorticoid and lipid metabolism in the 3T3-L1 adipocytes. Results. Based on the docking model and structure-activity relationships, tetrahydrothiazolopyridine derivatives TR-02 and TR-04 showed the highest potency against 11β-HSD1 by dose-dependently inhibiting conversion of cortisone to cortisol (IC50 values of 1.8 μM and 0.095 μM, resp.. Incubation of fat cells with 0.1–10 μM TR-01–4 significantly decreased cortisone-induced lipid accumulation in adipocytes and suppressed 11β-HSD1 mRNA expression. Observed reduction in adipocyte fat stores could be partially explained by decreased expression levels of adipogenic markers (PPAR-γ, aP2 and key enzymes of lipid metabolism, including fatty acid synthase (FAS, hormone sensitive lipase (HSL, and lipoprotein lipase (LPL. Conclusions. The tetrahydrothiazolopyridine moiety served as an active pharmacophore for inhibiting 11β-HSD1 and offered a novel therapeutic strategy to ameliorate metabolic alterations found in obesity and diabetes.

  8. Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.

    Science.gov (United States)

    Wendisch, V F; Spies, M; Reinscheid, D J; Schnicke, S; Sahm, H; Eikmanns, B J

    1997-10-01

    In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

  9. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  10. Glycogen synthase kinase 3 beta: can it be a target for oral cancer

    Directory of Open Access Journals (Sweden)

    Mishra Rajakishore

    2010-06-01

    Full Text Available Abstract Despite progress in treatment approaches for oral cancer, there has been only modest improvement in patient outcomes in the past three decades. The frequent treatment failure is due to the failure to control tumor recurrence and metastasis. These failures suggest that new targets should be identified to reverse oral epithelial dysplastic lesions. Recent developments suggest an active role of glycogen synthase kinase 3 beta (GSK3 β in various human cancers either as a tumor suppressor or as a tumor promoter. GSK3β is a Ser/Thr protein kinase, and there is emerging evidence that it is a tumor suppressor in oral cancer. The evidence suggests a link between key players in oral cancer that control transcription, accelerated cell cycle progression, activation of invasion/metastasis and anti-apoptosis, and regulation of these factors by GSK3β. Moreover, the major upstream kinases of GSK3β and their oncogenic activation by several etiological agents of oral cancer support this hypothesis. In spite of all this evidence, a detailed analysis of the role of GSK3β in oral cancer and of its therapeutic potential has yet to be conducted by the scientific community. The focus of this review is to discuss the multitude of roles of GSK3β, its possible role in controlling different oncogenic events and how it can be targeted in oral cancer.

  11. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  12. Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

    Science.gov (United States)

    Kholodar, Svetlana A; Kohen, Amnon

    2016-07-06

    Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the de novo biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead.

  13. Physical Activity: A Tool for Improving Health (Part 3--Recommended Amounts of Physical Activity for Optimal Health)

    Science.gov (United States)

    Gallaway, Patrick J.; Hongu, Nobuko

    2016-01-01

    By promoting physical activities and incorporating them into their community-based programs, Extension professionals are improving the health of individuals, particularly those with limited resources. This article is the third in a three-part series describing the benefits of physical activity for human health: (1) biological health benefits of…

  14. Improvement of coal fly ash pozzolanic activity by different physical methods

    Directory of Open Access Journals (Sweden)

    Jovanović I.

    2014-01-01

    Full Text Available This paper presents the results of laboratory investigations performed in order to determine the possibility of improving the pozzolanic activity of coal fly ash from two Serbian Thermal Power Plants. The initial samples of fly ash were treated by different physical processes (mechanical activation, grinding and classification. On the obtained samples, the pozzolanic activity was determined using standard methods (with lime and Portland cement. It was found that the above procedures can significantly improve the pozzolanic activity of fly ash, and the best results were achieved when the fly ash was treated by mechanical activation process in the laboratory ring mill.

  15. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

    Science.gov (United States)

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2012-12-05

    Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

  16. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

    Science.gov (United States)

    Brodelius, Maria; Lundgren, Anneli; Mercke, Per; Brodelius, Peter E

    2002-07-01

    A clone encoding farnesyl diphosphate