WorldWideScience
 
 
1

Malate synthase a membrane protein?  

International Nuclear Information System (INIS)

Malate synthase (MS) is generally regarded as a peripheral membrane protein, and believed by some to be ontogenetically associated with ER. However, immuno- and cyto-chemical in situ localizations show MS throughout the matrix of cotton (and cucumber) glyoxysomes, not specifically near their boundary membranes, nor in ER. Only a maximum of 50% MS can be solubilized from cotton glyoxysomes with 1% Triton X-100, 2mM Zwittergen 14, or 10mM DOC +/- salts. Cotton MS does not incorporate 3H-glucosamine in vivo, nor does it react with Con A on columns or blots. Cotton MS banded with ER in sucrose gradients (20-40%) in Tricine after 3h, but not after 22h in Tricine or Hepes, or after 3h in Hepes or K-phosphate. Collectively the authors data are inconsistent with physiologically meaningful MS-membrane associations in ER or glyoxysomes. It appears that experimentally-induced aggregates of MS migrate in ER gradients and occur in isolated glyoxysomes. These data indicate that ER is not involved in synthesis or modification of cottonseed MS prior to its import into the glyoxysomal matrix

2

Importance of malate synthase in the glyoxylate cycle of Ashbya gossypii for the efficient production of riboflavin.  

Science.gov (United States)

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption. PMID:19343342

Sugimoto, Takashi; Kanamasa, Shin; Kato, Tatsuya; Park, Enoch Y

2009-06-01

3

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.  

Science.gov (United States)

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389-394. © 2014 Wiley Periodicals, Inc. PMID:25524525

Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

2015-02-01

4

The localization of nitrite reductase, glutamate synthase and malate metabolism enzymes in Pisum arvense L. roots  

Directory of Open Access Journals (Sweden)

Full Text Available Centrifugation of a homogenate made from Pisum arvense L. roots in a sucrose density gradient enabled the separation of the plastid fraction from mitochondria and microsomes. The presence of nitrite reductase and glutamate synthase was demonstrated in the plastids. Malic enzyme activity was not linked with any organelle fraction and was found only in the cytosol. High malate dehydrogenase activity was found in the mitochondria fraction, although its activity was also determined in plastids. The results suggest that malic acid metabolism in plastids may be the source of reduced pyridine nucleotides for reactions catalysed by nitrite reductase and glutamate synthase.

Genowefa Kubik-Dobosz

1985-03-01

5

Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates of normozoosp...

Hulya Leventerler; Safiye Taga; Ibrahim Ferhat Urunsak; Ismail Atilla Aridogan; Suna Solmaz; Mehmet Turan Cetin; Nurten Dikmen

2013-01-01

6

Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates  

Directory of Open Access Journals (Sweden)

Full Text Available Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml of asthenoteratospermic (40.0±25.7 and azospermic (38.0±43.6 groups were significantly lower than normozoospermic, (93.9±52.1 males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x106 sperm of teratospermic group (136.8±61.8 was significantly higher compared to the normozoospermic (87.3±26.5 males. Glucose concentration (mg/dl in asthenoteratospermic (4.0±1.4 and azospermic (15.4±6.4 groups were significantly higher than fertile (2.0±2.1 males. Also fructose concentration (mg/dl in asthenoteratospermic (706.6±143.3 and azospermic (338.1±228.2 groups were significantly high compared to the normozoospermic (184.7±124.8 group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility. [Cukurova Med J 2013; 38(4.000: 648-658

Hulya Leventerler

2013-08-01

7

Characterization of the N-Acetyl-[alpha]-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis  

Energy Technology Data Exchange (ETDEWEB)

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce {alpha}-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase ({yields}GlcNAc-malate) and the BaBshB deacetylase ({yields}GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 {angstrom} resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other {alpha}-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.

Parsonage, Derek; Newton, Gerald L.; Holder, Robert C.; Wallace, Bret D.; Paige, Carleitta; Hamilton, Chris J.; Dos Santos, Patricia C.; Redinbo, Matthew R.; Reid, Sean D.; Claiborne, Al (Wake Forest); (UNC); (East Anglia); (UCSD)

2012-02-21

8

Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test  

International Nuclear Information System (INIS)

Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect /sup D/(V/K) of 1.3 +/- 0.1 has been found when [2H3]acetyl-CoA is the substrate. The 13C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO2 for isotope ratio mass spectrometric analysis. The 13C isotope effect with [1H3]acetyl-CoA is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of 13(V/K)/sub H/, the value of the 13C isotope effect when deuteriated [2H3]acetyl-CoA is the substrate can be predicted for three possible mechanisms. The results show clearly that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence oflike condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic

9

Long-range effects in anion-? interactions: their crucial role in the inhibition mechanism of Mycobacterium tuberculosis malate synthase.  

Science.gov (United States)

The glyoxylate shunt is an anaplerotic bypass of the traditional Krebs cycle. It plays a prominent role in Mycobacterium tuberculosis virulence, so it can be exploited for the development of antitubercular therapeutics. The shunt involves two enzymes: isocitrate lyase (ICL) and malate synthase (GlcB). The shunt bypasses two steps of the tricarboxylic acid cycle, allowing the incorporation of carbon, and thus, refilling oxaloacetate under carbon-limiting conditions. The targeting of ICL is complicated; however, GlcB, which accommodates the pantothenate tail of acetyl-CoA in the active site, is easier to target. A catalytic Mg(2+) unit is located at the bottom of the cavity, and plays a very important role. Recently, the development of effective antituberculosis drugs based on phenyldiketo acids (PDKAs) has been reported. Interestingly, all the crystal structures of GlcB-inhibitor complexes exhibit close contact between the carboxylate of Asp633 and the face of the aromatic ring of the inhibitor. Remarkably, the replacement of the phenyl ring in PDKA by aliphatic moieties yields inactive inhibitors, suggesting that the aromatic moiety is crucial for inhibition. However, the aromatic ring of PDKA is not electron-deficient, and consequently, the anion-? interaction is expected to be very weak (dominated only by polarization effects). Herein, through a combination analysis of the recent X-ray structures of GlcB-PDKA complexes retrieved from the protein data bank (PDB) and computational ab?initio studies (RI-MP2/def2-TZVP level of theory), we demonstrate the prominent role of the Mg(2+) ion in the active site, which promotes long-range enhancement of the anion-? interaction. PMID:24740694

Bauzá, Antonio; Quiñonero, David; Deyà, Pere M; Frontera, Antonio

2014-06-01

10

Malate synthase gene AoMls in the nematode-trapping fungus Arthrobotrys oligospora contributes to conidiation, trap formation, and pathogenicity.  

Science.gov (United States)

Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the ?AoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the ?AoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the ?AoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the ?AoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora. PMID:24323290

Zhao, Xinying; Wang, Yunchuan; Zhao, Yong; Huang, Yan; Zhang, Ke-Qin; Yang, Jinkui

2014-03-01

11

Electrophoretic and chemical studies on the X-ray damage of malate synthase  

International Nuclear Information System (INIS)

1) Both X-irradiation and treatment with H2O2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H2O2 induced inactivation. Moreover, addition of dithiothreitol after X-irradiation or H2O2 treatment allowed a considerable repair of enzymic activity. 2) Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotection by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregates themselves, however, were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman's reagent prevented both covalent cross-linking and enzyme aggregation. 3) Similar electrophoretic patterns as found for the X-irradiated enzymeterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H2O2. The similarity of the electropherograms, as well as the reversible diminution of enzymic activity and the loss of sulfhydryls in the presence of H2O2, suggest an involvement of H2O2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H2O2 treatment. (orig./AJ)

12

Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.  

Science.gov (United States)

The enzymes of the ?-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

Vorobieva, Anastassia A; Khan, Mohammad Shahneawz; Soumillion, Patrice

2014-10-17

13

Effects of Al(III) and Nano-Al13 Species on Malate Dehydrogenase Activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) ...

Rong Fu Chen; Huihui Li; Xiaodi Yang; Yu Peng; Ling Cai; Ren Fang Shen

2011-01-01

14

Analysis of hyaluronan synthase activity.  

Science.gov (United States)

Hyaluronan (HA) is a component of the extracellular matrix that is involved in many physiological and pathological processes. As HA modulates several functions (i.e., cell proliferation and migration, inflammation), its presence in the tissues can have positive or negative effects. HA synthases (HAS) are a family of three isoenzymes located on the plasma membrane that are responsible for the production of such polysaccharide and, therefore, their activity is critical to determine the accumulation of HA in tissues. Here, we describe a nonradioactive method to quantify the HAS enzymatic activity in crude cellular membrane preparation. PMID:25325955

Vigetti, Davide; Karousou, Evgenia; Viola, Manuela; Passi, Alberto

2015-01-01

15

Malate transport in Schizosaccharomyces pombe.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of ...

Osothsilp, C.; Subden, R. E.

1986-01-01

16

Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity  

Science.gov (United States)

Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level. PMID:22693256

Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J.; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee

2012-01-01

17

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance  

Science.gov (United States)

Aluminum (Al) activated root malate and citrate exudation plays an important role in Al tolerance in many plant species. Here, we report on the identification and characterization of AtMATE, a homolog of the recently discovered sorghum and barley Al tolerance genes, here shown to encode an Al-activ...

18

Malate inhibition of phosphoenolpyruvate carboxylase from crassula.  

Science.gov (United States)

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate(2-) concentration. Of the reaction components only HCO(3) (-) increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K(m), but no effect on V(max). Enzyme preincubated with malate shows the same effect of malate on K(m), but in addition V(max) is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate. PMID:16665178

Wedding, R T; Black, M K

1986-12-01

19

Transhydrogenase activities and malate dismutation linked to fumarate reductase system in the filarial parasite Setaria digitata.  

Science.gov (United States)

Setaria digitata, a cattle filarial parasite, similar to human filarial parasites, possesses significant activities of the 4 transhydrogenases namely NADH-NAD+, NADPH-NAD+, NADH-NADP+, and NADPH-NADP+ in the sonicated mitochondria like particles. The transhydrogenases appear to regulate the metabolic pathways of the parasite in response to the presence of adenyl nucleotides and are non-energy linked. Observations on the transhydrogenase and fumarate reductase activities show the existence of a protein bound NAD in the MLP and a linkage between the fumarate reductase system and malic enzyme through transhydrogenases. The malate dismutation reaction is the result of malic and fumarase enzyme activities. Fumarase and fumarate reductase activities result in succinate formation under anaerobic conditions showing major energy production at the fumarate reductase site. The existence of acetate kinase, phosphotransacetylase, pyruvate carboxylase, propionyl CoA carboxylase and CoA transferase enzymes in the mitochondrial system shows the presence of other energy producing sites in the parasite. The transhydrogenase system, NAD+/NADP+ malic enzyme, fumarase and fumarate reductase are the key enzymes of, production of reducing power for synthetic reactions and regulation of oxidative and reductive stages of the mitochondrial system. Hence, specific drugs targeted against this interconnected complex enzyme system, will be very effective in the control of filarial parasites. PMID:7558563

Unnikrishnan, L S; Raj, R K

1995-07-01

20

Unique animal prenyltransferase with monoterpene synthase activity.  

Science.gov (United States)

Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C(5)) and isopentenyl diphosphate (C(5)) to produce geranyl diphosphate (GDP; C(10)). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway. PMID:19277597

Gilg, Anna B; Tittiger, Claus; Blomquist, Gary J

2009-06-01

 
 
 
 
21

Malate metabolism by Desulfovibrio gigas and its link to sulfate and fumarate reduction: purification of the malic enzyme and detection of NAD(P)+ transhydrogenase activity.  

Science.gov (United States)

Malate metabolism was investigated in lactate grown cells of Desulfovibrio gigas ; 3 mol of malate are converted into 2 mol succinate and 1 mol acetate. The malic enzyme (L-malate:NADP+ oxidoreductase) was purified to homogeneity and partially characterized. The enzyme is monomeric with molecular weight of 45 kDa. Its spectrum has no visible absorption and the activity is stimulated by K+ and Mg2+. The presence of an NAD(P)+ transhydrogenase, the observation of partial reduction of adenylylsulfate reductase by NADH (via NADH-rubredoxin oxidoreductase) and evidence for NADH-linked fumarate reductase activity support the involvement of pyridine nucleotides in the electron pathway toward the reduction of sulfur compounds and/or fumarate. An electron transfer chain to fumarate is proposed, taking into consideration these results and the stoichiometry of end-products derived from malate dismutation. PMID:16887531

Chen, L; LeGall, J; Fareleira, P; Santos, H; Xavier, A V

1995-08-01

22

The role of malate in the synthesis of glutamate in Pisum arvense roots  

Directory of Open Access Journals (Sweden)

Full Text Available The in vivo and in vitro activities of NADH-dependent glutamate synthase in excised Pisum arvense roots increased several-fold under the influence of malate while pyruvate oxaloacctate. citrate and succinate inhibited this entyme. The plastids isolated from Pisum arvense root,. ahen incubated with glutamine and ?-ketoglutarate, released glutamate into the medium Malate clearly stimulated this process. Albizziin (25 mM completely reduced the presence of glutamate in the incubation mixture. These results indicate that reduced pyridine nucleotides arising in P. arvense root plastids during oxidation of malic acid may constitute the indispensable source of electrons for glutamic acid synthesis.

Genowefa Kubik-Dorosz

1986-12-01

23

Activation of LTBP3 gene by a long noncoding RNA (lncRNA) MALAT1 transcript in mesenchymal stem cells from multiple myeloma.  

Science.gov (United States)

Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules in tumor suppressor and oncogenic pathways. However, the magnitude of the contribution of lncRNA expression to normal human tissues and cancers has not been investigated in a comprehensive manner. Here we explored the biology of the lncRNA MALAT1 and considered the potential significance in mesenchymal stem cells from myeloma patients. By using assays such as RNA interference, luciferase, chromatin immunoprecipitation, and RNA immunoprecipitation, we showed that in mesenchymal stem cells MALAT1 promoted the activation effect of the key transcription factor Sp1 on LTBP3 promoter by modulating recruitment of Sp1 to the LTBP3 gene that regulated the bioavailability of TGF-? in particular. Our data suggested that lncRNA MALAT1 directly interacted with Sp1 and LTBP3 promoter to increase expression of LTBP3 gene. The specificity and efficiency of activation were ensured by the formation of a stable complex between MALAT1 and the LTBP3 promoter, direct interaction of MALAT1 with Sp1, and recruitment of Sp1 to the promoter. In this study, we showed that the mechanism of transcriptional activation of LTBP3 promoter depended on MALAT1 initiated from neighboring gene LTBP3 and involved both the direct interaction of the Sp1 and promoter-specific activation. Our knowledge of the role of MALAT1 in cellular transformation is pointing toward its potential use as a biomarker and a target for novel therapeutic approaches in multiple myeloma. PMID:25187517

Li, Bingzong; Chen, Ping; Qu, Jing; Shi, Lei; Zhuang, Wenyue; Fu, Jinxiang; Li, Jun; Zhang, Xiaohui; Sun, Yu; Zhuang, Wenzhuo

2014-10-17

24

Malate Inhibition of Phosphoenolpyruvate Carboxylase from Crassula1  

Science.gov (United States)

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate2? concentration. Of the reaction components only HCO3? increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in Km, but no effect on Vmax. Enzyme preincubated with malate shows the same effect of malate on Km, but in addition Vmax is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate. PMID:16665178

Wedding, Randolph T.; Black, M. Kay

1986-01-01

25

Extramitochondrial citrate synthase activity in bakers' yeast.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-typ...

Rickey, T. M.; Lewin, A. S.

1986-01-01

26

Characterization of putative capsaicin synthase promoter activity.  

Science.gov (United States)

Capsaicin is a very important secondary metabolite that is unique to Capsicum. Capsaicin biosynthesis is regulated developmentally and environmentally in the placenta of hot pepper. To investigate regulation of capsaicin biosynthesis, the promoter (1,537 bp) of pepper capsaicin synthase (CS) was fused to GUS and introduced into Arabidopsis thaliana (Col-0) via Agrobacterium tumefaciens to produce CSPRO::GUS transgenic plants. The CS was specifically expressed in the placenta tissue of immature green fruit. However, the transgenic Arabidopsis showed ectopic GUS expressions in the leaves, flowers and roots, but not in the stems. The CSPRO activity was relatively high under light conditions and was induced by both heat shock and wounding, as CS transcripts were increased by wounding. Exogenous capsaicin caused strong suppression of the CSPRO activity in transgenic Arabidopsis, as demonstrated by suppression of CS expression in the placenta after capsaicin treatment. Furthermore, the differential expression levels of Kas, Pal and pAmt, which are associated with the capsaicinoid biosynthetic pathway, were also suppressed in the placenta by capsaicin treatment. These results support that capsaicin, a feedback inhibitor, plays a pivotal role in regulating gene expression which is involved in the biosynthesis of capsaicinoids. PMID:19809800

Kim, June-Sik; Park, Minkyu; Lee, Dong Ju; Kim, Byung-Dong

2009-10-31

27

Effects of SO/sub 2/ on organic acid content and malate dehydrogenase activity in jack pine needles  

Energy Technology Data Exchange (ETDEWEB)

Sulphur dioxide fumigation (0.34 ppm for 96 h and 0.51 ppm for 72 h) of young jack pine (Pinus banksiana Lamb.) seedlings produced needle tip burning and caused a reduction in their organic acid content. Quinic acid, the major component, declined under both concentrations, whereas shikimic acid, second to quinic acid in abundance, declined under 0.51 ppm exposure but remained unchanged at 0.34 ppm exposure. Syringic acid increased at both SO/sub 2/ concentration levels, but the increase was lower at 0.51 ppm than at 0.34 ppm. Malic acid dehydrogenase activity declined long before the development of visual symptoms of SO/sub 2/ toxicity. The enzyme activity was reduced to almost 50% of the control value after 92 h fumigation with 0.34 ppm SO/sub 2/, at which point the visual symptoms began to appear. The malate dehydrogenase assay appears to have potential as a tool to detect SO/sup 2/ injury to vegetation prior to visual symptom development.

Sarkar, S.K.; Malhotra, S.S.

1979-01-01

28

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

Science.gov (United States)

Chrysanthemyl diphosphate synthase (CDS) is the first pathway-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 (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 ?m. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ?100 ?m, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 ?g h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

2014-12-26

29

[Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians].  

Science.gov (United States)

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica. PMID:3962529

Vykhrestiuk, N P; Burenina, E A; Iarygina, G V

1986-01-01

30

Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitric oxide, the product of nitric oxide synthase in inflammatory cells, may have a role in tissue injury through its oxidative metabolism. Nitric oxide may have a role in the pathogenesis of duodenal ulcer and may be one of the mechanisms responsible for the association between gastric infection with Helicobacter pylori and peptic disease. In this study, calcium independent nitric oxide synthase activity was detected in human gastric mucosa suggesting expression of the inducible isoform. In...

Rachmilewitz, D.; Karmeli, F.; Eliakim, R.; Stalnikowicz, R.; Ackerman, Z.; Amir, G.; Stamler, J. S.

1994-01-01

31

Modulation of Hyaluronan Synthase Activity in Cellular Membrane Fractions*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in euk...

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; Luca, Giancarlo; Hascall, Vincent C.; Passi, Alberto

2009-01-01

32

Study of oxygen activation mechanism by nitric-oxide synthases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitric oxide is exclusively synthesized by NO-Synthases in mammals. The heart of the NO-synthase activity is oxygen activation, which corresponds to the activation of the FeIIO2 intermediate. This step depends on the heme electronic properties and on the electron and proton transfers. Oxygen activation has to be well mastered to control exactly the nature of the end-product. Understanding the oxygen activation step is necessary to better understand the biological/pathological role of the mamm...

Brunel, Albane

2012-01-01

33

Profile of Enzyme Activity And Growth of Wood Rotting Fungi In Metal Ion Containing Media  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Key enzyme of the glyoxylate cycle (isocilrate lyase and malate synthase), tricarboxylic acid cycle (isocitrate dehydrogenase), and GABA route (glutamate dehydrogenase) were measured in mycelia of Fomitopsis palustris grown on metal ion containing media. A higher isocitrate lyase activity was observed on the initial stage of cultivation at all linds of media tested. while malate synthase was constant throughout the incubation period. The activity of isocitrate dehydrogenase and glutamate dehy...

Munir, Erman; Hattori, Takefumi; Shimada, Mikio

2008-01-01

34

Structure of starch synthase I from barley: insight into regulatory mechanisms of starch synthase activity.  

Science.gov (United States)

Starch, a polymer of glucose, is the major source of calories in the human diet. It has numerous industrial uses, including as a raw material for the production of first-generation bioethanol. Several classes of enzymes take part in starch biosynthesis, of which starch synthases (SSs) carry out chain elongation of both amylose and amylopectin. Plants have five classes of SS, each with different roles. The products of the reaction of SS are well known, but details of the reaction mechanism remain obscure and even less is known of how different SSs select different substrates for elongation, how they compete with each other and how their activities are regulated. Here, the first crystal structure of a soluble starch synthase is presented: that of starch synthase I (SSI) from barley refined to 2.7 Å resolution. The structure captures an open conformation of the enzyme with a surface-bound maltooligosaccharide and a disulfide bridge that precludes formation of the active site. The maltooligosaccharide-binding site is involved in substrate recognition, while the disulfide bridge is reflective of redox regulation of SSI. Activity measurements on several SSI mutants supporting these roles are also presented. PMID:23695246

Cuesta-Seijo, Jose A; Nielsen, Morten M; Marri, Lucia; Tanaka, Hidenori; Beeren, Sophie R; Palcic, Monica M

2013-06-01

35

A second Escherichia coli protein with CL synthase activity.  

Science.gov (United States)

The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned. Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL. A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity. Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion. PMID:10634942

Guo, D; Tropp, B E

2000-01-17

36

Phytochelatin synthase activity as a marker of metal pollution  

International Nuclear Information System (INIS)

Highlights: ? New tool for determination of phytochelatin synthase activity. ? The optimization of experimental condition for determination of the enzyme activity. ? First evaluation of Km for the enzyme. ? The effects of cadmium (II) not only on the activity of the enzyme but also on Km. -- Abstract: The synthesis of phytochelatins is catalyzed by ?-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO3)2 for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 oC for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. Km for PCS was estimatKm for PCS was estimated as 2.3 mM.

37

Changes in Activities of Sucrose Synthase and Sucrose Phosphate Synthase and Sugar Content During Postharvest Senescence in Two Broccoli Cultivars  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Changes in sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities and sugar content during postharvest senescence in broccoli (Brassica oleracea L. cvs. `Hartland` and `Sairin`) were studied. The first change in color became apparent between 2 and 3 d but whole head yellowed after 5 d of storage. The SS activity increased gradually in all portions although with few fluctuations in branchlets of both cultivars at the end of the storage time. In case of SPS, there w...

Bimal Kumar Pramanik; Toshiyuki Matsui; Haruo Suzuki; Yusuke Kosugi

2004-01-01

38

Brain fatty acid synthase activates PPAR? to maintain energy homeostasis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Central nervous system control of energy balance affects susceptibility to obesity and diabetes, but how fatty acids, malonyl-CoA, and other metabolites act at this site to alter metabolism is poorly understood. Pharmacological inhibition of fatty acid synthase (FAS), rate limiting for de novo lipogenesis, decreases appetite independently of leptin but also promotes weight loss through activities unrelated to FAS inhibition. Here we report that the conditional genetic inactivation of FAS in p...

Chakravarthy, Manu V.; Zhu, Yimin; Lo?pez, Miguel; Yin, Li; Wozniak, David F.; Coleman, Trey; Hu, Zhiyuan; Wolfgang, Michael; Vidal-puig, Antonio; Lane, M. Daniel; Semenkovich, Clay F.

2007-01-01

39

Nitric oxide synthase expression and enzymatic activity in multiple sclerosis.  

DEFF Research Database (Denmark)

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 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 iNOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age 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

Broholm, H; Andersen, B

2004-01-01

40

Energetic aspects of malate and lactate fermentation by Acetobacterium malicum  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Acetobacterium malicum ferments L-malate and L-lactate to acetate (and CO.,) as sole fermentation product. Molar growth yieIds we, re 14.5 g and 6.8 g dry cell matter, respectively. With a different Acetobacterium sp. strain, similar results were obtained. No oxaloacetate-decarboxylating enzyme activity was detected in cell-free extracts. u-Malate was oxidized by an unusual NAD-dependent, oxygen-sensitive malic enzyme. No oxatoacetate decarboxylase or malate dehydrogenase activity was foun...

Strohha?cker, Joachim; Schink, Bernhard

1991-01-01

 
 
 
 
41

Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.  

Science.gov (United States)

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. PMID:24584238

Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

2014-05-01

42

Cilofungin (LY121019) inhibits Candida albicans (1-3)-beta-D-glucan synthase activity.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cilofungin (LY121019) inhibited Candida albicans growth and activity of (1-3)-beta-glucan synthase, for which it was a noncompetitive inhibitor with a Ki-app of 2.5 microM. Cilofungin had no effect on chitin synthase activity. Based on these and other data, it seems likely that cilofungin inhibits fungal growth by inhibiting (1-3)-beta-glucan synthase activity.

Taft, C. S.; Stark, T.; Selitrennikoff, C. P.

1988-01-01

43

Human blood platelets lack nitric oxide synthase activity.  

Science.gov (United States)

Abstract Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography-mass spectrometry (GC-MS) method to detect NOS activity in human platelets. The method measures simultaneously [(15)N]nitrite and [(15)N]nitrate formed from oxidized (15)N-labeled nitric oxide ((15)NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-(15)N2]-arginine. Using this GC-MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes. PMID:25360996

Böhmer, Anke; Gambaryan, Stepan; Tsikas, Dimitrios

2014-10-31

44

PHALLONOPTIC MALATE: A CRITICAL GAZE AT SPACE, VISUAL CULTURE AND PERFORMANCE IN THE STREETS OF OROSA AND BOCOBO, MALATE, MANILA  

Directory of Open Access Journals (Sweden)

Full Text Available Phallonopticism comes from the confluence of the term “phallus” and “panopticon.” I define Phallonotpicism as the configuration of gaze, power and desires specific to but not limited to male performances. By utilizing the term phallonopticism as a heuristic category for analysis, this paper attempts to analyze how Malate’s urban spaces of consumption are gendered. It attempts to map the topography of male homosocial intersubjectivities and the production of desire, power and gaze through male performances, visual culture and structures. Orosa Street is known as the GLTB consumption district of Malate while just a block away, is Bocobo Street, where a “straight” and “heterosexual” male population dwell. The dominance of male homosociality along the said streets has been established through research findings from 2002 - 2009. Through the confluence of political- economic discourse and psychoanalysis this paper will argue that Malate’s urban spaces inform the male subject construction and formation. Furthermore, male homosocialities in both streets activate the production and consumption of gendered spatial politics through complex forms of surveillance, discipline, resistance, and subversion from its stakeholders such as state institutions, the market and Malate’s public.

Clod Marlan Krister V. Yambao

2009-12-01

45

Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo  

DEFF Research Database (Denmark)

The aim of the present study was to investigate the effect of methylene blue, a guanylate cyclase inhibitor, on the hippocampal nitric oxide synthase activity in vivo. We used a microdialysis-based technique of measuring conversion of [3H]l-arginine to [3H]l-citrulline in freely moving rats. The administration of methylene blue (0.1 and 1 mM) via the microdialysis probe caused a dose-dependent decrease in [3H]l-citrulline efflux comparable with the effect of unselective NOS inhibitor NG-nitro-L-arginine (2 mM). We conclude that methylene blue inhibits brain NOS activity in vivo and thus interferes with NO-cGMP cascade in different levels.

Volke, V; Wegener, Gregers

1999-01-01

46

Inhibition of pulmonary thromboxane A2 synthase activity and airway responses by CGS 13080.  

Science.gov (United States)

The effects of CGS 13080, a thromboxane (TXA2) synthase inhibitor, on airway responses to arachidonic acid (AA) were investigated in the anesthetized cat. Feline and human lung microsomal fraction exhibited prostaglandin I2 (PGI2, prostacyclin), and TXA2 synthase activities, and human platelet microsomal fractions exhibited TXA2 synthase activity. Cat and human lung microsomal fractions, but not human platelets, exhibited the presence of GSH-dependent PGE2 isomerase activity. CGS 13080 inhibited TXA2 synthase activity in all three microsomal fractions in a concentration-dependent manner. The increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance in response to AA were decreased significantly by CGS 13080. These data suggest that the bronchoconstrictor actions of AA are mediated in large part by the formation of TXA2. The data further indicate that cyclooxygenase products other than TXA2 are involved in the bronchoconstrictor response to AA since meclofenamate had greater inhibitory activity than did CGS 13080. Moreover, the effects of CGS 13080 were due to inhibition of TXA2 synthase rather than an effect on TXA2 receptors, since airway responses to the TXA2 mimic, U46619, were not altered. The present data show that CGS 13080 inhibits TXA2 synthase activity without altering cyclooxygenase, PGI2 synthase, or GSH-dependent PGE2 isomerase activities. The data further indicate that in vivo administration of CGS 13080 may selectively increase PGI2 synthase activity. PMID:2725478

McNamara, D B; Harrington, J K; Bellan, J A; Graybar, G B; Underwood, D C; Kadowitz, P J

1989-01-23

47

CDP-diacylglycerol synthase activity in Clostridium perfingens  

Energy Technology Data Exchange (ETDEWEB)

CTP: phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimium between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition on the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C. (Refs. 24).

Carmen, G.M.; Zaniewski, R.L.; Cousminer, J.J.

1982-01-01

48

Malate-Induced Hysteresis of Phosphoenolpyruvate Carboxylase from Crassula argentea.  

Science.gov (United States)

The hysteretic behavior of phosphoenolpyruvate (PEP) carboxylase from Crassula argentea has been investigated. Incubation of the purified enzyme with the inhibitor malate prior to starting the reaction by the addition of PEP resulted in a kinetic lag of several minutes duration. The length of the lag was inversely proportional to the enzyme concentration, suggesting subunit association-dissociation as the hysteretic mechanism, rather than a mechanism based on a slow conformational change in the enzyme. Dynamic laser light scattering measurements also support this conclusion, showing that the diffusion coefficient of malate-incubated enzyme slowly decreased after the reaction was started by the addition of PEP. Lags were observed only at pH values of 7.5 or lower. Maximum lags were observed after 10 min of preincubation with malate. Fumarate and succinate, which like malate caused mixed inhibition, also caused lags. In contrast, no lag was induced by malate in the presence of PEP or by the competitive inhibitor phosphoglycolate. The activators glucose 6-phosphate and malonate decreased the malate-induced lag. PMID:16667161

Ngam-Ek, A; Seery, T A; Amis, E J; Grover, S D

1989-11-01

49

Nitrooleate mediates nitric oxide synthase activation in endothelial cells.  

Science.gov (United States)

Nitrated lipids such as nitrooleate (OLA-NO2) can act as endogenous peroxisome proliferator-activated receptor gamma (PPAR?) ligands to exert vascular protective effects. However, the molecular mechanisms regarding nitric oxide (NO) production and its regulation are not fully defined in the vasculature. Here, we show that OLA-NO2 increased endothelial NO release by modulating activation of endothelial nitric oxide synthase (eNOS) in endothelial cells. Treatment with OLA-NO2 (3 ?M) increased NO release in a time-dependent manner. OLA-NO2 decreased protein expression of eNOS and caveolin-1 (Cav-1) but increased heat shock protein 90 (Hsp90) expression. Immunoprecipitation analysis confirmed that OLA-NO2 replaced eNOS/Cav-1 with eNOS/Hsp90 interaction, resulting in increasing eNOS activity. OLA-NO2 also induced eNOS phosphorylation at Ser633 and Ser1177 and eNOS dephosphorylation at Ser113 and Thr495. In addition, OLA-NO2 induced phosphorylation of Akt and extracellular signal-regulated protein kinase (ERK1/2), which might contribute to eNOS activation. Collectively, these results substantiate a new functional role for nitrated fatty acid, demonstrating that OLA-NO2 exerts vascular protective effects by increasing NO bioavailability through eNOS phosphorylation/dephosphorylation and interaction with associated proteins such as Hsp90 and Cav-1. PMID:24664541

Shin, Eunju; Yeo, Eunju; Lim, Jihye; Chang, Yun Hee; Park, Haeryun; Shim, Eugene; Chung, Haeyon; Hwang, Hye Jin; Chun, Jiyeon; Hwang, Jinah

2014-05-01

50

Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.  

Science.gov (United States)

Central nervous system control of energy balance affects susceptibility to obesity and diabetes, but how fatty acids, malonyl-CoA, and other metabolites act at this site to alter metabolism is poorly understood. Pharmacological inhibition of fatty acid synthase (FAS), rate limiting for de novo lipogenesis, decreases appetite independently of leptin but also promotes weight loss through activities unrelated to FAS inhibition. Here we report that the conditional genetic inactivation of FAS in pancreatic beta cells and hypothalamus produced lean, hypophagic mice with increased physical activity and impaired hypothalamic PPARalpha signaling. Administration of a PPARalpha agonist into the hypothalamus increased PPARalpha target genes and normalized food intake. Inactivation of beta cell FAS enzyme activity had no effect on islet function in culture or in vivo. These results suggest a critical role for brain FAS in the regulation of not only feeding, but also physical activity, effects that appear to be mediated through the provision of ligands generated by FAS to PPARalpha. Thus, 2 diametrically opposed proteins, FAS (induced by feeding) and PPARalpha (induced by starvation), unexpectedly form an integrative sensory module in the central nervous system to orchestrate energy balance. PMID:17694178

Chakravarthy, Manu V; Zhu, Yimin; López, Miguel; Yin, Li; Wozniak, David F; Coleman, Trey; Hu, Zhiyuan; Wolfgang, Michael; Vidal-Puig, Antonio; Lane, M Daniel; Semenkovich, Clay F

2007-09-01

51

Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy  

DEFF Research Database (Denmark)

OBJECTIVE: Ceramides are precursors of complex sphingolipids (SLs), which are important for normal functioning of both the developing and mature brain. Altered SL levels have been associated with many neurodegenerative disorders, including epilepsy, although few direct links have been identified between genes involved in SL metabolism and epilepsy. METHODS: We used quantitative real-time PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed 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 composition was also reflected in a reduction in cholera toxin B immunofluorescence, indicating that membrane composition and function are altered. INTERPRETATION: We propose that reduced levels of CERS2, and consequently diminished levels of ceramides and SLs containing very long-chain fatty acids, lead to development of PME.

Mosbech, Mai-Britt; Olsen, Anne S B

2014-01-01

52

Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activit...

Rouzer, C. A.; Matsumoto, T.; Samuelsson, B.

1986-01-01

53

Enhancing human spermine synthase activity by engineered mutations.  

Science.gov (United States)

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine ? spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. PMID:23468611

Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

2013-01-01

54

L-malate production by metabolically engineered Escherichia coli.  

Science.gov (United States)

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts. PMID:21097588

Zhang, X; Wang, X; Shanmugam, K T; Ingram, L O

2011-01-01

55

Changes in Activities of Sucrose Synthase and Sucrose Phosphate Synthase and Sugar Content During Postharvest Senescence in Two Broccoli Cultivars  

Directory of Open Access Journals (Sweden)

Full Text Available Changes in sucrose synthase (SS and sucrose phosphate synthase (SPS activities and sugar content during postharvest senescence in broccoli (Brassica oleracea L. cvs. `Hartland` and `Sairin` were studied. The first change in color became apparent between 2 and 3 d but whole head yellowed after 5 d of storage. The SS activity increased gradually in all portions although with few fluctuations in branchlets of both cultivars at the end of the storage time. In case of SPS, there was no inclining or declining pattern of the activity in any portion of the two cultivars at the end of the storage period. In both cultivars, branchlets showed higher SS and SPS activity than florets. Sucrose content gradually decreased in both portions of the two cultivars with time. Fructose content was higher than glucose and sucrose in the florets as well as branchlets of both cultivars. There was a highly significant negative correlation observed between the SS activity and sucrose content in the florets and branchlets of both cultivars. There was no significant correlation between sugar contents and SPS activity in any portions of both cultivars.

Bimal Kumar Pramanik

2004-01-01

56

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, ca...

Maniam, S.; Coutts, As; Stratford, MR; Mcgouran, J.; Kessler, B.; La Thangue, Nb

2014-01-01

57

Microbodies in germinating fern spores: evidence for glyoxysomal activity.  

Science.gov (United States)

Enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, are active during the germination of spores of the fern Dryopteris filix-mas. Increases in activity of both enzymes are correlated with the breakdown of lipid reserves. The occurrence of these enzymes suggests that the microbodies previously described in these spores are glyoxysomes. PMID:17759428

Demaggio, A E; Greene, C; Unal, S; Stetler, D A

1979-11-01

58

Malate Synthesis and Secretion Mediated by a Manganese-Enhanced Malate Dehydrogenase Confers Superior Manganese Tolerance in Stylosanthes guianensis.  

Science.gov (United States)

Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in Stylosanthes spp. has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in Stylosanthes guianensis was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two S. guianensis genotypes, which revealed that the Fine-stem genotype had higher Mn tolerance than the TPRC2001-1 genotype, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn-stimulated increases in malate concentrations and exudation rates were observed only in the Fine-stem genotype. Proteomic analysis of Fine-stem roots revealed that S. guianensis Malate Dehydrogenase1 (SgMDH1) accumulated in response to Mn toxicity. Western-blot and quantitative PCR analyses showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1-mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, as well as in vivo increased malate concentrations in yeast (Saccharomyces cerevisiae), soybean (Glycine max) hairy roots, and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of S. guianensis is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels. PMID:25378694

Chen, Zhijian; Sun, Lili; Liu, Pandao; Liu, Guodao; Tian, Jiang; Liao, Hong

2015-01-01

59

Spermine synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Spermine is present in many organisms including animals, plants, some fungi, some archaea, and some bacteria. It is synthesized by spermine synthase, a highly specific aminopropyltransferase. This review describes spermine synthase structure, genetics, and function. Structural and biochemical studies reveal that human spermine synthase is an obligate dimer. Each monomer contains a C-terminal domain where the active site is located, a central linking domain that also forms the lid of the catal...

Pegg, Anthony E.; Michael, Anthony J.

2009-01-01

60

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli  

Directory of Open Access Journals (Sweden)

Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

Bechard Matthew E.

2003-01-01

 
 
 
 
61

Changes in Carbohydrate Content and Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Broccoli During Short Term Storage at Low Temperature  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This study investigated the changes in carbohydrate content and activities of invertase, sucrose synthase and sucrose phosphate synthase in two broccoli cultivar (Brassica oleracea L. Cvs. `Hartland` and `Sairin`) stored at 5?C. Respiration rate rapidly declined after 1 day of storage and gradually decreased at the end of the storage period. Color did not change significantly at the end of the storage period. In both cultivars and portions, the invertase activity increased gradually ...

Bimal Kumar Pramanik; Toshiyuki Matsui; Haruo Suzuki; Yusuke Kosugi

2004-01-01

62

A High-Throughput Colorimetric Screening Assay for Terpene Synthase Activity Based on Substrate Consumption  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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. W...

Furubayashi, Maiko; Ikezumi, Mayu; Kajiwara, Jun; Iwasaki, Miki; Fujii, Akira; Li, Ling; Saito, Kyoichi; Umeno, Daisuke

2014-01-01

63

Modification of a thiol at the active site of the Ascaris suum NAD-malic enzyme results in changes in the rate-determining steps for oxidative decarboxylation of L-malate  

International Nuclear Information System (INIS)

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in KNAD and Ki metal and decreases in Vmax as the metal size increases from Mg2+ to Mn2+ to Cd2+, indicative of crowding in the site. The Kmalate value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/Kmalate increase with all three metal ions compared to the native enzyme concomitant with a decrease in the 13C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The 13C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary 13C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd2+. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd2+ as the divalent metal activator. Upon inactivation, only Cd2+ ions are bound at high stoichiometry to the enzyme,bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd2+

64

Malate metabolism in Hoya carnosa mitochondria and its role in photosynthesis during CAM phase III.  

Science.gov (United States)

This study investigated the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant, Hoya carnosa, in malate metabolism during CAM phase III. The mitochondria showed high malate dehydrogenase (mMDH) and aspartate amino transferase (mAST), and a significant amount of malic enzyme (mME) activities. H. carnosa readily oxidized malate via mME and mMDH in the presence of some cofactors such as thiamine pyrophosphate (TPP), coenzyme A (CoA) or NAD(+). A high respiration rate of malate oxidation was observed at pH 7.2 with NAD(+) and glutamate (Glu). Providing AST and Glu simultaneously into the respiratory medium strongly increased the rates of malate oxidation, and this oxidation was gradually inhibited by an inhibitor of alpha-ketoglutarate (alpha-KG) carrier, pyridoxal-5'-phosphate (PLP). The mitochondria readily oxidized aspartate (Asp) or alpha-KG individually with low rates, while they oxidized Asp and alpha-KG simultaneously with high rates, and this simultaneous oxidation was also inhibited by PLP. By measuring the capacity of the mitochondrial shuttle, it was found that the OAA produced via mMDH seemed not to be transported outside the mitochondria, but mAST interconverted OAA and Glu to Asp and alpha-KG, respectively, and exported them out via a malate-aspartate (malate-Asp) shuttle. The data in this research suggest that during phase III of PCK-CAM, H. carnosa mitochondria oxidized malate via both mME and the mMDH systems depending on metabolic requirements. However, malate metabolism by the mMDH system did not operate via a malate-OAA shuttle similarly to Ananas comosus mitochondria, but it operated via a malate-Asp shuttle similarly to Kalanchoë daigremontiana mitochondria. PMID:18403382

Hong, Hoang Thi Kim; Nose, Akihiro; Agarie, Sakae; Yoshida, Takayuki

2008-01-01

65

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with ?-Opioid Agonist Activity.  

Science.gov (United States)

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the ?-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the ?-opioid GPCR was predicated on the modulatory role of nitric oxide on ?-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 ?M), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent ?-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 ?M). This work represents a novel approach in the development of new analgesics for the treatment of pain. PMID:24900459

Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

2012-03-01

66

MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9.  

Science.gov (United States)

Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer (CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression of MALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and protein levels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9. PMID:25446987

Yang, Min-Hui; Hu, Zhi-Yan; Xu, Chuan; Xie, Lin-Ying; Wang, Xiao-Yan; Chen, Shi-You; Li, Zu-Guo

2015-01-01

67

Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3??  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3?) protected cells from ET-induced changes in the cell cycle. GSK-3? inhibi...

Larabee, Jason L.; Degiusti, Kevin; Regens, James L.; Ballard, Jimmy D.

2008-01-01

68

Studies on the active site of deacetoxycephalosporin C synthase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS wa...

Lloyd, Md; Lee, Hj; Harlos, K.; Zhang, Zh; Baldwin, Je; Schofield, Cj; Charnock, Jm; Garner, Cd; Hara, T.; Terwisscha Scheltinga, Ac; Valega?rd, K.; Viklund, Ja; Hajdu, J.; Andersson, I.; Danielsson, A.

1999-01-01

69

Enhancing Human Spermine Synthase Activity by Engineered Mutations  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by e...

Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

2013-01-01

70

Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis.  

Science.gov (United States)

The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results. PMID:1706759

Goosen, N K; Broers, C A; Hombergen, E J; Stumm, C K; Vogels, G D

1990-11-01

71

Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation  

Science.gov (United States)

Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

Paliyath, G.; Poovaiah, B. W.

1988-01-01

72

Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0, and tobacco (BY-2, were permeabilized with the channel-forming peptide alamethicin. Results Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. Conclusion Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed, during which plasmodesmata are transiently left open.

Rasmusson Allan G

2009-03-01

73

Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds.  

Science.gov (United States)

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III. PMID:11509967

Hwang, E I; Ahn, B T; Lee, H B; Kim, Y K; Lee, K S; Bok, S H; Kim, Y T; Kim, S U

2001-08-01

74

Studies of the mechanism by which hepatic citrate synthase activity increases in vitamin B12 deprivation.  

Science.gov (United States)

Hepatic citrate synthase activity has been shown to be increased 2- to 3-fold in vitamin B12 deficiency. Immunochemical titrations of the affinity chromatography-purified enzyme obtained from liver of animals with B12 deprivation demonstrated that this increase in activity was the result of a true increase in enzyme protein content. When fixed ratios of aliquots of normal and B12-deprived rat liver homogenates were mixed, the activity measured showed no change from the expected total citrate synthase activity based on the admixture ratios. Partial purification of the enzyme resulted in the expected recovery of the enzyme at each of the purification steps. Thus, it is unlikely that the change in enzyme activity in B12 deprivation was due to the presence of a soluble or easily dissociable normally occurring activator or inhibitor. Ouchterlony double diffusion studies, immunochemical titration, and determination of Km vlaues for exalacetate and acetyl-CoA (substrates for citrate synthase) and Ki values for ATP (inhibitor of citrate synthase) all indicated that the enzyme from the B12-deprived livers was structurally the same as that from normal liver. Hepatic citrate synthase degradation rate constants were shown to be essentially unchanged in B12deficiency. The rate of hepatic citrate synthase synthesis, under steady state conditions, was shown to be 2.8-fold greater in the B12-deficient animal than in the normal animal. The increased rate of synthesis appeared to explian the increased enzyme content. Finally, no change in specific activity of the enzyme was seen in brain, heart, or kidney in the B12-deprived animal. PMID:818082

Mukherjee, A; Srere, P A; Frenkel, E P

1976-04-10

75

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active  

Energy Technology Data Exchange (ETDEWEB)

Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

Schoenitzer, Veronika [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Eichner, Norbert [Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Clausen-Schaumann, Hauke [Munich University of Applied Sciences, Lothstrasse 34, D-80335 Muenchen, Germany, and Center for NanoScience (CeNS), Geschwister-Scholl-Platz 1, D-80539 Muenchen (Germany); Weiss, Ingrid M., E-mail: ingrid.weiss@inm-gmbh.de [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany)

2011-12-02

76

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active  

International Nuclear Information System (INIS)

Highlights: ? Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. ? Chitin synthase activity releases chitin, partly associated with the cell surface. ? Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. ? Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. ? This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA? cell lines are shown.

77

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

78

Calcium-Dependent Nitric Oxide Synthase Activity in Rat Thymocytes  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We examined the conversion of L-[3H]arginine to L-[3H]citrulline in lysate from rat thymocytes, which was dependent on Ca2+and cofactors (FAD, BH4, NADPH). Removal of Ca2+of the medium, reduced the total L-[3H]citrulline formation by about 97%. The L-[3H]citrulline formation was completely inhibited by the NO synthase inhibitors, NG-nitro-L-arginine and NG-monomethyl-L-arginine, with values for IC50of 1.2 [mu]M and 19.4 [mu]M, respectively. In intact thymocytes, the L-[3H]citrulline formation...

Cruz, M. T.; Carmo, A.; Carvalho, A. P.; Lopes, M. C.

1998-01-01

79

Changes in Carbohydrate Content and Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Broccoli During Short Term Storage at Low Temperature  

Directory of Open Access Journals (Sweden)

Full Text Available This study investigated the changes in carbohydrate content and activities of invertase, sucrose synthase and sucrose phosphate synthase in two broccoli cultivar (Brassica oleracea L. Cvs. `Hartland` and `Sairin` stored at 5?C. Respiration rate rapidly declined after 1 day of storage and gradually decreased at the end of the storage period. Color did not change significantly at the end of the storage period. In both cultivars and portions, the invertase activity increased gradually at the end of the storage time. The acid invertase in cell wall-bound fraction (CWBF showed a higher activity than that in soluble fraction (SF. Again, the branchlets showed a significantly higher invertase activity than that of florets. In both cultivars, sucrose synthase (SS activity gradually increased in the florets with some fluctuations in the branchlets at the end of the storage time. Sucrose phosphate synthase activity (SPS activity did not change markedly in any portion of the two cultivars at the end of the storage period. Sucrose content gradually decreased in both portions of the two cultivars with time. It was negatively correlated with the acid invertase and SS activities in both portions accounting well for the relation between the substrate and the activity. Fructose and glucose content were higher than sucrose in the florets as well as branchlets in both cultivars.

Bimal Kumar Pramanik

2004-01-01

80

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

2011-08-17

 
 
 
 
81

Synthesis of digalactosyl diacylglycerols and their structure-inhibitory activity on human lanosterol synthase.  

Science.gov (United States)

Digalactosyl and monogalactocyl diacylglycerols (DGDG and MGDG), which were identified as anti-hyperlipemia active components in Colocasia esculenta (Taro), were synthesized. The inhibitory activity of DGDG, MGDG and related compounds on human lanosterol synthase was evaluated as anti-hyperlipemic activity. DGDG with two myristoyl groups at both sn-1 and sn-2 positions and with an oleoyl group at the sn-1 position showed the most potent activity. PMID:15582431

Tanaka, Rie; Sakano, Yuichi; Nagatsu, Akito; Shibuya, Masaaki; Ebizuka, Yutaka; Goda, Yukihiro

2005-01-01

82

Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Asparagus Spears During Storage  

Directory of Open Access Journals (Sweden)

Full Text Available We held asparagus (Asparagus officinalis L. spears at 25?C for up to 5 days after harvest and examined changes in soluble carbohydrates and the activities of enzymes concerned with carbohydrate breakdown in both top and bottom portions of the spears. The acid invertase in soluble fraction showed a higher activity than that in cell wall bound fraction and the top portion of the spear showed a significantly higher soluble acid invertase activity than the bottom portion. But the activity of cell wall bound acid invetase was higher in bottom portion. In both the top and bottom portions soluble acid invertase activity increased during first day of storage and after that it started to decrease gradually up to five days. In case of cell wall bound acid invertase the activity increased during first two days and after that it also started to decrease. Sucrose synthase activity, found higher in bottom portion than top, started to decline from the first day of storage and continued up to five days. But the activity declined rapidly on third day. In case of sucrose phosphate synthase there was no specific inclining or declining pattern of activity in any portion of the spear. Among the soluble sugars fructose and glucose were predomonant and fructose content was significantly higher than the glucose and sucrose. All the three sugars started to decrease from the first day and continued up to five days. Sucrose content was negatively correlated with invertase and sucrose synthase in both portions and accounted well for the relation between the substrate and enzyme activity. But sucrose phohphate synthase activity remained almost constant during the storage period and there was no significant correlation between sugar content and sucrose phosphate synthase activity.

Pankaj Kumar Bhowmik

2001-01-01

83

Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase.  

Science.gov (United States)

When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.Between 60 and 90 minutes after increasing the medium osmolarity the isofloridoside pool in cells approached a high steady-state level. About this time it was observed that isofloridoside-phosphate synthase activity passed transiently through a much higher level than before, and a higher molecular weight form of the active enzyme could be observed on gel filtration chromatography. PMID:16660747

Kauss, H; Thomson, K S; Thomson, M; Jeblick, W

1979-03-01

84

Ethylmalonic acid impairs brain mitochondrial succinate and malate transport.  

Science.gov (United States)

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) occur in ethylmalonic encephalopathy (EE) and short chain acyl-CoA dehydrogenase deficiency (SCADD). Although these autosomal recessive disorders are clinically characterized by neurological abnormalities, the mechanisms underlying the brain damage are poorly known. Considering that little is known about the neurotoxicity of EMA and that hyperlacticacidemia occurs in EE and SCADD, we evaluated the effects of this metabolite on important parameters of oxidative metabolism in isolated rat brain mitochondria. EMA inhibited either ADP-stimulated or uncoupled mitochondrial respiration supported by succinate and malate, but not by glutamate plus malate. In addition, EMA mildly stimulated oxygen consumption by succinate-respiring mitochondria in resting state. Methylmalonic acid (MMA), malonic acid (MA) and butylmalonic acid (BtMA) had a similar effect on ADP-stimulated or uncoupled respiration. Furthermore, EMA-, MMA- and BtMA-induced inhibitory effects on succinate oxidation were significantly minimized by nonselective permeabilization of the mitochondrial membranes by alamethicin, whereas MA inhibitory effect was not altered. In addition, MA was the only tested compound that reduced succinate dehydrogenase activity. We also observed that EMA markedly inhibited succinate and malate transport through the mitochondrial dicarboxylate carrier. Mitochondrial membrane potential was also reduced by EMA and MA, but not by MMA, using succinate as electron donor, whereas none of these compounds was able to alter the membrane potential using glutamate plus malate as electron donors. Taken together, our results strongly indicate that EMA impairs succinate and malate uptake through the mitochondrial dicarboxylate carrier. PMID:22133302

Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Busanello, Estela Natasha Brandt; Ribeiro, César Augusto João; Melo, Daniela Rodrigues; Leipnitz, Guilhian; Castilho, Roger Frigério; Wajner, Moacir

2012-01-01

85

Lubeluzole, a novel long-term neuroprotectant, inhibits the glutamate-activated nitric oxide synthase pathway.  

Science.gov (United States)

The novel drug lubeluzole, but not its (-)-R-isomer, protects against sensorimotor deficits provoked by photochemical stroke in rats. We studied the mechanism of protection of lubeluzole against glutamate toxicity in primary hippocampal cell cultures. In a model for glutamate antagonism, i.e., treatment of the cultures with compound during the glutamate trigger, lubeluzole was not protective. In contrast, after prolonged pretreatment, i.e., administration of compound to the culture for 7 days before glutamate, lubeluzole was neuroprotective. It had an IC50 of 48 nM and its R-isomer was nine times less active. Under these conditions, lubeluzole inhibited glutamate-stimulated guanosine 3',5'-cyclic monophosphate production (IC50 37 nM). Again the R-isomer was seven times less active. The compounds did not affect nitric oxide synthase activity, guanylate cyclase activity or arginine uptake. After prolonged pretreatment, lubeluzole attenuated citrulline production in the culture, which could not be compensated for by excess arginine. Because prolonged lubeluzole treatment does not inhibit glutamate-activated [Ca+2]i rise in these cultures, the findings may indicate that expression of nitric oxide synthase or levels of its cofactors were reduced. Treatment of C6 glioma cells with lubeluzole did not affect lipopolysaccharide/gamma interferon induced guanosine 3',5'-cyclic monophosphate levels, suggesting that lubeluzole does not inhibit the glial nitric oxide synthase pathway. In conclusion, the long-term neuroprotective property of lubeluzole against glutamate toxicity in hippocampal cultures is reflected by the fact of interference with the glutamateactivated nitric oxide synthase pathway. Prolonged treatment may reduce expression of nitric oxide synthase or levels of its cofactors. PMID:8930181

Lesage, A S; Peeters, L; Leysen, J E

1996-11-01

86

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)

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. PMID:23243312

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

87

Ethylene negatively regulates aluminium-induced malate efflux from wheat roots and tobacco cells transformed with TaALMT1.  

Science.gov (United States)

An important mechanism for Al(3+) tolerance in wheat is exudation of malate anions from the root apex through activation of malate-permeable TaALMT1 channels. Here, the effect of ethylene on Al(3+)-activated efflux of malate was investigated using Al(3+)-tolerant wheat genotype ET8, which has high expression of TaALMT1. Exposure of ET8 plants to Al(3+) enhanced ethylene evolution in root apices. Treatment with the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene gas suppressed Al(3+)-induced malate efflux from root apices, whereas the intracellular malate concentrations in roots were not affected. Malate efflux from root apices was enhanced in the presence of Al(3+) by two antagonists of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and 2-aminoisobutyric acid (AIB). An increase in Al accumulation in root apices was observed when treated with ACC, whereas AVG and AIB suppressed Al accumulation in root apices. Al(3+)-induced inhibition of root elongation was ameliorated by pretreatment with AIB. In addition, ethylene donor (Ethrel) also inhibited Al(3+)-induced malate efflux from tobacco cells transformed with TaALMT1. ACC and the anion-channel blocker niflumate had a similar and non-additive effect on Al-induced malate efflux from root apices. Treatment of ET8 plants with ACC enhanced expression of TaALMT1, suggesting that the inhibitory effect of ethylene on Al-induced malate efflux is unlikely to occur at the transcriptional level. These findings indicate that ethylene may behave as a negative regulator of Al(3+)-induced malate efflux by targeting TaALMT1-mediated malate efflux by an unknown mechanism. PMID:24668874

Tian, Qiuying; Zhang, Xinxin; Ramesh, Sunita; Gilliham, Matthew; Tyerman, Stephen D; Zhang, Wen-Hao

2014-06-01

88

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of no...

GONNET SUSANA; DÍAZ PEDRO

2000-01-01

89

A Noncatalytic Domain of Glycogen Synthase Kinase-3 (GSK-3) Is Essential for Activity*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3? and GSK-3?, are serine/threonine kinases involved in numerous cellular processes and diverse diseases, including Alzheimer disease, cancer, and diabetes. GSK-3 isoforms function redundantly in some settings, while, in others, they exhibit distinct activities. Despite intensive investigation into the physiological roles of GSK-3 isoforms, the basis for their differential activities remains unresolved. A more comprehensive understanding of t...

Buescher, Jessica L.; Phiel, Christopher J.

2010-01-01

90

Anaerobic Toluene Activation by Benzylsuccinate Synthase in a Highly Enriched Methanogenic Culture  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic photot...

Beller, Harry R.; Edwards, Elizabeth A.

2000-01-01

91

Constitutive nitric oxide synthase (cNOS) activity in Langerhans islets from streptozotocin diabetic rats  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the p [...] resence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

T.M., Fonovich de Schroeder; M.D., Carattino; M., Frontera; O.L., Catanzaro.

1998-05-01

92

Constitutive nitric oxide synthase (cNOS activity in Langerhans islets from streptozotocin diabetic rats  

Directory of Open Access Journals (Sweden)

Full Text Available Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

Fonovich de Schroeder T.M.

1998-01-01

93

Malate dehydrogenase in plants: Its genetics, structure, localization and use as a marker  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This article is an overview of literature data on the structure, properties, functions and genetic control of the enzyme malate dehydrogenase (MDH) in plants. In most of the plant entities studied, this enzyme is highly polymorphic, which means that malate dehydrogenase has multiple molecular forms. It has been found that MDH polymorphism in each species is genetically determined by several loci with multiple alleles. A readily identifiable phenotypic manifestation and a high activity of mala...

Rimma Sergeevna Yudina

2012-01-01

94

Identification of a class of sulfonamides highly active against dihydropteroate synthase form Toxoplasma gondii, Pneumocystis carinii, and Mycobacterium avium.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Sulfanilanilides with 3',5'-halogen substitutions had Ki values 6- to 57-fold lower than the Ki of sulfamethoxazole when tested against dihydropteroate synthase from Toxoplasma gondii. The compounds acted as competitive inhibitors. These compounds were also active against dihydropteroate synthase from Pneumocystis carinii, Mycobacterium avium, and Escherichia coli but were not significantly more active than sulfamethoxazole. The compounds were significantly more active in culture than were st...

Chio, L. C.; Bolyard, L. A.; Nasr, M.; Queener, S. F.

1996-01-01

95

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase.  

Science.gov (United States)

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration. PMID:25168243

Maniam, S; Coutts, A S; Stratford, M R; McGouran, J; Kessler, B; La Thangue, N B

2015-01-01

96

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3), a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and re...

Price Brendan D; Turenne Gaetan A

2001-01-01

97

Constitutive nitric oxide synthase (cNOS) activity in Langerhans islets from streptozotocin diabetic rats  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pan...

Fonovich de Schroeder T.M.; Carattino M.D.; Frontera M.; Catanzaro O.L.

1998-01-01

98

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells1[S  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increas...

Martin, Maria Laura; Liebisch, Gerhard; Lehneis, Stefan; Schmitz, Gerd; Alonso-sande, Mari?a; Bestard-escalas, Joan; Lopez, Daniel H.; Garci?a-verdugo, Jose? Manuel; Soriano-navarro, Mario; Busquets, Xavier; Escriba?, Pablo V.; Barcelo?-coblijn, Gwendolyn

2013-01-01

99

Growth and sucrose synthase activity of developing chickpea (Cicer arietinum L.) seeds under field conditions  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Seed growth characteristics and sucrose synthase activity in chickpea (Cicer arietinum L.) were examined in a field experiment at Merredin, Western Australia. ‘Sona’, a small-seeded desi cultivar, and ‘Kaniva’, a large-seeded kabuli cultivar, were grown after flowering with irrigation and under a rainout shelter, used to induce terminal drought. Seed and pod wall dry weight followed a similar pattern in the two cultivars with terminal drought significantly reducing the dry weight of t...

Ashok Kumar(University of Delhi, Delhi, India); Turner, Neil C.

2009-01-01

100

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

1995-01-01

 
 
 
 
101

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarea...

Laurini Ricardo; Andolf Ellika; Al-Hijji J; Batra Satish

2003-01-01

102

Glycogen synthase 2 is a novel target gene of peroxisome proliferator-activated receptors.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Glycogen synthase 2 (Gys-2) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue, yet little is known about regulation of Gys-2 transcription. The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well. Here, we show that Gys-2 is a direct target gene of PPARalpha, PPARbeta/delta and PPARgamma. Expression of Gys-2 is sign...

Mandard, Ste?phane; Stienstra, Rinke; Escher, Pascal; Tan, Nguan Soon; Kim, Insook; Gonzalez, Frank; Wahli, Walter; Desvergne, Be?atrice; Mu?ller, Michael; Kersten, Sander

2007-01-01

103

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

International Nuclear Information System (INIS)

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 [14C]-L-arginine to [14C]-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 [14C]-L-arginine to [14C]-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.

104

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

105

Malate-Induced Hysteresis of Phosphoenolpyruvate Carboxylase from Crassula argentea1  

Science.gov (United States)

The hysteretic behavior of phosphoenolpyruvate (PEP) carboxylase from Crassula argentea has been investigated. Incubation of the purified enzyme with the inhibitor malate prior to starting the reaction by the addition of PEP resulted in a kinetic lag of several minutes duration. The length of the lag was inversely proportional to the enzyme concentration, suggesting subunit association-dissociation as the hysteretic mechanism, rather than a mechanism based on a slow conformational change in the enzyme. Dynamic laser light scattering measurements also support this conclusion, showing that the diffusion coefficient of malate-incubated enzyme slowly decreased after the reaction was started by the addition of PEP. Lags were observed only at pH values of 7.5 or lower. Maximum lags were observed after 10 min of preincubation with malate. Fumarate and succinate, which like malate caused mixed inhibition, also caused lags. In contrast, no lag was induced by malate in the presence of PEP or by the competitive inhibitor phosphoglycolate. The activators glucose 6-phosphate and malonate decreased the malate-induced lag. PMID:16667161

Ngam-ek, Apinya; Seery, Thomas A.P.; Amis, Eric J.; Grover, Scott D.

1989-01-01

106

Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes  

International Nuclear Information System (INIS)

Here we demonstrate that human keratinocytes possess a Ca2+/ calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[3H]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent witnduced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation. (Author)

107

Active intermediates of polyhydroxyalkanoate synthase from Aeromonas caviae in polymerization reaction.  

Science.gov (United States)

Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae FA440 (PhaC(Ac), BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a practical bioplastic, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaC(Ac) have not been clarified until now. In the present study, a gene of PhaC(Ac) was cloned and overexpressed by a cell-free protein expression system. Both the polymerization activity and oligomerization behavior of the purified PhaC(Ac) were characterized in order to clarify the active intermediates of PhaC(Ac) based on the hydrodynamic diameters and specific activities of PhaC(Ac). The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaC(Ac) (7.5 ?M) were also investigated, and then the Hill coefficient (n = 2.6 ± 0.4) and the microscopic dissociation constant (K(m) = 77 ± 5 ?M) were determined. Based on the results, the active intermediate of PhaC(Ac) was concluded to be the dimeric PhaC(Ac) containing 3HB-CoA as an activator for its dimerization. This information is critical for revealing the relationships between its dimerization and function in PHA synthesis. PMID:23043466

Numata, Keiji; Motoda, Yoko; Watanabe, Satoru; Tochio, Naoya; Kigawa, Takanori; Doi, Yoshiharu

2012-11-12

108

Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca(2+)-independent conversion of L-arginine to L-citrulline that can be c...

Galea, E.; Feinstein, D. L.; Reis, D. J.

1992-01-01

109

A novel aphrodisiac compound from an orchid that activates nitric oxide synthases.  

Science.gov (United States)

Nitric oxide (NO) is known to have roles in several crucial biological functions including vasodilation and penile erection. There are neuronal, endothelial and inducible NO synthases that influence the levels of NO in tissues and blood. NO activates guanylate cyclase and thereby increases the levels of cyclic GMP (cGMP). Viagra (sildenafil), a top selling drug in the world for erectile dysfunction, inhibits phosphodiesterase-5, which hydrolyses cGMP to GMP. Thus, it fosters an NO-mediated increase in the levels of cGMP, which mediates erectile function. Here, we show the aphrodisiac activity of a novel chemical isolate from the flowers of an epiphytic orchid, Vanda tessellata (Roxb.) ex Don, which activates neuronal and endothelial, but not inducible, NO synthases. The aphrodisiac activity is caused by an increase in the level of NO in corpus cavernosum. The drug increases blood levels of NO as early as 30 min after oral administration. The active compound was isolated by column chromatography. Based on the spectral data, the active compound is found to be a new compound, 2,7,7-tri methyl bicyclo [2.2.1] heptane. We anticipate that our findings could lead to the development of a commercially viable and valuable drug for erectile dysfunction. PMID:23595103

Subramoniam, A; Gangaprasad, A; Sureshkumar, P K; Radhika, J; Arun, K B; Arun, B K

2013-01-01

110

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after...

Rest, Michel E.; Frank, Christian; Molenaar, Douwe

2000-01-01

111

An application of RP-HPLC for determination of the activity of cystathionine ?-synthase and ?-cystathionase in tissue homogenates.  

Science.gov (United States)

The RP-HPLC-based method of determination of the activity of cystathionine ?-synthase and ?-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, ?-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine ?-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure ?-ketobutyrate. The results confirmed high cystathionine ?-synthase activity and no ?-cystathionase activity in brain, and high ?-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue. PMID:25307719

Bronowicka-Adamska, Patrycja; Zagajewski, Jacek; Wróbel, Maria

2014-10-13

112

Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase.  

Science.gov (United States)

Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS. PMID:16367761

Abe, Ikuro; Watanabe, Tatsuya; Lou, Weiwei; Noguchi, Hiroshi

2006-01-01

113

A soybean plastid-targeted NADH-malate dehydrogenase: cloning and expression analyses.  

Science.gov (United States)

A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ?34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene. PMID:21669645

Imsande, J; Berkemeyer, M; Scheibe, R; Schumann, U; Gietl, C; Palmer, R G

2001-12-01

114

The metabolism of malate by cultured rat brain astrocytes  

International Nuclear Information System (INIS)

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-[U-14C]malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucospartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain

115

The metabolism of malate by cultured rat brain astrocytes  

Energy Technology Data Exchange (ETDEWEB)

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

1990-12-01

116

Naphthoquinones and Bioactive Compounds from Tobacco as Modulators of Neuronal Nitric Oxide Synthase Activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone c...

Venkatakrishnan, Priya; Gairola, C. Gary; Castagnoli, Neal; Miller, R. Timothy

2009-01-01

117

Protein kinase C? regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In this study, we explore the roles of the delta isoform of PKC (PKC?) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKC? with either rottlerin or with the peptide, ?V1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKC? inhibition using either rottlerin or the ...

Sud, Neetu; Wedgwood, Stephen; Black, Stephen M.

2008-01-01

118

Higher Activity of the Inducible Nitric Oxide Synthase Contributes to Very Early Onset Inflammatory Bowel Disease  

Digital Repository Infrastructure Vision for European Research (DRIVER)

OBJECTIVES: The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early ...

Dhillon, Sandeep S.; Mastropaolo, Lucas A.; Murchie, Ryan; Griffiths, Christopher; Tho?ni, Cornelia; Elkadri, Abdul; Xu, Wei; Mack, Amanda; Walters, Thomas; Guo, Conghui; Mack, David; Huynh, Hien; Baksh, Shairaz; Silverberg, Mark S.; Brumell, John H.

2014-01-01

119

Effect of vitamin B12-deficiency on the activity of hepatic cystathionine beta-synthase in rats.  

Science.gov (United States)

The effect of vitamin B12(B12)-deficiency on the activities of hepatic methionine synthase, homocysteine methyltransferase, and cystathionine beta-synthase was investigated in rats. The rats bred from B12-deficient dams were fed the B12-deficient diets for 150 days after weaning. Growth retardation of the B12-deficient rats was already observed on day 30 and continued through 150 days. But dietary supplementation of 0.5% DL-methionine slightly improved the growth retardation. Urinary excretion of methylmalonic acid increased to about 15 mg/mg creatinine and hepatic B12 concentration declined to about 2 ng/g liver after a 150-day feeding of the B12-deficient diets. Hepatic methionine synthase activity in rats fed the B12-deficient diets supplemented with or without methionine decreased to about 5% of B12-supplemented controls. Hepatic betaine-homocysteine methyltransferase activity showed no significant change caused by B12-deficiency. Hepatic cystathionine beta-synthase activity in rats fed the B12-deficient diets supplemented with or without methionine decreased to about 61% and 27% of their B12-supplemented controls, respectively, but the decrease was partially improved by methionine supplementation. In conclusion, the rats bred from B12-deficient dams showed a severe B12-deficiency after a 150-day feeding of the B12-deficient diets. The decrease of hepatic cystathionine beta-synthase activity was supposed to be due to the adaptation by the defect of methionine resynthesis. PMID:2732804

Doi, T; Kawata, T; Tadano, N; Iijima, T; Maekawa, A

1989-04-01

120

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

Directory of Open Access Journals (Sweden)

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%.

Silva José Carlos da

2003-01-01

 
 
 
 
121

Yeast chitin synthase 2 activity is modulated by proteolysis and phosphorylation.  

Science.gov (United States)

Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division. PMID:18823281

Martínez-Rucobo, Fuensanta W; Eckhardt-Strelau, Luise; Terwisscha van Scheltinga, Anke C

2009-01-15

122

Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase  

Energy Technology Data Exchange (ETDEWEB)

Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M. (UW)

2009-01-12

123

[Characterization of Escherichia hermannii by electrophoresis of esterases, acid phosphatase and glutamate and malate dehydrogenases].  

Science.gov (United States)

Esterases, acid phosphatase and glutamate and malate dehydrogenases of 11 strains of Escherichia hermannii were analysed by horizontal electrophoresis in polyacrylamide agarose gel. Seven esterase bands were defined by their range of activity on synthetic substrates and their sensitivity or resistance to di-isopropyl fluorophosphate. These bands were different in activity and in mobility from those produced by E. coli strains. On the basis of variations in mobility of glutamate and malate dehydrogenases and in the number and mobility of esterases, the strains were divided into 3 zymotypes. PMID:3426158

Goullet, P; Picard, B; Richard, C

1986-01-01

124

Functional characterization of a Plagiochasma appendiculatum flavone synthase I showing flavanone 2-hydroxylase activity.  

Science.gov (United States)

FNS I is a 2-oxoglutarate dependent dioxygenase (2-ODD) found mainly in species of the Apiaceae family. Here, an FNS I cDNA sequence was isolated from the liverwort Plagiochasma appendiculatum (Aytoniaceae) and characterized. The recombinant protein exhibited high FNS I activity catalyzing the conversion of naringenin to apigenin and 2-hydroxynaringenin. The critical residue for flavanone-2-hydroxylation activity was Tyr240, as identified from homology modeling and site-directed mutagenesis. The recombinant protein also showed some flavonol synthase activity, as it can convert dihydrokaempferol to kaempferol. When the Leu311 residue was mutated to Phe, the enzyme's capacity to convert dihydrokaempferol to kaempferol was substantially increased. PaFNS I represents a 2-ODD in which a hydrophobic ?-stacking interaction between the key residue and the naringenin A-ring determines 2-hydroxyflavanone formation. PMID:24859082

Han, Xiao-Juan; Wu, Yi-Feng; Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Lou, Hong-Xiang; Cheng, Ai-Xia

2014-06-27

125

Human gene encoding prostacyclin synthase (PTGIS): Genomic organization, chromosomal localization, and promoter activity  

Energy Technology Data Exchange (ETDEWEB)

The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13.11-q13.13. The 1.5-kb sequence of the 5{prime} of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-{gamma} response element, GATA, NF-{kappa}B, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5{prime}-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3{prime}-untranslated region as probe indicated that the sizes of the 3{prime}-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs. 54 refs., 7 figs.

Yokoyama, Chieko; Yabuki, Tomoko; Inoue, Hiroyasu [National Cardiovascular Center Research Institute, Osaka (Japan)] [and others

1996-09-01

126

Modulation of fatty acid synthase enzyme activity and expression during hepatitis C virus replication.  

Science.gov (United States)

The hepatitis C virus (HCV) induces alterations of host cells to facilitate its life cycle. Fatty acid synthase (FASN) is a multidomain enzyme that plays a key role in the biosynthesis of fatty acids and is upregulated during HCV infection. Herein, we applied activity-based protein profiling (ABPP) that allows for the identification of differentially active enzymes in complex proteomic samples, to study the changes in activity of FASN during HCV replication. For this purpose, we used an activity-based probe based on the FASN inhibitor Orlistat, and observed an increase in the activity of FASN in the presence of a subgenomic and a genomic HCV replicon as well as in chimeric SCID/Alb-uPA mice infected with HCV genotype 1a. To study the molecular basis for this increase in FASN activity, we overexpressed individual HCV proteins in Huh7 cells and observed increased expression and activity of FASN in the presence of core and NS4B, as measured by western blots and ABPP, respectively. Triglyceride levels were also elevated in accordance with FASN expression and activity. Lastly, immunofluorescence and ABPP imaging analyses demonstrated that while the abundance and activity of FASN increases significantly in the presence of HCV, its localization does not change. Together these data suggest that the HCV-induced production of fatty acids and neutral lipids is provided by an increase in FASN abundance and activity that is sufficient to allow HCV propagation without transporting FASN to the replication complexes. PMID:23601646

Nasheri, Neda; Joyce, Michael; Rouleau, Yanouchka; Yang, Pengyu; Yao, Shao; Tyrrell, D Lorne; Pezacki, John Paul

2013-04-18

127

Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening.  

Science.gov (United States)

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

2012-01-01

128

A three-step assay for ceramide synthase activity using a fluorescent substrate and HPLC.  

Science.gov (United States)

Ceramides are a family of signalling lipids with diverse physiological functions that include pro-differentiative and pro-apoptotic signalling. Ceramides and their derivatives are major constituents of myelin, maintaining neuronal conductivity. Ceramides are synthesized by ceramide synthases, of which there are six isoforms in mammals (CERS1-6). These enzymes catalyse the transfer of a variable length fatty acid to a sphingoid base, typically sphingosine or dihydrosphingosine. We previously reported a fluorescent thin-layer chromatography assay for ceramide synthase activity. In this paper we describe an improved fluorescent assay, using HPLC to achieve clear resolution of closely related ceramide species and to facilitate easy quantification of both product and substrate. Our HPLC assay protocol eliminates the need for a chloroform extraction step. Instead a simple three-step procedure is used: (1) reactions are run; (2) reactions are terminated with addition of methanol and centrifuged; (3) products are quantified with HPLC. HPLC resolution enables assays in which multiple fatty acid substrates are used in the same reaction. Using this approach, we show that CERS2 demonstrates a preference for the monounsaturated C24:1 fatty acid substrate compared to the saturated C24:0 substrate, potentially explaining why myelin is enriched in ceramides containing the monounsaturated form of very long chain fatty acids. PMID:25403920

Couttas, Timothy A; Lim, Xin Y; Don, Anthony S

2015-01-01

129

The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity.  

Science.gov (United States)

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants. PMID:12068104

Xu, Changcheng; Härtel, Heiko; Wada, Hajime; Hagio, Miki; Yu, Bin; Eakin, Chris; Benning, Christoph

2002-06-01

130

Ferutinin stimulates nitric oxide synthase activity in median eminence of the rat.  

Science.gov (United States)

Several species of Ferula genus have been used in folk medicine in digestive disorders, rheumatism, headache, arthritis, and as tranquilizers, antispasmodic and aphrodisiac. From the dry and powdered roots of Ferula hermonis Boiss was extracted the oxygenated sesquiterpene 1,5-trans-daucane type: ferutinine (1). The structure of (1) was established by spectroscopic methods as: IR, (1)H RMN, (13)C RMN, COSY, HMBC, HMQC, NOESY, EIMS, and CIMS. The possible signaling pathway of ferutinin (1) in nervous tissue in vitro was assessed and the results showed that this compound is able to increase nitric oxide synthase activity and inositol monophosphate accumulation (49%, each) in the median eminence of the rat brain, suggesting that compound (1) is associated to the activation of phosphoinositide breakdown and nitric oxide production (NO), the last is a gaseous intercellular messenger known to play a broad role in human biology from homeostasis to pathology. PMID:16574358

Colman-Saizarbitoria, Trina; Boutros, Paulo; Amesty, Angel; Bahsas, Alí; Mathison, Yaira; Garrido, Maria del Rosario; Israel, Anita

2006-07-19

131

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp.  

Directory of Open Access Journals (Sweden)

Full Text Available Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW, the nodule fresh weight (FW, the nitrogenase activity, the nodule glutamine synthetase (GS and glutamate synthase (GOGAT activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of nodules was measured in L. pedunculatus. Nitrogenase activity in L. tenuis, L. pedunculatus and L. subbiflorus was six fold the activity in L. corniculatus. Nodule GS and GOGAT activities did not follow this same pattern. L. tenuis had the highest values of GS and GOGAT activities in the nodule, and a high nitrogenase activity which is consistent with its high plant DW. The four species of Lotus were compared and no correlation between nitrogen fixation parameters and ammonia assimilation enzymes was found, but the GS/GOGAT ratio has a positive and significant correlation (r²=0.82** with the amino acid content in stems.

GONNET SUSANA

2000-01-01

132

Antiproliferative activity of guava leaf extract via inhibition of prostaglandin endoperoxide H synthase isoforms.  

Science.gov (United States)

Prostaglandin endoperoxide H synthase (PGHS) is a key enzyme for the synthesis of prostaglandins (PGs) which play important roles in inflammation and carcinogenesis. Because the extract from Psidium guajava is known to have a variety of beneficial effects on our body including the anti-inflammatory, antioxidative and antiproliferative activities, we investigated whether the extract inhibited the catalytic activity of the two PGHS isoforms using linoleic acid as an alternative substrate. The guava leaf extract inhibited the cyclooxygenase reaction of recombinant human PGHS-1 and PGHS-2 as assessed by conversion of linoleic acid to 9- and 13-hydroxyoctadecadienoic acids (HODEs). The guava leaf extract also inhibited the PG hydroperoxidase activity of PGHS-1, which was not affected by nonsteroidal anti-inflammatory drugs (NSAIDs). Quercetin which was one of the major components not only inhibited the cyclooxygenase activity of both isoforms but also partially inhibited the PG hydroperoxidase activity. Overexpression of human PGHS-1 and PGHS-2 in the human colon carcinoma cells increased the DNA synthesis rate as compared with mock-transfected cells which did not express any isoforms. The guava leaf extract not only inhibited the PGE(2) synthesis but also suppressed the DNA synthesis rate in the PGHS-1- and PGHS-2-expressing cells to the same level as mock-transfected cells. These results demonstrate the antiproliferative activity of the guava leaf extract which is at least in part caused by inhibition of the catalytic activity of PGHS isoforms. PMID:19457650

Kawakami, Yuki; Nakamura, Tomomi; Hosokawa, Tomoko; Suzuki-Yamamoto, Toshiko; Yamashita, Hiromi; Kimoto, Masumi; Tsuji, Hideaki; Yoshida, Hideki; Hada, Takahiko; Takahashi, Yoshitaka

2009-01-01

133

Rho 1 GTPase activates the (1-3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The Schizosaccharomyces pombe Cdc42 and Rho1 GTPases were tested for their ability to complement the cwg2-1 mutant phenotype of a decrease in (1-3)beta-D-glucan synthase activity when grown at the non-permissive temperature. Only Rho1 is able to partly complement the defect in glucan synthase associated with the cwg2-1 mutation. Moreover, overexpression of the rho1 gene in wild-type S.pombe cells causes aberrant morphology with loss of polarity and cells with several septa. Under this conditi...

Arellano, M.; Dura?n, A.; Pe?rez, P.

1996-01-01

134

Helicobacter pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3?.  

Science.gov (United States)

Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Helicobacter pylori (H. pylori) infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking. Here we explored the effect of H. pylori infection on tau phosphorylation. The results showed that H. pylori filtrate induced significant tau hyperphosphorylation at several AD-related tau phosphorylation sites, such as Thr205, Thr231, and Ser404, both in mouse neuroblastoma N2a cells and rat brains with activation of glycogen synthase kinase-3? (GSK-3?). Application of GSK-3 inhibitors efficiently attenuated the H. pylori-induced tau hyperphosphorylation. Our data provide evidence supporting the role of H. pylori infection in AD-like tau pathology, suggesting that H. pylori eradication may be beneficial in the prevention of tauopathy. PMID:25079798

Wang, Xiu-Lian; Zeng, Ji; Yang, Yang; Xiong, Yan; Zhang, Zhi-Hua; Qiu, Mei; Yan, Xiong; Sun, Xu-Ying; Tuo, Qing-Zhang; Liu, Rong; Wang, Jian-Zhi

2015-01-01

135

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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 meta [...] bolism. 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.

Vijay, Venugopal; Robert E., Leggett; Catherine, Schuler; Robert M., Levin.

2010-12-01

136

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)

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.

Vijay Venugopal

2010-12-01

137

Reductive Heme-Dependent Activation of the N-Oxide Prodrug AQ4N by Nitric Oxide Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Anaerobic reduction of anticancer prodrugs is a promising route to achieve targeting and selectivity in anticancer drug design. Most reductive prodrug activations involve simple electron transfer from a flavoprotein and are not amenable to specific targeting. Here we report that the N-oxide AQ4N is reduced by a nitric oxide synthase. This reduction involves interaction with the h...

Nishida, Clinton R.; Ortiz Montellano, Paul R.

2008-01-01

138

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

DEFF Research Database (Denmark)

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 depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP 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

Willemoës, Martin; MØlgaard, Anne

2005-01-01

139

Activation of Glycogen Synthase Kinase 3? Ameliorates Diabetes-induced Kidney Injury.  

Science.gov (United States)

Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3? (GSK3?) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3? by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3? and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3?, indicating that NO may mediate SNP stimulation of GSK3?. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3? by activation of Src, Pyk2, Akt, and ERK; GSK3? inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3? by SNP ameliorates kidney injury induced by diabetes. PMID:25339176

Mariappan, Meenalakshmi M; Prasad, Sanjay; D'Silva, Kristin; Cedillo, Esteban; Sataranatarajan, Kavithalakshmi; Barnes, Jeffrey L; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

2014-12-19

140

Systemic administration of pramiracetam increases nitric oxide synthase activity in the cerebral cortex of the rat.  

Science.gov (United States)

The effect of systemic administration of pramiracetam on neuronal type nitric oxide synthase (NOS) activity and NOS mRNA expression were studied in the hippocampus and cerebral cortex in rats. A dose of 300 mg/kg (i.p.) of this nootropic produced an approximately 20% increase in NOS activity in rat brain cortical homogenates but not in hippocampal homogenates; no significant changes were observed in NOS mRNA expression in the cortex and hippocampus. A lower dose of pramiracetam (100 mg/kg i.p.) was ineffective on NOS mRNA expression and enzyme activity. Interestingly, administration of pramiracetam (300 mg/kg i.p.) in rats pretreated (24 h before) with lithium chloride (LiCl) (3 mEq/kg i.p.) yielded a 40% increase in cortical NOS activity. However, in LiCl-pretreated rats this nootropic failed to affect cortical NOS mRNA expression; LiCl (3 mEq/kg i.p.) given alone produced no effect. In conclusion, the present data demonstrate that pramiracetam given alone or in combination with LiCl increases NOS activity in brain cortical homogenates of rats and this may contribute to the mechanisms underlying learning and memory improvement produced by this nootropic. PMID:8557218

Corasaniti, M T; Paoletti, A M; Palma, E; Granato, T; Navarra, M; Nisticò, G

1995-01-01

 
 
 
 
141

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

Science.gov (United States)

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. PMID:21203393

Qureshi, Asfia; Subathra, Marimuthu; Grey, Angus; Schey, Kevin; Del Poeta, Maurizio; Luberto, Chiara

2010-01-01

142

Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels  

Science.gov (United States)

BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, Prat aortic rings (60+/-6% versus 39+/-6%, Prings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Berkowitz, Dan E.; White, Ron; Li, Dechun; Minhas, Khalid M.; Cernetich, Amy; Kim, Soonyul; Burke, Sean; Shoukas, Artin A.; Nyhan, Daniel; Champion, Hunter C.; Hare, Joshua M.

2003-01-01

143

[A case of respiratory dyskinesia due to clebopride malate].  

Science.gov (United States)

Clebopride malate is therapeutically used for the treatment of peptic ulcer. This drug has potent antidopaminergic activity that causes acute dystonic reaction, parkinsonism and tardive dyskinesia as adverse effects. Here, we have reported an 86-year-old man who developed abnormal involuntary movement of respiratory muscles and lower limb muscles after this drug had been given for four months. This involuntary movement appeared spontaneously at resting state and disappeared during sleep. Surface EMG demonstrated a synchronous grouping discharge in m. orbicularis oris, m. sternocleidomastoideus and m. interstales which synchronized with diaphragmatic movement on cinefluorography. Involuntary movement of the lower limbs was synchronous bilaterally and had little relationship with diaphragmatic movement. This involuntary movement was irregular not only in rhythm but also in duration. According to this irregular nature, we diagnosed this involuntary movement as respiratory dyskinesia with limb dyskinesia that belongs to tardive dyskinesia. After cessation of clebopride malate limb dyskinesia disappeared rapidly and respiratory dyskinesia markedly decreased. We emphasize that respiratory dyskinesia should be differentiated from psychogenic hyperventilation as easily misdiagnosed on initial examination. PMID:1764865

Kawasaki, H; Yamamoto, M; Okayasu, H; Wakayama, Y

1991-08-01

144

Synthesis, anti-fungal and 1,3-?-D-glucan synthase inhibitory activities of caffeic and quinic acid derivatives.  

Science.gov (United States)

New derivatives of caffeic acid and quinic acid were synthesized and their anti-fungal and inhibitory activities on fungal 1,3-?-glucan synthase were determined in comparison with those of the corresponding chlorogenic acid derivatives. All the chlorogenic, quinic and caffeic acid derivatives that were coupled with an H(2)N-orn-4-(octyloxy) aniline group (1, 1b and 1c) displayed potent activities in both anti-fungal and inhibition of 1,3-glucan synthase assays. Compounds 1 and 1c inhibited the fungal membrane enzyme with the potency comparable to that of a known 1,3-?-D-glucan synthase inhibitor, aculeacin A. The results revealed that the anti-fungal activity of the chlorogenic acid derivative with a free amino group was at least partly due to inhibition of the fungal 1,3-?-glucan synthase. These results suggest that further investigation on caffeic acid derivatives may lead to the discovery of novel anti-fungal agents with drug-like properties. PMID:20813534

Ma, Chao-Mei; Abe, Takashi; Komiyama, Tadazumi; Wang, Wei; Hattori, Masao; Daneshtalab, Mohsen

2010-10-01

145

Activation of Phosphotyrosine Phosphatase Activity Attenuates Mitogen-Activated Protein Kinase Signaling and Inhibits c-FOS and Nitric Oxide Synthase Expression in Macrophages Infected with Leishmania donovani  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Intracellular protozoan parasites of the genus Leishmania antagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact of Leishmania donovani on mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PM...

Nandan, Devki; Lo, Raymond; Reiner, Neil E.

1999-01-01

146

Acetoacetyl-CoA reductase activity of lactating bovine mammary fatty acid synthase.  

Science.gov (United States)

Fatty acid synthase, purified from lactating bovine mammary gland, utilizes coenzyme A esters of acetoacetic, 3-hydroxybutyric, and crotonic acids as substrates for its partial reactions at micromolar concentrations. The NADPH:acetoacetyl-CoA reductase had a Km of 5 microM acetoacetyl-CoA and a Vmax of about 4 mumol of NADPH oxidized min-1 mg-1. In contrast, the Km for the model compound, acetoacetyl pantetheine was 820 microM and that of S-acetoacetyl-N-acetylcysteamine was over 40 mM. The reduction of acetoacetyl-CoA was observed with the enzyme from rat tissues also but not with those from avian tissues or yeast. With the bovine mammary enzyme, the reaction was found to oxidize 2 mol of NADPH for every mol of acetoacetyl-CoA consumed. Butyrate was the major product of reduction. The reductase activity was susceptible to inhibition by several sulfhydryl reagents; it was lost when the synthase was dissociated into one-half molecular weight subunits or when the incubation mixture was depleted of CoA. It was competitively inhibited by acetyl-CoA, butyryl-CoA, methylmalonyl-CoA, and 2-methylcrotonyl-CoA. These results as well as its use as a primer in fatty acid synthesis by the enzyme suggest that the acetoacetyl group from acetoacetyl-CoA is transferred to the enzyme, presumably to its 4'-phosphopantheine prosthetic group. The acyl group is then expected to remain attached to the enzyme while it is reduced, dehydrated, and reduced again to form a butyryl group which can either undergo chain elongation, if malonyl-CoA is present, or be released from the enzyme by hydrolysis or transfer to free CoA. PMID:7016867

Dodds, P F; Guzman, M G; Chalberg, S C; Anderson, G J; Kumar, S

1981-06-25

147

A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Prostaglandin H2 synthase (PGHS is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2 prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2 are the target for nonsteroidal anti-inflammatory drugs (NSAIDs. Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel in vitro electron paramagnetic resonance (EPR-based assay for measuring the activity of PGHS-1. Methods We validated a novel in vitro PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH to 3-carboxy-proxy (CP under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR spectrum. Results The assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 ?M, 1 mM caused significant inhibition of spin-adduct formation (72 ± 11 and 100 ± 16% inhibition of control respectively; P 0.05. Conclusion We have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays.

Rossi Adriano G

2006-09-01

148

Spermine synthase activity affects the content of decarboxylated S-adenosylmethionine.  

Science.gov (United States)

dcAdoMet (decarboxylated S-adenosylmethionine) is an essential intermediate in the synthesis of polyamines. Its content is normally very low, amounting to less than 5% of that of S-adenosylmethionine itself. It was found that in mice lacking spermine synthase there was a large increase in dcAdoMet and that overexpression of spermine synthase reduced the amount of this nucleoside. There was also an increase in dcAdoMet in cells derived from patients with Snyder-Robinson syndrome, a rare X-linked recessive human disease caused by SMS gene mutations that greatly reduce the content of spermine synthase. These results suggest that there is an inverse relationship between the amount of spermine synthase protein and the content of dcAdoMet and raise the possibility that some of the abnormalities seen in mammals deficient in spermine synthase might be due to changes in dcAdoMet pools. PMID:20950271

Pegg, Anthony E; Wang, Xiaojing; Schwartz, Charles E; McCloskey, Diane E

2011-01-01

149

Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation.  

Science.gov (United States)

During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (-P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO2 fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to -P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO2 fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency. PMID:25151130

Le Roux, Marcellous; Phiri, Ethel; Khan, Wesaal; Sakiro?lu, Muhammet; Valentine, Alex; Khan, Sehaam

2014-11-01

150

Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats.  

Science.gov (United States)

1. Metformin is an activator of AMP-activated protein kinase (AMPK). Recent studies suggest that pharmacological activation of AMPK inhibits cardiac hypertrophy. In the present study, we examined whether long-term treatment with metformin could attenuate ventricular hypertrophy in a rat model. The potential involvement of nitric oxide (NO) in the effects of metformin was also investigated. 2. Ventricular hypertrophy was established in rats by transaortic constriction (TAC). Starting 1 week after the TAC procedure, rats were treated with metformin (300 mg/kg per day, p.o.), N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg per day, p.o.) or both for 8 weeks prior to the assessment of haemodynamic function and cardiac hypertrophy. 3. Cultured cardiomyocytes were used to examine the effects of metformin on the AMPK-endothelial NO synthase (eNOS) pathway. Cells were exposed to angiotensin (Ang) II (10?? mol/L) for 24 h under serum-free conditions in the presence or absence of metformin (10?³ mol/L), compound C (10?? mol/L), L-NAME (10?? mol/L) or their combination. The rate of incorporation of [³H]-leucine was determined, western blotting analyses of AMPK-eNOS, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) were undertaken and the concentration of NO in culture media was determined. 4. Transaortic constriction resulted in significant haemodynamic dysfunction and ventricular hypertrophy. Myocardial fibrosis was also evident. Treatment with metformin improved haemodynamic function and significantly attenuated ventricular hypertrophy. Most of the effects of metformin were abolished by concomitant L-NAME treatment. L-NAME on its own had no effect on haemodynamic function and ventricular hypertrophy in TAC rats. 5. In cardiomyocytes, metformin inhibited AngII-induced protein synthesis, an effect that was suppressed by the AMPK inhibitor compound C or the eNOS inhibitor L-NAME. The improvement in cardiac structure and function following metformin treatment was associated with enhanced phosphorylation of AMPK and eNOS and increased NO production. 6. The findings of the present study indicate that long-term treatment with metformin could attenuate ventricular hypertrophy induced by pressure overload via activation of AMPK and a downstream signalling pathway involving eNOS-NO. PMID:21083698

Zhang, Cheng-Xi; Pan, Si-Nian; Meng, Rong-Sen; Peng, Chao-Quan; Xiong, Zhao-Jun; Chen, Bao-Lin; Chen, Guang-Qin; Yao, Feng-Juan; Chen, Yi-Li; Ma, Yue-Dong; Dong, Yu-Gang

2011-01-01

151

Acetohydroxyacid synthase activity and transcripts profiling reveal tissue-specific regulation of ahas genes in sunflower.  

Science.gov (United States)

Acetohydroxyacid synthase (AHAS) is the target site of several herbicides and catalyses the first step in the biosynthesis of branched chain amino acid. Three genes coding for AHAS catalytic subunit (ahas1, ahas2 and ahas3) have been reported for sunflower. The aim of this work was to study the expression pattern of ahas genes family and AHAS activity in sunflower (Helianthus annuus L.). Different organs (leaves, hypocotyls, roots, flowers and embryos) were evaluated at several developmental stages. The transcriptional profile was studied through RT-qPCR. The highest expression for ahas1 was shown in leaves, where all the induced and natural gene mutations conferring herbicide resistance were found. The maximal expression of ahas2 and ahas3 occurred in immature flowers and embryos. The highest AHAS activity was found in leaves and immature embryos. Correlation analysis among ahas gene expression and AHAS activity was discussed. Our results show that differences in ahas genes expression are tissue-specific and temporally regulated. Moreover, the conservation of multiple AHAS isoforms in sunflower seems to result from different expression requirements controlled by tissue-specific regulatory mechanisms at different developmental stages. PMID:24908515

Ochogavía, Ana C; Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana A; Nestares, Graciela

2014-07-01

152

Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation  

International Nuclear Information System (INIS)

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with [?-32P]ATP. Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25 degrees C before assay. The spontaneous inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32P]Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrateusing SPS as substrate

153

Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. [Spinacia oleracea  

Energy Technology Data Exchange (ETDEWEB)

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ({gamma}-{sup 32}P)ATP. Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25{degrees}C before assay. The spontaneous inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg{sup 2+} and was relatively insensitive to Ca{sup 2+} and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with ({sup 32}P)Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate.

Huber, J.L. (North Carolina State Univ., Raleigh (United States)); Huber, S.C. (Dept. of Agriculture, Raleigh, NC (United States) North Carolina State Univ., Raleigh (United States)); Hite, D.R.C.; Outlaw, W.H. Jr. (Florida State Univ., Tallahassee (United States))

1991-01-01

154

15-hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery.  

Science.gov (United States)

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time. PMID:16220200

Ye, Hong; Bi, Hai-Rong; Lü, Chang-Lian; Tang, Xiao-Bo; Zhu, Da-Ling

2005-10-25

155

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

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background ?-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.

Veenhuis Marten

2008-03-01

156

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu c...

Leiper, James; Murray-rust, Judith; Mcdonald, Neil; Vallance, Patrick

2002-01-01

157

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis.  

Science.gov (United States)

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed. PMID:17892446

Bernal, Adriana Jimena; Jensen, Jakob Krüger; Harholt, Jesper; Sørensen, Susanne; Moller, Isabel; Blaukopf, Claudia; Johansen, Bo; de Lotto, Robert; Pauly, Markus; Scheller, Henrik Vibe; Willats, William G T

2007-12-01

158

UDP-[14C]glucose-labelable polypeptides from pea: Possible components of glucan synthase I activity  

International Nuclear Information System (INIS)

A membrane-bound polypeptide doublet of about 40 kD can be rapidly labeled with UDP-[14C]glucose under the assay conditions for glucan synthase I (GS-I). Label seems covalently bound, and chases when unlabeled UDPG is added; it might represent a covalent intermediate in polysaccharide synthesis. Labeling and GS-I activity show several common features: they co-sediment with Golgi membranes in sucrose gradients; they depend similarly on Mg2+ or Mn2+ (not Ca2+); they decrease dramatically from stem apex to base, and are higher in epidermis than internal tissue; they show similar sensitivities to several inhibitors. But the doublet still labels after polysaccharide-synthesizing activity has been destroyed by Triton X-100. The doublet polypeptides might be glucosyl tranferases whose ability to transfer glucose units to a glucan chain is detergent-sensitive, but to accept glucose from UDPG is not; or they might be detergent-insensitive primary glucose acceptors, from which a distinct, detergent-sensitive transferase(s) move(s) these units to glucan chains

159

Activation of macrophage nuclear factor-?B and induction of inducible nitric oxide synthase by LPS  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Chronic lung disease (CLD of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS to stimulate rat alveolar macrophages to produce nitric oxide (NO, express inducible nitric oxide synthase (iNOS and activate nuclear factor-?B (NF-?B in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes. Methods Griess reaction was used to measure the nitrite level. Western blot and a semi-quantitative RT-PCR were performed to detect iNOS expression. Electrophoretic mobility shift assay (EMSA was performed to analyze the activation of NF-?B. Results We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (?10 ng/ml and time dependent manner (p -4–10-10 M and dexamethasone (10-4–10-6 M (p Conclusion Our findings imply that Gram-negative bacterial infection and the inflammatory responses are important factors in the development of CLD. The down-regulatory effect of steroids on iNOS expression and NO production might explain the beneficial effect of steroids in neonates with CLD.

Yan Zhong-Qun

2002-09-01

160

Insulin activation of mouse diaphragm glycogen synthase (GS) involves generation of electrophoretically distinct subunit species  

International Nuclear Information System (INIS)

Glycogen synthase, the rate limiting enzyme for glycogen synthesis, was analyzed in mouse diaphragm extracts both by immunoprecipitation and immunoblotting using specific antibodies raised to the rabbit muscle enzyme. Diaphragms, with the supporting ribs attached, were incubated either with or without [32P]P/sub i/ in the medium. In extracts from unincubated, rapidly frozen diaphragms, immunoblotting indicated the presence of 3 distinct species, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, phosphorylation of immunoprecipitated GS with the kinase F/sub A//GSK-3 converted the higher mobility forms into the low mobility species. In diaphragms incubated with [32P]P/sub i/, 32P was incorporated only into one of the GS species, that of lowest mobility, indicating differential labelling among the multiple subunit forms. Insulin action, which increased the -/+ glucose-6-P activity ratio from 0.2 to 0.4, converted the low mobility species to the two higher mobility forms. The authors propose that this effect of insulin can be explained by dephosphorylation in the proline/serine rich site 3 region of GS, which has potent influence on both mobility on SDS-PAGE and activity

 
 
 
 
161

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells.  

Science.gov (United States)

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised. PMID:23471028

Martin, Maria Laura; Liebisch, Gerhard; Lehneis, Stefan; Schmitz, Gerd; Alonso-Sande, María; Bestard-Escalas, Joan; Lopez, Daniel H; García-Verdugo, José Manuel; Soriano-Navarro, Mario; Busquets, Xavier; Escribá, Pablo V; Barceló-Coblijn, Gwendolyn

2013-05-01

162

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells1[S  

Science.gov (United States)

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised. PMID:23471028

Martin, Maria Laura; Liebisch, Gerhard; Lehneis, Stefan; Schmitz, Gerd; Alonso-Sande, María; Bestard-Escalas, Joan; Lopez, Daniel H.; García-Verdugo, José Manuel; Soriano-Navarro, Mario; Busquets, Xavier; Escribá, Pablo V.; Barceló-Coblijn, Gwendolyn

2013-01-01

163

Piceatannol-3'-O-?-D-glucopyranoside as an active component of rhubarb activates endothelial nitric oxide synthase through inhibition of arginase activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Arginase competitively inhibits nitric oxide synthase (NOS) via use of the common substrate L-arginine. Arginase II has recently reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. Here, we demonstrate that piceatannol-3'-O-?-D-glucopyranoside (PG), a potent component of stilbenes, inhibits the activity of arginase I and II prepared from mouse liver and kidney lysates, respectively, in a dose-dependent manner. In human umbilical vein e...

Woo, Ainieng; Min, Byungsun; Ryoo, Sungwoo

2010-01-01

164

Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels  

Science.gov (United States)

BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Berkowitz, Dan E.; White, Ron; Li, Dechun; Minhas, Khalid M.; Cernetich, Amy; Kim, Soonyul; Burke, Sean; Shoukas, Artin A.; Nyhan, Daniel; Champion, Hunter C.; Hare, Joshua M.

2003-01-01

165

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX  

Science.gov (United States)

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes. PMID:23671075

Laptenok, Sergey P.; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Myllykallio, Hannu; Liebl, Ursula; Vos, Marten H.

2013-01-01

166

Materials and methods for efficient succinate and malate production  

Energy Technology Data Exchange (ETDEWEB)

Genetically engineered microorganisms have been constructed to produce succinate and malate in mineral salt media in pH-controlled batch fermentations without the addition of plasmids or foreign genes. The subject invention also provides methods of producing succinate and malate comprising the culture of genetically modified microorganisms.

Jantama, Kaemwich; Haupt, Mark John; Zhang, Xueli; Moore, Jonathan C; Shanmugam, Keelnatham T; Ingram, Lonnie O' Neal

2014-04-08

167

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture  

Directory of Open Access Journals (Sweden)

Full Text Available The visual system is a potential target for methylmercury (MeHg intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05. Maximum values (P < 0.05 were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates. MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Herculano A.M.

2006-01-01

168

Interleukin-33 Increases Antibacterial Defense by Activation of Inducible Nitric Oxide Synthase in Skin  

Science.gov (United States)

Interleukin-33 (IL-33) is associated with multiple diseases, including asthma, rheumatoid arthritis, tissue injuries and infections. Although IL-33 has been indicated to be involved in Staphylococcus aureus (S. aureus) wound infection, little is known about how IL-33 is regulated as a mechanism to increase host defense against skin bacterial infections. To explore the underlying intricate mechanism we first evaluated the expression of IL-33 in skin from S. aureus-infected human patients. Compared to normal controls, IL-33 was abundantly increased in skin of S. aureus-infected patients. We next developed a S. aureus cutaneous infection mouse model and found that IL-33 was significantly increased in dermal macrophages of infected mouse skin. The expression of IL-33 by macrophages was induced by staphylococcal peptidoglycan (PGN) and lipoteichoic acid (LTA) via activation of toll-like receptor 2(TLR2) –mitogen-activated protein kinase (MAPK)-AKT-signal transducer and activator of transcription 3(STAT3) signaling pathway as PGN and LTA failed to induce IL-33 in Tlr2-deficient peritoneal macrophages, and MAPK,AKT, STAT3 inhibitors significantly decreased PGN- or LTA-induced IL-33. IL-33, in turn, acted on macrophages to induce microbicidal nitric oxygen (NO) release. This induction was dependent on inducible nitric oxide synthase (iNOS) activation, as treatment of macrophages with an inhibitor of iNOS, aminoguanidine, significantly decreased IL-33-induced NO release. Moreover, aminoguanidine significantly blocked the capacity of IL-33 to inhibit the growth of S. aureus, and IL-33 silencing in macrophages significantly increased the survival of S. aureus in macrophages. Furthermore, the administration of IL-33-neutralizing antibody into mouse skin decreased iNOS production but increased the survival of S. aureus in skin. These findings reveal that IL-33 can promote antimicrobial capacity of dermal macrophages, thus enhancing antimicrobial defense against skin bacterial infections. PMID:24586149

Jiang, Ziwei; Zhang, Tian; Wang, Yue; Li, Zhiheng; Wu, Yelin; Ji, Shizhao; Xiao, Shichu; Ryffel, Bernhard; Radek, Katherine A.; Xia, Zhaofan; Lai, Yuping

2014-01-01

169

Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes.  

Science.gov (United States)

Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization. PMID:8616221

Kurvari, V; Qian, F; Snell, W J

1995-12-01

170

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The enzyme porphobilinogen synthase (PBGS, which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. Results The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of ~6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. Conclusion The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Fairman Robert

2004-11-01

171

Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.  

Science.gov (United States)

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis. PMID:24453164

Beeler, Seraina; Liu, Hung-Chi; Stadler, Martha; Schreier, Tina; Eicke, Simona; Lue, Wei-Ling; Truernit, Elisabeth; Zeeman, Samuel C; Chen, Jychian; Kötting, Oliver

2014-03-01

172

Iridoid Synthase Activity Is Common among the Plant Progesterone 5?-Reductase Family.  

Science.gov (United States)

Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, among which the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5?-reductase (P5?R) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5?R genes. Characterisation of recombinant CrP5?R proteins demonstrates that all but CrP5?R3 can reduce progesterone, and thus can be classified as P5?Rs. Three of them, namely CrP5?R1, CrP5?R2 and CrP5?R4, could also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5?R5) in secoiridoid synthesis. In depth functional analysis by subcellular protein localisation, gene expression analysis, in situ hybridisation and virus-induced gene silencing, indicates that besides IS, CrP5?R4 may also participate in secoiridoid biosynthesis. Finally, we cloned a set of P5?R genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that 'IS activity' is intrinsic to angiosperm P5?R proteins and has evolved early during evolution. PMID:25239067

Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

2014-09-19

173

Iridoid Synthase Activity Is Common among the Plant Progesterone 5?-Reductase Family.  

Science.gov (United States)

Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5?-reductase (P5?R) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5?R genes. Characterization of recombinant CrP5?R proteins demonstrates that all but CrP5?R3 can reduce progesterone and thus can be classified as P5?Rs. Three of them, namely CrP5?R1, CrP5?R2, and CrP5?R4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5?R5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5?R4 may also participate in secoiridoid biosynthesis. We cloned a set of P5?R genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5?R proteins and has evolved early during evolution. PMID:25578278

Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

2015-01-01

174

Distinct parts of leukotriene C4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein  

International Nuclear Information System (INIS)

Leukotriene C4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C4 synthase (LTC4S) participate in its biosynthesis. We report evidence that LTC4S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC4S. FLAP bound to the N-terminal part/first hydrophobic region of LTC4S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC4S. Fluorescent FLAP- and LTC4S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC4S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC4S. Direct interaction of 5-LO and LTC4S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC4S.

175

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.  

Science.gov (United States)

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases. PMID:25368106

Tidhar, Rotem; Sims, Kacee; Rosenfeld-Gur, Eden; Shaw, Walter; Futerman, Anthony H

2015-01-01

176

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess col...

Otero, Miguel; Lago, Roci?o; Lago, Francisca; Reino, Juan Jesu?s Gomez; Gualillo, Oreste

2005-01-01

177

Contractile Activity Regulates Inducible Nitric Oxide Synthase Expression and NOi Production in Cardiomyocytes via a FAK-Dependent Signaling Pathway  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Intracellular nitric oxide (NOi) is a physiological regulator of excitation-contraction coupling, but is also involved in the development of cardiac dysfunction during hypertrophy and heart failure. To determine whether contractile activity regulates nitric oxide synthase (NOS) expression, spontaneously contracting, neonatal rat ventricular myocytes (NRVM) were treat with L-type calcium channel blockers (nifedipine and verapamil) or myosin II ATPase inhibitors (butanedione monoxime (BDM) and ...

Miensheng Chu; Yevgeniya Koshman; Rekha Iyengar; Taehoon Kim; Brenda Russell; Samarel, Allen M.

2012-01-01

178

Cyclooxygenase 2 Promotes Cell Survival by Stimulation of Dynein Light Chain Expression and Inhibition of Neuronal Nitric Oxide Synthase Activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cyclooxygenase 2 (COX-2) inhibits nerve growth factor (NGF) withdrawal apoptosis in differentiated PC12 cells. The inhibition of apoptosis by COX-2 was concomitant with prevention of caspase 3 activation. To understand how COX-2 prevents apoptosis, we used cDNA expression arrays to determine whether COX-2 regulates differential expression of apoptosis-related genes. The expression of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase [PIN]) was signifi...

Chang, Yu-wen E.; Jakobi, Rolf; Mcginty, Ann; Foschi, Marco; Dunn, Michael J.; Sorokin, Andrey

2000-01-01

179

Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ER?) within the plasma membrane of endothelial cells (EC). ER? is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ER? to the plasma membrane. However, the role played by caveolin 1 in regulating 17?-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of...

Sud, Neetu; Wiseman, Dean A.; Black, Stephen M.

2010-01-01

180

Inhibition of Thromboxane A Synthase Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StA...

Wang, Xingjia; Yin, Xiangling; Schiffer, Randolph B.; King, Steven R.; Stocco, Douglas M.; Grammas, Paula

2007-01-01

 
 
 
 
181

Interrogation of Global Active Site Occupancy of a Fungal Iterative Polyketide Synthase Reveals Strategies for Maintaining Biosynthetic Fidelity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using ...

Vagstad, Anna L.; Bumpus, Stefanie B.; Belecki, Katherine; Kelleher, Neil L.; Townsend, Craig A.

2012-01-01

182

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

DEFF Research Database (Denmark)

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 dephosphorylation at sites 2+2a, 3a, and 3a + 3b. Furthermore, we report the existence of several glycogen metabolism regulatory mechanisms based on GS intracellular compartmentalization. After exhausting exercise, epinephrine-induced protein kinase A activation leads to GS site 1b phosphorylation targeting the enzyme to intramyofibrillar glycogen particles, which are preferentially used during muscle contraction. On the other hand, when phosphorylated at sites 2+2a, GS is preferentially associated with subsarcolemmal and intermyofibrillar glycogen particles. Finally, we verify the existence in human vastus lateralis muscle of the previously reported mechanism of glycogen metabolism regulation in rabbit tibialis anterior muscle. After overnight low muscle glycogen level and/or in response to exhausting exercise-induced glycogenolysis, GS is associated with spherical structures at the I-band of sarcomeres.

Prats, Clara; Helge, JØrn W

2009-01-01

183

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3, a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. Results The 2 isoforms of GSK3, GSK3? and GSK3?, phosphorylate the sequence Ser-X-X-X-Ser(P when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3? phosphorylation at serine 33 in vitro. GSK3? did not phosphorylate p53 under any condition. GSK3? increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3? to regulate p53 transcriptional activity. GSK3? is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3? kinase activity was inhibited in response to DNA damage, suggesting that GSK3? regulation of p53 is not involved in the p53-DNA damage response. Conclusions GSK3? can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3? does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3? may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

Price Brendan D

2001-07-01

184

Higher Activity of the Inducible Nitric Oxide Synthase Contributes to Very Early Onset Inflammatory Bowel Disease  

Science.gov (United States)

OBJECTIVES: The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early onset IBD (VEO-IBD). METHODS: Seventeen tag single nucleotide polymorphisms (SNPs) in the NOS2 gene were successfully genotyped in VEO-IBD patients. Genetic associations were replicated in an independent VEO-IBD cohort. Functional analysis for iNOS activity was performed on the most significantly associated functional variant. RESULTS: The NOS2 rs2297518 SNP was found to be associated in VEO-IBD in two independent cohorts. Upon combined analysis, a coding variant (S608L) showed the strongest association with VEO-IBD (Pcombined=1.13 × 10?6, OR (odds ratio)=3.398 (95% CI (confidence interval) 2.02–5.717)) as well as associations with VEO-Crohn's disease and VEO-ulcerative colitis (UC). This variant also showed an association with UC diagnosed between 11 and 17 years of age but not with adult-onset IBD (>17 years). B-cell lymphoblastoid cell lines genotyped for the risk variant as well as Henle-407 cells transfected with a plasmid construct with the risk variant showed higher NO production. Colonic biopsies of VEO-IBD patients showed higher immunohistochemical staining of nitrotyrosine, indicating more nitrosative stress and tissue damage. CONCLUSIONS: These studies suggest the importance of iNOS in genetic susceptibility to younger IBD presentation due to higher NO production. PMID:24430113

Dhillon, Sandeep S; Mastropaolo, Lucas A; Murchie, Ryan; Griffiths, Christopher; Thöni, Cornelia; Elkadri, Abdul; Xu, Wei; Mack, Amanda; Walters, Thomas; Guo, Conghui; Mack, David; Huynh, Hien; Baksh, Shairaz; Silverberg, Mark S; Brumell, John H; Snapper, Scott B; Muise, Aleixo M

2014-01-01

185

Functional regulation of neuronal nitric oxide synthase expression and activity in the rat retina.  

Science.gov (United States)

In the nervous system within physiological conditions, nitric oxide (NO) production depends on the activity of nitric oxide synthases (NOSs), and particularly on the expression of the neuronal isoform (nNOS). In the sensory systems, the role of NO is poorly understood. In this study, we identified nNOS-positive cells in the inner nuclear layer (INL) of the rat retina, with distinct characteristics such as somata size, immunolabeling level and location. Employing mathematical cluster analysis, we determined that nNOS amacrine cells are formed by two distinct populations. We next investigated the molecular identity of these cells, which did not show colocalization with calbindin (CB), choline acetyltransferase (ChAT), parvalbumin (PV) or protein kinase C (PKC), and only partial colocalization with calretinin (CR), revealing the accumulation of nNOS in specific amacrine cell populations. To access the functional, circuitry-related roles of these cells, we performed experiments after adaptation to different ambient light conditions. After 24h of dark-adaptation, we detected a subtle, yet statistically significant decrease in nNOS transcript levels, which returned to steady-state levels after 24h of normal light-dark cycle, revealing that nNOS expression is governed by ambient light conditions. Employing electron paramagnetic resonance (EPR), we demonstrated that dark-adaptation decreases NO production in the retina. Furthermore, nNOS accumulation changed in the dark-adapted retinas, with a general reduction in the inner plexiform layer. Finally, computational analysis based on clustering techniques revealed that dark-adaptation differently affected both types of nNOS-positive amacrine cells. Taken together, our data disclosed functional regulation of nNOS expression and activity, disclosing new circuitry-related roles of nNOS-positive cells. More importantly, this study indicated unsuspected roles for NO in the sensory systems, particularly related to adaptation to ambient demands. PMID:25116452

Walter, Lais Takata; Higa, Guilherme Shigueto Vilar; Schmeltzer, Christian; Sousa, Erica; Kinjo, Erika Reime; Rüdiger, Sten; Hamassaki, Dânia Emi; Cerchiaro, Giselle; Kihara, Alexandre Hiroaki

2014-11-01

186

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

International Nuclear Information System (INIS)

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. [1-14C]-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 [14C]-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

187

Cloning and characterization of a Neurospora crassa gene required for (1,3) beta-glucan synthase activity and cell wall formation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The glucan synthase 1 gene (gs-1) is required for (1,3) beta-glucan synthase activity [E.C. 2.4.1.34; UDP glucose:1,3-beta-D-glucan 3-beta-D-glucosyltransferase] and for cell wall formation. The gs-1 gene was cloned by functional complementation of the cell-wall-less defect of the (1,3) beta-glucan synthase-deficient mutant, TM1, by using a genomic Neurospora crassa cosmid library. A 2568-nucleotide gs-1 cDNA sequence revealed a 532-amino acid open reading frame encoding a polypeptide of 59 k...

Enderlin, C. S.; Selitrennikoff, C. P.

1994-01-01

188

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

DEFF Research Database (Denmark)

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, 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 and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P < 0.001) between the relative change in V.O2max and the relative change in CS activity. All reported absolute values of CS and V.O2max did not correlate (r =- 0.07, n = 148, P = 0.4). 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

VigelsØ Hansen, Andreas; Andersen, Nynne Bjerre

2014-01-01

189

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

Science.gov (United States)

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 the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K(m) values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V(max) value as 2.20 mmol (minxmg of protein)(-1). ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M.jannaschii enzyme. The M.jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B.subtilis PRPP synthase. Soaking of the crystals with 5'-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases. PMID:16288921

Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva; McGuire, Jim; Larsen, Sine; Hove-Jensen, Bjarne

2005-12-01

190

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN- gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 wa...

1995-01-01

191

Inducible Nitric Oxide Synthase Deficiency Impairs Matrix Metalloproteinase-9 Activity and Disrupts Leukocyte Migration in Hepatic Ischemia/Reperfusion Injury  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Matrix metalloproteinase 9 (MMP-9) is a critical mediator of leukocyte migration in hepatic ischemia/reperfusion (I/R) injury. To test the relevance of inducible nitric oxide synthase (iNOS) expression on the regulation of MMP-9 activity in liver I/R injury, our experiments included both iNOS-deficient mice and mice treated with ONO-1714, a specific iNOS inhibitor. The inability of iNOS-deficient mice to generate iNOS-derived nitric oxide (NO) profoundly inhibited MMP-9 activity and depressed...

Hamada, Takashi; Duarte, Sergio; Tsuchihashi, Seiichiro; Busuttil, Ronald W.; Coito, Ana J.

2009-01-01

192

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recom...

Faraci, W. S.; Nagel, A. A.; Verdries, K. A.; Vincent, L. A.; Xu, H.; Nichols, L. E.; Labasi, J. M.; Salter, E. D.; Pettipher, E. R.

1996-01-01

193

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase  

Energy Technology Data Exchange (ETDEWEB)

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-05-01

194

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase  

International Nuclear Information System (INIS)

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

195

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions.  

Science.gov (United States)

Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable. PMID:25100160

Harmer, Jenny E; Hiscox, Martyn J; Dinis, Pedro C; Fox, Stephen J; Iliopoulos, Andreas; Hussey, James E; Sandy, James; Van Beek, Florian T; Essex, Jonathan W; Roach, Peter L

2014-11-15

196

Serotonin 5HT2A receptor activation inhibits inducible nitric oxide synthase activity in C6 glioma cells.  

Science.gov (United States)

C6-glioma cells endogenously express both 5HT2A receptors and inducible nitric oxide synthase (iNOS). iNOS can be induced by transcriptional activation to produce nitric oxide (NO) in response to a challenge with lipopolysaccharide (LPS). Experiments were conducted to determine whether 5HT2A receptor activation could modify the production of NO in response to LPS. Incubation of 10 microg/ml LPS with C6-glioma cells for a period of 24 hours resulted in a 2.6 fold increase in nitrite levels, as a measure of NO levels, over vehicle treated controls. Co-incubation with the selective 5HT2A receptor partial agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) produced a dose-dependent inhibition of the LPS-induced nitrite levels of 22% with an IC50 of 16 nM. The full agonists serotonin (5HT) and alpha-methyl-5HT produced an inhibition of approximately 30% at a concentration of 1 microM. The inhibitory effect of 1 microM DOI was blocked by the 5HT2A receptor antagonists spiperone and ritanserin (10 nM). Inhibition of protein kinase C (PKC) using 100 nM chelerythrine prevented the DOI-mediated decrease in LPS-induced nitrite levels. Addition of DOI to the cells after 1 hr following the LPS addition did not produce a decrease in nitrite levels indicating iNOS was not modified post-translationally. The data demonstrate that iNOS activity can be modulated by serotonin 5HT2A receptor activation, most likely at the initiation of the induction process, via PKC. We therefore suggest that there may be a link between the serotonergic system and NO-mediated immune responses in the brain. PMID:9365229

Miller, K J; Mariano, C L; Cruz, W R

1997-01-01

197

A novel sex-specific and inducible monoterpene synthase activity associated with a pine bark beetle, the pine engraver, Ips pini.  

Science.gov (United States)

Ecological interactions of conifers and coniferophagous bark beetles are determined in part by terpenoids (isoprenoids), which are major defense metabolites of conifer oleoresin. Curiously, similar compounds are important aggregation pheromones of conifer-attacking bark beetles. Terpene synthases are responsible for generating the enormous variety of terpenoid carbon skeletons found in nature. These catalysts convert short-chain prenyl diphosphates to a diverse assortment of hemiterpene, monoterpene, sesquiterpene, and diterpene natural products. While terpene synthases have frequently been characterized from plant and microbial sources, they have not yet been described in animals. Here we report the discovery of a monoterpene synthase activity in an insect, the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae). Cell-free assays of I. pini revealed that geranyl diphosphate (GDP) is converted to the acyclic monoterpene myrcene in whole-body extracts from males, but not females. Furthermore, the monoterpene synthase activity in males can be induced by prior treatment with juvenile hormone III (JH III) or by feeding on phloem from the host trees, Jeffrey pine (Pinus jeffreyi Grev. & Balf.) or red pine (Pinus resinosa Ait.). The sex-specificity and endocrine induction of this activity argue for its involvement in the biosynthesis of monoterpenoid pheromones mediated by enzymes from insect tissue. This discovery is the first example of a monoterpene synthase in the Metazoa and evokes exciting new questions about the origin, evolution, and occurrence of terpene synthases. PMID:12712251

Martin, Diane; Bohlmann, Jörg; Gershenzon, Jonathan; Francke, Wittko; Seybold, Steven J

2003-04-01

198

Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352.  

Science.gov (United States)

A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin. PMID:9190827

Kawai, S; Suzuki, H; Yamamoto, K; Kumagai, H

1997-06-01

199

Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin.

Kawai, S.; Suzuki, H.; Yamamoto, K.; Kumagai, H.

1997-01-01

200

Endothelial Nitric-oxide Synthase Activation Generates an Inducible Nitric-oxide Synthase-like Output of Nitric Oxide in Inflamed Endothelium*  

Science.gov (United States)

High levels of NO generated in the vasculature under inflammatory conditions are usually attributed to inducible nitric-oxide synthase (iNOS), but the role of the constitutively expressed endothelial NOS (eNOS) is unclear. In normal human lung microvascular endothelial cells (HLMVEC), bradykinin (BK) activates kinin B2 receptor (B2R) signaling that results in Ca2+-dependent activation of eNOS and transient NO. In inflamed HLMVEC (pretreated with interleukin-1? and interferon-?), we found enhanced binding of eNOS to calcium-calmodulin at basal Ca2+ levels, thereby increasing its basal activity that was dependent on extracellular l-Arg. Furthermore, B2R stimulation generated prolonged high output eNOS-derived NO that is independent of increased intracellular Ca2+ and is mediated by a novel G?i-, MEK1/2-, and JNK1/2-dependent pathway. This high output NO stimulated with BK was blocked with a B2R antagonist, eNOS siRNA, or eNOS inhibitor but not iNOS inhibitor. Moreover, B2R-mediated NO production and JNK phosphorylation were inhibited with MEK1/2 and JNK inhibitors or MEK1/2 and JNK1/2 siRNA but not with ERK1/2 inhibitor. BK induced Ca2+-dependent eNOS phosphorylation at Ser1177, Thr495, and Ser114 in cytokine-treated HLMVEC, but these modifications were not dependent on JNK1/2 activation and were not responsible for prolonged NO output. Cytokine treatment did not alter the expression of B2R, G?q/11, G?i1,2, JNK, or eNOS. B2R activation in control endothelial cells enhanced migration, but in cytokine-treated HLMVEC it reduced migration. Both responses were NO-dependent. Understanding how JNK regulates prolonged eNOS-derived NO may provide new therapeutic targets for the treatment of disorders involving vascular inflammation. PMID:23255592

Lowry, Jessica L.; Brovkovych, Viktor; Zhang, Yongkang; Skidgel, Randal A.

2013-01-01

 
 
 
 
201

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carbosylation, oxaloacetate reduction and malate export  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO2-fixing pathway has a theoretical maximum yield of 2 mol malat...

Zelle, R. M.; Hulster, E.; Winden, W. A.; Waard, P.; Dijkema, C.; Winkler, A. A.; Geertman, J. M. A.

2008-01-01

202

l-Malate Production by Metabolically Engineered Escherichia coli? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactiv...

Zhang, X.; Wang, X.; Shanmugam, K. T.; Ingram, L. O.

2011-01-01

203

Dissociation of dimeric 6-hydroxymellein synthase, a polyketide biosynthetic enzyme in carrot cell extracts, with loss of keto-reducing activity.  

Science.gov (United States)

6-Hydroxymellein synthase, an inducible polyketide biosynthetic enzyme in carrot cell extracts, is composed of two identical subunits, and the homodimer is dissociated to monomeric peptides under high-ionic-strength conditions with loss of the synthase activity. Appreciable radioactivities were associated with the synthase proteins when the monomer enzyme was incubated with the radiolabeled substrates, acetyl-coenzyme A (CoA) and malonyl-CoA. Therefore, it appeared that the synthase does not lose the ability of binding the substrate even after the dissociation to monomers. The monomeric synthase liberated triacetic acid lactone as the derailment product instead of 6-hydroxymellein from the enzyme-attached triketomethylene chain which is the immediate precursor of an NADPH-dependent keto-reducing reaction involved in 6-hydroxymellein biosynthesis. These observations strongly suggest that the monomeric synthase retains the ability of ketomethylene chain elongation by the condensation of acyl-CoAs, but is lacking in an NADPH-dependent keto-reducing activity toward the triketide intermediate. Results obtained in the present experiments imply that the catalytic domain of acyl-CoA condensation is able to associate with that of keto reduction, possibly belonging to another subunit, only in the homodimeric structure to organize the multicatalytic reaction center. PMID:7639527

Kurosaki, F

1995-08-01

204

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

Directory of Open Access Journals (Sweden)

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-3?/? and pGSK-3?/?Tyr279/216 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-3?/? and pGSK-3?/?Tyr279/216 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-3?/? and pGSK-3?/?Tyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3?/? 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-3?/?Tyr279/216 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-3?/? or pGSK-3?/?Tyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3?/? expression, and pGSK-3?/?Tyr279/216 expression were independent prognostic factors for overall survival. GSK-3 inhibition by lithium chloride, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8, or GSK-3 small interfering RNA can decrease viability of SKOV3 and SKOV3-TR30 ovarian cancer cells. Additionally, lithium chloride-treated SKOV3 xenograft mice had a significant reduction in tumor growth compared with control-treated animals. Conclusion: Our findings suggest that overexpression and aberrant activation of GSK-3 may contribute to progression and poor prognosis in ovarian cancer. Inhibition of GSK-3 may be a potential therapy for ovarian cancer.Keywords: ovarian carcinoma, immunohistochemistry, lithium chloride, TDZD-8

Fu Y

2014-06-01

205

The reduction of arterial tension produced by stevioside is dependent on nitric oxide synthase activity when the endothelium is intact.  

Science.gov (United States)

In endothelium-intact rat aortic ring preparations pre-contracted with norepinephrine or KCl, NG-nitro L-arginine (L-NOARG, 0.1 mM) and 1H-[1,2,4] oxidiazolo [4,3-a] quinoxalin-1-one (ODQ, 10 microM) antagonized the reduction of the vascular tone induced by stevioside, but this antagonism did not occur when the experiment was performed with endothelium-denuded aortic rings. The data indicates that the vasodilatation produced by stevioside is dependent on nitric oxide synthase and guanylate cyclase activities when the endothelium is not damaged. PMID:18480593

Bornia, Elaine Campana Sanches; do Amaral, Valéria; Bazotte, Roberto Barbosa; Alves-Do-Prado, Wilson

2008-02-01

206

Synthesis, anticandidal activity of N(3)-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic amide derivatives - Novel inhibitors of glucosamine-6-phosphate synthase.  

Science.gov (United States)

Novel FMDP amides 4-6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans. PMID:25497131

Pawlak, Dorota; Stolarska, Magdalena; Wojciechowski, Marek; Andruszkiewicz, Ryszard

2015-01-27

207

Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhi...

Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, Wayne F.; Koppisch, Andrew T.; Freel Meyers, Caren L.

2013-01-01

208

Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ß-Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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-dependen...

Gherasim, Carmen; Yadav, Pramod K.; Kabil, Omer; Niu, Wei-ning; Banerjee, Ruma

2014-01-01

209

Central nervous system nitric oxide synthase activity regulates insulin secretion and insulin action.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Systemic inhibition of nitric oxide synthase (NOS) with NG-monomethyl-L-arginine (L-NMMA) causes acute insulin resistance (IR), but the mechanism is unknown. We tested whether L-NMMA-induced IR occurs via NOS blockade in the central nervous system (CNS). Six groups of Sprague-Dawley rats were studied after chronic implantation of an intracerebroventricular (ICV) catheter into the lateral ventricle and catheters into the carotid artery and jugular vein. Animals were studied after overnight foo...

Shankar, R.; Zhu, J. S.; Ladd, B.; Henry, D.; Shen, H. Q.; Baron, A. D.

1998-01-01

210

Molecular mechanism of endothelial nitric-oxide synthase activation by Platycodon grandiflorum root-derived saponins.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) has antithrombotic and antiatherosclerotic properties in the vasculature. Previously, we demonstrated that saponins derived from the roots of Platycodon grandiflorum (CKS) inhibited the tumor necrosis factor-alpha-induced expression of adhesion molecules in human endothelial cells. In this study, we found that CKS increased eNOS phosphorylation and NO production in human endothelial cells. Treatment with CKS increased the ...

Kim, Hg; Hien, Tt; Han, Eh; Chung, Yc; Jeong, Hg

2010-01-01

211

Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site.  

Science.gov (United States)

Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro. PMID:23108268

Manos-Turvey, Alexandra; Cergol, Katie M; Salam, Noeris K; Bulloch, Esther M M; Chi, Gamma; Pang, Angel; Britton, Warwick J; West, Nicholas P; Baker, Edward N; Lott, J Shaun; Payne, Richard J

2012-12-14

212

Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cas...

Daher, Zeinab; Boulay, Pierre-luc; Desjardins, Fanny; Gratton, Jean-philippe; Claing, Audrey

2010-01-01

213

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation  

Directory of Open Access Journals (Sweden)

Full Text Available 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.

Helena H. Chowdhury

2014-10-01

214

Volume regulation in poterioochromonas: involvement of calmodulin in the ca-stimulated activation of isofloridoside-phosphate synthase.  

Science.gov (United States)

In Poterioochromonas malhamensis Peterfi (syn. Ochromonas malhamensis Pringsheim) osmotically induced shrinkage is reversed by an accumulation of isofloridoside. Addition of Ca(2+) ions to homogenates from standard volume cells initiates an enzyme system for the activation of isofloridoside-phosphate synthase. This process is stimulated in the presence of Ca(2+) by calmodulin, isolated from the same alga or from bovine brain, and requires the presence of membranes. The stimulation observed when Ca(2+) is added without exogenous calmodulin is inhibited by the calmodulin-binding substance R 24571. These results show that the effect of Ca(2+) is mediated by calmodulin. The Ca(2+)/calmodulin-dependent activation is enhanced when fluoride or molybdate ions are present in the homogenization buffer. This might indicate the involvement of a phosphorylated compound in the activation mechanism. PMID:16662779

Kauss, H

1983-01-01

215

Doubling the CO{sub 2} concentration enhanced the activity of carbohydrate-metabolism enzymes, source carbohydrate production, photoassimilate transport, and sink strength for Opuntia ficus-indica  

Energy Technology Data Exchange (ETDEWEB)

After exposure to a doubled CO{sub 2} concentration of 750 {mu}mol mol{sup -1} air for about 3 months, glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO{sub 2} concentration of 370 {mu}mol mol{sup -1}, but sucrose content was virtually unaffected. Doubling the CO{sub 2} concentration increased the noncturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32% soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO{sub 2} accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO{sub 2} increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO{sub 2} concentrations occurs for O. ficus-indica, consistent with its higher source capacity and sink strength than under current CO{sub 2}. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops.

Wang, Ning; Nobel, P.S. [Univ. of California, Los Angeles, CA (United States)

1996-03-01

216

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

International Nuclear Information System (INIS)

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 3? (GSK-3?) 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-? (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

217

Influence of ionizing radiation and 12-crown-4 on L-arginine-dependent synthesis of nitrogen oxide and NO-synthase activity  

International Nuclear Information System (INIS)

The influence of ionizing radiation and 12-crown-4 may be considered as modulatory at the respect of nitrogen oxide production and NO-synthase activity which is connected with the functional activity of monooxygenase system. Similar character of the alterations caused by ionized radiation and 12-crown-4 suggests the presence of radiomimetic properties of the latter

218

Electrogenic L-malate transport by Lactobacillus plantarum: a basis for energy derivation from malolactic fermentation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a drivin...

Olsen, E. B.; Russell, J. B.; Henick-kling, T.

1991-01-01

219

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 ?) and complex of endogenous subs...

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

2009-01-01

220

Involvement of glutamate, oxidative stress and inducible nitric oxide synthase in the convulsant activity of ciprofloxacin in mice.  

Science.gov (United States)

This study investigated the potential convulsive activity of ciprofloxacin in mice and the possible mechanism(s) of this activity. Intraperitoneal (i.p.) administration of ciprofloxacin into mice resulted in convulsive seizures in a dose-dependent manner. The clonic median convulsant dose (CD(50)) of ciprofloxacin in mice was increased by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine, not changed by pretreatment with 7-nitroindazole and decreased by pretreatment with L-arginine and fenbufen. The increase in nitric oxide (NO) production and malondialdehyde (MDA) level as well as the decrease in intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity induced by the estimated clonic CD(50) of ciprofloxacin in mice brain was inhibited by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine. These biochemical alterations were not changed by pretreatment with 7-nitroindazole but enhanced by pretreatment with L-arginine. The elevation induced by the clonic CD(50) of ciprofloxacin in brain glutamate level was not changed by pretreatment with MK-801, alpha-lipoic acid, aminoguanidine or L-arginine. Combined treatment of mice with fenbufen and ciprofloxacin produced elevation of brain NO production and glutamate and MDA levels as well as inhibition of brain intracellular GSH level and GSH-Px activity. In addition, i.p. administration of the clonic CD(50) of ciprofloxacin produced an increase in inducible but not in neuronal NO synthase mRNA and protein expressions in mice brain. These results suggest that elevation of brain glutamate levels with consequent oxidative stress and increase in the expression and activity of brain inducible NO synthase may play a pivotal role in ciprofloxacin-induced convulsive seizures. PMID:22542655

Abdel-Zaher, Ahmed O; Afify, Abdel-Halim M; Kamel, Sohair M; Farghaly, Hanan M; El-Osely, Gehan M; El-Awaad, Ehab A M

2012-06-15

 
 
 
 
221

Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site  

Energy Technology Data Exchange (ETDEWEB)

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by {sup 13}C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using (2-{sup 13}C)PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP and EPSP bound to the enzyme in a ternary complex with phosphate. These peak assignments account for the quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal. The rate of formation of the EPSP ketal was very slow establishing that it is a side product of the normal enzymatic reaction. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, and NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups. All of these results reaffirm identification of the tetrahedral species as the only observable intermediate in the EPSP synthase reaction.

Anderson, K.S.; Sammons, R.D.; Leo, G.C.; Sikorski, J.A. (Monsanto Agricultural Company, St. Louis, MO (USA)); Benesi, A.J.; Johnson, K.A. (Pennsylvania State Univ., University Park (USA))

1990-02-13

222

Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site  

International Nuclear Information System (INIS)

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by 13C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using [2-13C]PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP and EPSP bound to the enzyme in a ternary complex with phosphate. These peak assignments account for the quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal. The rate of formation of the EPSP ketal was very slow establishing that it is a side product of the normal enzymatic reaction. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, and NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups. All of these results reaffirm identification of the tetrahedral species as the only observable intermediate in the EPSP synthase reaction

223

Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in capsicum under drought stress.  

Science.gov (United States)

Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined. PMID:24984087

Phimchan, Paongpetch; Chanthai, Saksit; Bosland, Paul W; Techawongstien, Suchila

2014-07-23

224

Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase  

International Nuclear Information System (INIS)

Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol 32P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ? subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn2+ or Ni2+ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn2+ or Ni2+ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni2+ was a more potent activator of phosphorylated calcineurin compared to Mn2+. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni2+ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca2+

225

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

Science.gov (United States)

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. PMID:24373751

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

2014-03-01

226

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)

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. PMID:15542052

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

2004-12-15

227

Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export:  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol mal...

Zelle, R. M.; Hulster, E.; Winden, W. A.; Waard, P.; Dijkema, C.; Winkler, A. A.; Geertman, J. M.; Dijken, J. P.; Pronk, J. T.; Maris, A. J. A.

2008-01-01

228

Relayed 13C magnetization transfer. Detection of malate dehydrogenase reaction in vivo  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here we report indire...

Yang, Jehoon; Shen, Jun

2007-01-01

229

EDC4 interacts with and regulates the dephospho-CoA kinase activity of CoA synthase.  

Science.gov (United States)

Coenzyme A synthase (CoAsy) is a bifunctional enzyme which facilitates the last two steps of Coenzyme A biogenesis in higher eukaryotes. Here we describe that CoAsy forms a complex with enhancer of mRNA-decapping protein 4 (EDC4), a central scaffold component of processing bodies. CoAsy/EDC4 complex formation is regulated by growth factors and is affected by cellular stresses. EDC4 strongly inhibits the dephospho-CoA kinase activity of CoAsy in vitro. Transient overexpression of EDC4 decreases cell proliferation, and further co-expression of CoAsy diminishes this effect. Here we report that EDC4 might contribute to regulation of CoA biosynthesis in addition to its scaffold function in processing bodies. PMID:22982864

Gudkova, Daria; Panasyuk, Ganna; Nemazanyy, Ivan; Zhyvoloup, Alexander; Monteil, Pascale; Filonenko, Valeriy; Gout, Ivan

2012-10-19

230

Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity.  

Science.gov (United States)

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation. PMID:22452347

Vagstad, Anna L; Bumpus, Stefanie B; Belecki, Katherine; Kelleher, Neil L; Townsend, Craig A

2012-04-18

231

Inhibition of nitric oxide synthase activity improves focal cerebral damage induced by cerebral ischemia/reperfusion in normotensive rats  

Directory of Open Access Journals (Sweden)

Full Text Available Introduction: Nitric oxide seems to play a dual role in ischemia/reperfusion injury. Few studies have investigated whether it exacerbates or improves brain edema. In the present study, we inhibited the activity of nitric oxide synthase by L-NAME and evaluated the cerebral infarct volume, tissue swelling and brain edema, alongside the measurement of blood flow of the ischemic region. Methods: Transient focal cerebral ischemia was induced by 60 min middle cerebral artery occlusion followed by 12 hours reperfusion in rat. Experiments were performed in three groups of rats (n=12 each Sham, control ischemic, and L-NAME pretreated (1 mg/kg IP. Laser Doppler flowmetry was used to measure the regional blood flow. After neurological deficit score (NDS testing, the brains were prepared for TTC staining or brain water content technique to measure the infarct volume and brain edema. Results: Pretreatment with L-NAME significantly reduced NDS (3.66 ± 0.33 to 1.5 ± 0.34, infarct volume of cortex (374 ± 34 to 160 ± 41 mm3 and striatum (158 ± 15 to 87 ± 16 mm3, tissue swelling (7.35 ± 1.27% to 4.05 ± 0.91% and brain edema (3.5 ± 0.48% to 1.6 ± 0.6% without significant alteration of blood flow of the ischemic region. Conclusion: The findings of this study indicate that inhibition of nitric oxide synthase activity reduces infarct volume and brain edema of the ischemic region induced during 60 min middle cerebral artery occlusion. This effect is not accompanied with any alteration in the blood flow of the ischemic region.

Mohammad Taghi Mohammadi

2010-03-01

232

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 induc...

Ehsan Karimi; Jaafar, Hawa Z. E.; Ali Ghasemzadeh

2012-01-01

233

Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin - Functional, evidence against reductive oxygen activation by the pterin cofactor  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Tetrahydrobiopterin ((6R) -5,6,7,8-tetrahydro-L-biopterin (H(4)biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H(4)biopterin, ...

Riethmuller, C.; Gorren, A. C. F.; Pitters, E.; Hemmens, B.; Habisch, H. J.; Heales, S. J. R.; Schmidt, K.; Werner, E. R.; Mayer, B.

1999-01-01

234

Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes  

International Nuclear Information System (INIS)

Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ?g/ml) permeabilized rat adipocytes. Incorporation of 3H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10-9M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different

235

Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes  

Energy Technology Data Exchange (ETDEWEB)

Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ..mu..g/ml) permeabilized rat adipocytes. Incorporation of /sup 3/H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10/sup -9/M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different.

Mooney, R.A.; Wisniewski, J.L.

1986-05-01

236

High-cholesterol diet increases xanthine oxidase and decreases nitric oxide synthase activities in erythrocytes from rats.  

Science.gov (United States)

The aim of this study was to investigate the effects of a high-cholesterol diet on oxidant/antioxidant status and nitric oxide synthase (NOS) activity in erythrocytes from rats. Sixteen Sprague-Dawley-type albino male rats were used in the study. The rats were randomly divided into 2 groups: the control group (group 1) was fed a standard rat diet, and the treated group (group 2) was fed a high-cholesterol diet (4% cholesterol, 1% cholic acid, and 0.5% thiouracil) in addition to standard pellet rat diet for 3 months. At the end of the study period, blood samples were obtained from the rats under ether anesthesia. Oxidant (malondialdehyde level, sensitivity to oxidation value, and xanthine oxidase [XO] activity) and antioxidant parameters (antioxidant potential value, superoxide dismutase, catalase, and glutathione peroxidase activities) were studied in erythrocyte preparations. Activities of erythrocyte NOS and arginase enzymes and serum total cholesterol levels were also measured. We observed that serum total cholesterol levels, erythrocyte XO activities, and sensitivity to oxidation values significantly increased in group 2 (cholesterol fed) compared with the control group (group 1). Erythrocyte NOS activities were also found to decrease in group 2. In conclusion, our results suggest that cholesterol feeding causes an increase in XO activity and a decrease in NOS activity in the erythrocytes from rats. The increase in XO activity may render the erythrocyte membranes sensitive to oxidant stress, and the decrease in NOS activity in the erythrocytes may increase cardiovascular disease risk via reduced endothelial relaxation. PMID:19083410

Devrim, Erdinç; Ergüder, Imge B; Ozbek, Hanefi; Durak, Ilker

2008-03-01

237

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.  

Science.gov (United States)

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas. PMID:25294105

Saigo, Mariana; Golic, Adrián; Alvarez, Clarisa E; Andreo, Carlos S; Hogenhout, Saskia A; Mussi, María A; Drincovich, María F

2014-12-01

238

Role of dietary fish oil on nitric oxide synthase activity and oxidative status in mice red blood cells.  

Science.gov (United States)

The consumption of n-3 polyunsaturated fatty acids (PUFAs) derived from fish oil concomitant with a reduced intake of saturated fats is associated with cardiovascular benefits, which may result from the participation of nitric oxide (NO). In contrast, PUFAs are vulnerable to peroxidation, which could affect the oxidative stability of the cell and reduce NO bioavailability. Therefore, we investigated the effects of high fat diets with increasing amounts of fish oil (0-40% of energy) in place of lard on the l-arginine-NO pathway, the arginase pathway and oxidative status in mice red blood cells (RBC). We found that l-arginine transport, as well as NO synthase (NOS) expression and activity, was enhanced by the highest doses of fish oil (30 and 40%). In contrast, diets rich in lard led to NOS expression and activity impairment. Arginase expression was not significantly affected by any of the dietary regimens. No significant difference in protein and lipid oxidative markers was observed among any of the fish-oil fed mice; only lard feeding induced protein damage in addition to a decreased superoxide dismutase activity. These data suggest that a substantial dose of fish oil, but not low doses, activates the RBC l-arginine-NO pathway without resulting in oxidative damage. PMID:25317541

Martins, Marcela A; Moss, Monique B; Mendes, Iara K S; Águila, Márcia B; Mandarim-de-Lacerda, Carlos Alberto; Brunini, Tatiana M C; Mendes-Ribeiro, Antônio Cláudio

2014-12-01

239

2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.  

Science.gov (United States)

There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

Bitok, J Kipchirchir; Meyers, Caren Freel

2012-10-19

240

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.  

Science.gov (United States)

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors. PMID:21729692

Huang, Xinyi; Hernick, Marcy

2011-10-15

 
 
 
 
241

New insights into activation and substrate recognition of polyhydroxyalkanoate synthase from Ralstonia eutropha.  

Science.gov (United States)

The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC(Re). In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279-6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC(Re) was conducted in the presence of TritonX-100, and PhaC(Re) obeyed Michaelis-Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3'-dephospho CoA had no inhibitory effect. Furthermore, PhaC(Re) showed a considerably reduced reaction rate for 3'-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis-Menten kinetics. These results suggest that the 3'-phosphate group of CoA plays a critical role in substrate recognition by PhaC(Re). PMID:22543354

Ushimaru, Kazunori; Sangiambut, Smith; Thomson, Nicholas; Sivaniah, Easan; Tsuge, Takeharu

2013-02-01

242

Interactions among mitochondrial aspartate aminotransferase, malate dehydrogenase, and the inner mitochondrial membrane from heart, hepatoma, and liver.  

Science.gov (United States)

The inner mitochondrial membranes from bovine heart, rat liver, and Morris hepatoma 7777 all bound the mitochondrial isozymes of aspartate aminotransferase and malate dehydrogenase with comparable affinities and binding ratios (mg of enzyme bound per mg of membrane protein). A low molecular weight fraction separated from a detergent extract of the heart membrane by chromatography on Sephacryl S-300 contained most of the binding activity of the extract for the aminotransferase and had a dissociation constant for the aminotransferase of 0.2 microM. The protein component of the membrane binding sites for the aminotransferase was apparently present in this fraction because binding activity was largely eliminated by proteolysis with trypsin. When this fraction was chromatographed on an aminotransferase affinity column, only the portion that was bound and eluted by 0.25 M KCl associated with added aminotransferase. Unlike the membrane, which was markedly inhibited by the non-ionic detergent Genapol but was inhibited only 20% by trypsin, the binding activity of this subfraction was completely inhibited by trypsin but not by Genapol. This suggests, on the membrane, that the aminotransferase binds to the binding protein and is then transferred to lipids specifically associated with the binding protein. These putative lipids are presumably removed on the affinity column. Although the yield of the binding protein was low, there is probably ample binding protein in mitochondria to accommodate the aminotransferase. In every case, binding of the aminotransferase to the membrane inactivated the malate dehydrogenase binding site whereas malate dehydrogenase had little effect on the binding of the aminotransferase and only associated with the higher molecular weight fractions from the Sephacryl column that contained Complex I activity. Inactivation of the malate dehydrogenase site by the aminotransferase, but not vice versa, could result from aminotransferase associating with the binding protein and malate dehydrogenase with Complex I followed by association of the enzymes with lipids located in the same region of the membrane. However, since aminotransferase is more cationic, it is not displaced readily from the lipids by malate dehydrogenase. The relevance of these interactions to the organization of the enzymes is discussed. PMID:2246239

Teller, J K; Fahien, L A; Valdivia, E

1990-11-15

243

New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs; Nouveaux procedes pour mesurer les activites synthase et phosphatase de la bisphosphoglycerate mutase. Interet pour le developpement de drogues therapeutiques  

Energy Technology Data Exchange (ETDEWEB)

In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

Ravel, P.; Garel, M.C. [Hopital Henri-Mondor, 94 - Creteil (France); Toullec, D. [Laboratoire Glaxo Wellcome, 91- Les Ulis (France)

1997-12-31

244

Contractile Activity Regulates Inducible Nitric Oxide Synthase Expression and NOi Production in Cardiomyocytes via a FAK-Dependent Signaling Pathway  

Science.gov (United States)

Intracellular nitric oxide (NOi) is a physiological regulator of excitation-contraction coupling, but is also involved in the development of cardiac dysfunction during hypertrophy and heart failure. To determine whether contractile activity regulates nitric oxide synthase (NOS) expression, spontaneously contracting, neonatal rat ventricular myocytes (NRVM) were treat with L-type calcium channel blockers (nifedipine and verapamil) or myosin II ATPase inhibitors (butanedione monoxime (BDM) and blebbistatin) to produce contractile arrest. Both types of inhibitors significantly reduced iNOS but not eNOS expression, and also reduced NOi production. Inhibiting contractile activity also reduced focal adhesion kinase (FAK) and AKT phosphorylation. Contraction-induced iNOS expression required FAK and phosphatidylinositol 3-kinase (PI(3)K), as both PF573228 and LY294002 (10??M, 24?h) eliminated contraction-induced iNOS expression. Similarly, shRNAs specific for FAK (shFAK) caused FAK knockdown, reduced AKT phosphorylation at T308 and S473, and reduced iNOS expression. In contrast, shRNA-mediated knockdown of PYK2, the other member of the FAK-family of protein tyrosine kinases, had much less of an effect. Conversely, overexpression of a constitutively active form of FAK (CD2-FAK) or AKT (Myr-AKT) reversed the inhibitory effect of BDM on iNOS expression and NOi production. Thus, contraction-induced iNOS expression and NOi production in NRVM are mediated via a FAK-PI(3)K-AKT signaling pathway. PMID:22900166

Chu, Miensheng; Koshman, Yevgeniya; Iyengar, Rekha; Kim, Taehoon; Russell, Brenda; Samarel, Allen M.

2012-01-01

245

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301 with Thromboxane Synthase Inhibitory Activity in Rats Subjected to Cerebral Ischemia  

Directory of Open Access Journals (Sweden)

Full Text Available ONO-1301 has been developed as a novel long-acting prostacyclin agonist with thromboxane synthase inhibitory activity. In the present study, we investigated the cerebroprotective effect of ONO-1301 on post-ischemic injury induced by cerebral ischemia in rats. ONO-1301 (1 and 10 mg/kg was administrated orally at reperfusion and then twice a day for 42 days. The cell damage induced by cerebral ischemia in the hippocampal CA1 was evaluated using both Nissl staining and proliferating cell nuclear antigen (PCNA staining on the 42 days after cerebral ischemia. Activated astrocytes were evaluated using immunofluorescence staining with GFAP on the 42 days after cerebral ischemia. Spatial learning was assessed using a Morris water maze (MWM task on the 56 days (i.e. after a 14 days washout period. ONO-1301- treated rats (1 and 10 mg/kg significantly improved cell death in the hippocampal CA1, the number of PCNA-positive cells and astrocyte activation. The spatial learning of ONO-1301-treated rats compared with vehicle- treated rats in the MWM task. These results suggest that repeated treatment with oral ONO-1301 could prevent or limit post-ischemic brain damage. In particular, treatment with ONO-1301 within 7 days after ischemia is most effective to improve ischemic damage.

Mai Hazekawa

2011-10-01

246

Relayed 13C magnetization transfer: Detection of malate dehydrogenase reaction in vivo  

Science.gov (United States)

Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9 ± 2 ?mol/g wet weight/min (means ± SD, n = 5) at 11.7 Tesla in anesthetized adult rats infused with [1,6- 13C 2]glucose.

Yang, Jehoon; Shen, Jun

2007-02-01

247

Broussochalcone A, a potent antioxidant and effective suppressor of inducible nitric oxide synthase in lipopolysaccharide-activated macrophages.  

Science.gov (United States)

The antioxidant properties of broussochalcone A (BCA) and its effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages were investigated in this study. BCA, isolated from Broussonetia papyrifera Vent., inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC(50) of 0.63 +/- 0.03 microM. It was as potent as butylated hydroxytoluene, a common antioxidant used for food preservation. In a diphenyl-2-picrylhydrazyl assay system, the radical-scavenging activity of BCA seemed to be more potent than that of alpha-tocopherol, its IC(0.200) being 7.6 +/- 0.8 microM. BCA could directly scavenge superoxide anion and hydroxyl radicals. These results indicated that BCA was a powerful antioxidant with versatile free radical-scavenging activity. On the other hand, we found that BCA suppressed NO production concentration-dependently, with an IC(50) of 11.3 microM in LPS-activated macrophages. This effect was not the consequence of a direct inhibitory action on the enzyme activity of inducible NO synthase (iNOS). Our results indicated that BCA exerts potent inhibitory effects on NO production, apparently mediated by its suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, nuclear factor-kappa B activation, and iNOS expression. Therefore, we conclude that the antioxidant activities of BCA and its inhibition of IkappaBalpha degradation and iNOS protein expression may have therapeutic potential, given that excessive free radicals and NO production have been associated with various inflammatory diseases. PMID:11286985

Cheng, Z; Lin, C; Hwang, T; Teng, C

2001-04-15

248

Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Platelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture. Methods Fetal membranes were collected from Caesarean sections at term. RNA was extracted ...

Seyffarth Gunter; Nelson Paul N; Dunmore Simon J; Rodrigo Nalinda; Murphy Damian J; Carson Ray J

2004-01-01

249

Correction of chronic hyperglycemia with vanadate, but not with phlorizin, normalizes in vivo glycogen repletion and in vitro glycogen synthase activity in diabetic skeletal muscle.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Vanadate has insulin-like activity in vitro and in vivo. To characterize the in vivo mechanism of action of vanadate, we examined meal tolerance, insulin-mediated glucose disposal, in vivo liver and muscle glycogen synthesis, and in vitro glycogen synthase activity in 90% partially pancreatectomized rats. Four groups were studied: group I, sham-operated controls; group II, diabetic rats; group III, diabetic rats treated with vanadate; and group IV, diabetic rats treated with phlorizin. Insuli...

Rossetti, L.; Lauglin, M. R.

1989-01-01

250

Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation. ?Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation,...

Kimura, H.; Hokari, R.; Miura, S.; Shigematsu, T.; Hirokawa, M.; Akiba, Y.; Kurose, I.; Higuchi, H.; Fujimori, H.; Tsuzuki, Y.; Serizawa, H.; Ishii, H.

1998-01-01

251

Significance of redox-active cysteines in human FAD synthase isoform 2.  

Science.gov (United States)

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain. PMID:25135855

Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

2014-08-15

252

Changes in lactate dehydrogenase and malate dehydrogenase activities during hypoxia and after temperature acclimation in the armored fish, Rhinelepis strigosa (Siluriformes, Loricariidae) / Mudanças na atividade da lactato desidrogenase e malato desidrogenase durante hipóxia e após aclimatação a diferentes temperaturas no cascudo, Rhinelepis strigosa (Siluriformes, Loricariidae)  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Foram estudadas a lactato desidrogenase (LDH) e malato desidrogenase (MDH) de músculo branco de peixes aclimatados a 20, 25 e 30°C em diferentes concentrações de substrato e submetidos à hipóxia. Diferenças significativas em atividade enzimática para LDH e MDH são descritas aqui pela primeira vez em [...] Rhinelepis strigosa em peixes aclimatados a 20°C e submetidos à hipóxia por seis horas. A LDH apresentou uma diminuição significativa na afinidade enzimática ao piruvato em hipóxia severa de peixes aclimatados a 20°C e um aumento significativo na afinidade enzimática ao piruvato em peixes aclimatados a 30°C. Abstract in english Lactate (LDH) and malate dehydrogenase (MDH) of white skeletal muscle of fishes acclimated to 20, 25 and 30°C and thereafter submitted to hypoxia were studied in different substrate concentrations. Significant differences for LDH and MDH of white muscle enzyme activities are described here for the f [...] irst time in Rhinelepis strigosa of fishes acclimated to 20°C and submitted to hypoxia for six hours. LDH presented a significant decrease in enzyme affinity for pyruvate in acute hypoxia, for fishes acclimated to 20°C and an increase for fishes acclimated to 30°C.

L., PANEPUCCI; M. N., FERNANDES; J. R., SANCHES; F. T., RANTIN.

2000-05-01

253

Changes in lactate dehydrogenase and malate dehydrogenase activities during hypoxia and after temperature acclimation in the armored fish, Rhinelepis strigosa (Siluriformes, Loricariidae Mudanças na atividade da lactato desidrogenase e malato desidrogenase durante hipóxia e após aclimatação a diferentes temperaturas no cascudo, Rhinelepis strigosa (Siluriformes, Loricariidae  

Directory of Open Access Journals (Sweden)

Full Text Available Lactate (LDH and malate dehydrogenase (MDH of white skeletal muscle of fishes acclimated to 20, 25 and 30°C and thereafter submitted to hypoxia were studied in different substrate concentrations. Significant differences for LDH and MDH of white muscle enzyme activities are described here for the first time in Rhinelepis strigosa of fishes acclimated to 20°C and submitted to hypoxia for six hours. LDH presented a significant decrease in enzyme affinity for pyruvate in acute hypoxia, for fishes acclimated to 20°C and an increase for fishes acclimated to 30°C.Foram estudadas a lactato desidrogenase (LDH e malato desidrogenase (MDH de músculo branco de peixes aclimatados a 20, 25 e 30°C em diferentes concentrações de substrato e submetidos à hipóxia. Diferenças significativas em atividade enzimática para LDH e MDH são descritas aqui pela primeira vez em Rhinelepis strigosa em peixes aclimatados a 20°C e submetidos à hipóxia por seis horas. A LDH apresentou uma diminuição significativa na afinidade enzimática ao piruvato em hipóxia severa de peixes aclimatados a 20°C e um aumento significativo na afinidade enzimática ao piruvato em peixes aclimatados a 30°C.

L. PANEPUCCI

2000-05-01

254

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

Science.gov (United States)

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

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

2002-10-15

255

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

DEFF Research Database (Denmark)

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<0.01) and normal glycogen (89.5±7.6 vs 152.6±8.1 mmol•kg(-1); p<0.01), but not significantly in muscles with low glycogen (90.0±5.0 vs 102.8±7.8 mmol•kg(-1); 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 withhigh compared to low glycogen. GS site 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where adrenaline decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where adrenaline injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independent of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where adrenaline injection decreased glycogen content. In conclusion, adrenaline stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low glycogen content. Adrenaline-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content.

Kolnes, Anders J; Birk, Jesper Bratz

2015-01-01

256

Adrenaline-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles  

DEFF Research Database (Denmark)

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<0.01) and normal glycogen (89.5±7.6 vs 152.6±8.1 mmol•kg(-1); p<0.01), but not significantly in muscles with low glycogen (90.0±5.0 vs 102.8±7.8 mmol•kg(-1); 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 withhigh compared to low glycogen. GS site 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where adrenaline decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where adrenaline injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independent of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where adrenaline injection decreased glycogen content. In conclusion, adrenaline stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low glycogen content. Adrenaline-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content.

Kolnes, Anders J; Birk, Jesper Bratz

2015-01-01

257

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

Science.gov (United States)

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 < 0.01) and normal glycogen (89.5 ± 7.6 vs. 152 ± 8.1 mmol/kg, P < 0.01), but not significantly in 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. PMID:25465888

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

2015-02-01

258

Changes in the Profile of NO Synthases Affect Coronary Blood Flow Autoregulation and Myocardial Contractile Activity during Restraint Stress in Rats.  

Science.gov (United States)

The efficiency of autoregulation of the coronary blood flow and contractile activity of the myocardium in the presence of inhibitors of constitutive and inducible NO synthases was studied in rats exposed to 6-h restraint stress. Intracoronary administration of S-methylisothiourea (10 ?mol/liter), but not L-NAME (60 ?mol/liter) fully prevented post-stress increase in the volume coronary blood flow rate, intensity of heart perfusion, and reduction of ventricular developed pressure at all levels of perfusion pressure. Real-time PCR showed 6-fold increased expression of inducible NO-synthase mRNA in the heart tissue against the background of unchanged expression of neuronal and endothelial NO synthases and 2-3-fold elevated content of transcripts of stress-inducible genes Hspa1a and Hspbp1. It was shown that the hypotension of coronary vessels and reduced contractile function of the myocardium are related to NO production by inducible NO synthase in endotheliocytes of coronary vessels and cardiomyocytes. PMID:25430647

Solodkov, A P; Lazuko, S S; Knyazev, E N; Nechaev, I N; Krainova, N A

2014-12-01

259

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis  

DEFF Research Database (Denmark)

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger

2007-01-01

260

Inter-domain Communication of Human Cystathionine ?-Synthase: STRUCTURAL BASIS OF S-ADENOSYL-l-METHIONINE ACTIVATION.  

Science.gov (United States)

Cystathionine ?-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis. PMID:25336647

McCorvie, Thomas J; Kopec, Jolanta; Hyung, Suk-Joon; Fitzpatrick, Fiona; Feng, Xidong; Termine, Daniel; Strain-Damerell, Claire; Vollmar, Melanie; Fleming, James; Janz, Jay M; Bulawa, Christine; Yue, Wyatt W

2014-12-26

 
 
 
 
261

Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.  

Science.gov (United States)

MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family. PMID:22607697

Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

2012-06-19

262

The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells.  

Science.gov (United States)

Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca2+ level, and BAPTA-AM, a Ca2+ chelator, reduced the Ca2+ surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production. PMID:25643147

Maiwulanjiang, Maitinuer; Bi, Cathy W C; Lee, Pinky S C; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M; Xiong, Aizhen; Li, Ning; Dong, Tina T X; Aisa, Haji A; Tsim, Karl W K

2015-01-01

263

Radiochemical nitric oxide synthase activity determination in rat brain with fast and accurate HPLC analysis.  

Science.gov (United States)

In the central nervous system, NOS activity is involved in several physiological events, such as refinement of afferent connections in development, or linking cerebral blood flow to neural activity in adulthood, and also in many pathological events, such as cell death in brain ischemia and regulation of vasospasm in hemorrhage. Therefore, we studied NOS activity in the CNS. We describe a fast and accurate method in which we use HPLC analysis to identify and quantify citrulline eluted by ion-exchange chromatography, thus implementing the current method to evaluate NOS activity. This technique could be readily applied for NOS activity determination not only in brain, but also in all other tissues. PMID:11356384

Repici, M; Cabella, C; Colombatto, S; Vercelli, A

2001-06-01

264

Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase.  

Science.gov (United States)

Reactive oxygen species (ROS) such as superoxide (O2*-) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Activation of NADPH-oxidase leads to dilatation of the basilar artery in vivo via production of H2O2, but the endogenous stimuli for this unique vasodilator mechanism are unknown. Shear stress is known to activate both NADPH-oxidase and phosphatidylinositol-3 kinase (PI3-K) in cultured cells. Hence, this study used a cranial window preparation in anesthetized rats to investigate whether increased intraluminal blood flow could induce cerebral vasodilatation via the activation of NADPH-oxidase and/or PI3-K. Bilateral occlusion of the common carotid arteries to increase basilar artery blood flow caused reproducible, reversible vasodilatation. Topical treatment of the basilar artery with the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) (0.5 and 5 micromol/L) inhibited flow-induced dilatation by up to 50% without affecting dilator responses to acetylcholine. Treatment with the H2O2 scavenger, catalase similarly attenuated flow-induced dilatation, suggesting a role for NADPH-oxidase-derived H2O2 in this response. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) partially reduced flow-induced dilatation, and combined treatment with a ROS inhibitor (DPI or catalase) and L-NAME caused a greater reduction in flow-induced dilatation than that seen with any of these inhibitors alone. Flow-induced dilatation was also markedly inhibited by the PI3-K inhibitor, wortmannin. Increased O2*- production in the endothelium of the basilar artery during acute increases in blood flow was confirmed using dihydroethidium. Thus, flow-induced cerebral vasodilatation in vivo involves production of ROS and nitric oxide, and is dependent on PI3-K activation. PMID:16222243

Paravicini, Tamara M; Miller, Alyson A; Drummond, Grant R; Sobey, Christopher G

2006-06-01

265

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats.  

Science.gov (United States)

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload. PMID:17890423

Loyer, Xavier; Oliviero, Patricia; Damy, Thibaud; Robidel, Estelle; Marotte, Françoise; Heymes, Christophe; Samuel, Jane-Lise

2007-11-01

266

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity  

Science.gov (United States)

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10(-8) M Dex reduced PGF2 alpha production 55-65% and PGE2 production 84-90% after 24-72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF2 alpha efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF2 alpha production 162% after 24 h, returning PGF2 alpha efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF2 alpha efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8-24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production.

Chromiak, J. A.; Vandenburgh, H. H.

1994-01-01

267

Coagulase-negative Staphylococci favor conversion of arginine into ornithine despite a widespread genetic potential for nitric oxide synthase activity.  

Science.gov (United States)

Within ecosystems that are poor in carbohydrates, alternative substrates such as arginine may be of importance to coagulase-negative staphylococci (CNS). However, the versatility of arginine conversion in CNS remains largely uncharted. Therefore, a set of 86 strains belonging to 17 CNS species was screened for arginine deiminase (ADI), arginase, and nitric oxide synthase (NOS) activities, in view of their ecological relevance. In fermented meats, for instance, ADI could improve bacterial competitiveness, whereas NOS may serve as an alternative nitrosomyoglobin generator to nitrate and nitrite curing. About 80% of the strains were able to convert arginine, but considerable inter- and intraspecies heterogeneity regarding the extent and mechanism of conversion was found. Overall, ADI was the most commonly employed pathway, resulting in mixtures of ornithine and small amounts of citrulline. Under aerobic conditions, which are more relevant for skin-associated CNS communities, several strains shifted toward arginase activity, leading to the production of ornithine and urea. The obtained data indeed suggest that arginase occurs relatively more in CNS isolates from a dairy environment, whereas ADI seems to be more abundant in strains from a fermented meat background. With some exceptions, a reasonable match between phenotypic ADI and arginase activity and the presence of the encoding genes (arcA and arg) was found. With respect to the NOS pathway, however, only one strain (Staphylococcus haemolyticus G110) displayed phenotypic NOS-like activity under aerobic conditions, despite a wide prevalence of the NOS-encoding gene (nos) among CNS. Hence, the group of CNS displays a strain- and condition-dependent toolbox of arginine-converting mechanisms with potential implications for competitiveness and functionality. PMID:25281381

Sánchez Mainar, María; Weckx, Stefan; Leroy, Frédéric

2014-12-01

268

Active site binding and catalytic role of bicarbonate in 1,4-dihydroxy-2-naphthoyl coenzyme A synthases from vitamin K biosynthetic pathways.  

Science.gov (United States)

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes a carbon-carbon bond formation reaction in the biosynthesis of both vitamin K1 and K2. Bicarbonate is crucial to the activity of a large subset of its orthologues but lacks a clearly defined structural and mechanistic role. Here we determine the crystal structure of the holoenzymes from Escherichia coli at 2.30 Å and Synechocystis sp. PCC6803 at 2.04 Å, in which the bicarbonate cofactor is bound to the enzyme active site at a position equivalent to that of the side chain carboxylate of an aspartate residue conserved among bicarbonate-insensitive DHNA-CoA synthases. Binding of the planar anion involves both nonspecific electrostatic attraction and specific hydrogen bonding and hydrophobic interactions. In the absence of bicarbonate, the anion binding site is occupied by a chloride ion or nitrate, an inhibitor directly competing with bicarbonate. These results provide a solid structural basis for the bicarbonate dependence of the enzymatic activity of type I DHNA-CoA synthases. The unique location of the bicarbonate ion in relation to the expected position of the substrate ?-proton in the enzyme's active site suggests a critical catalytic role for the anionic cofactor as a catalytic base in enolate formation. PMID:22606952

Sun, Yueru; Song, Haigang; Li, Jie; Jiang, Ming; Li, Yan; Zhou, Jiahai; Guo, Zhihong

2012-06-01

269

Radiochemical nitric oxide synthase activity determination in rat brain with fast and accurate HPLC analysis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In the central nervous system, NOS activity is involved in several physiological events, such as refinement of afferent connections in development, or linking cerebral blood flow to neural activity in adulthood, and also in many pathological events, such as cell death in brain ischemia and regulation of vasospasm in hemorrhage. Therefore, we studied NOS activity in the CNS. We describe a fast and accurate method in which we use HPLC analysis to identify and quantify citrulline eluted by ion-e...

Repici, Mariaelena; Vercelli, Alessandro; Colombatto, Sebastiano; Cabella, Claudia

2001-01-01

270

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cystathionine ?-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 ?L of plasma using a stable isotope substrate - 2,3,3-2H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-2H-cystathionine, using L...

Krijt, Jakub; Kopecka?, Jana; Hni?zda, Ales?; Moat, Stuart; Kluijtmans, Leo A. J.; Mayne, Philip; Koz?ich, Viktor

2011-01-01

271

The chimeric gene linked to glucocorticoid-suppressible hyperaldosteronism encodes a fused P-450 protein possessing aldosterone synthase activity.  

Science.gov (United States)

Glucocorticoid-suppressible hyperaldosteronism (GSH) is one variety of primary aldosteronism with hypertension and is inherited in an autosomal dominant mode. A recent report has indicated that GSH is caused by a gene duplication arising from unequal crossing over between the two genes, CYP11B1 and CYP11B2, encoding P-450(11 beta) and P-450C18, respectively (Lifton et al. Nature (1992) 355, 262-265). The nucleotide sequence analysis in the present study has demonstrated that unequal crossing over in the chimeric gene formed by the gene duplication occurs within the region from the 3'-portion of exon 4 through the 5'-portion of intron 4 in Australian GSH patients. Namely, the chimeric gene encodes a fused P-450 protein consisting of the amino-terminal side of P-450(11 beta) (encoded by exons 1-4 of CYP11B1) and the carboxyl-terminal side of P-450C18 (encoded by exons 5-9 of CYP11B2). When a cDNA corresponding to the chimeric gene is transfected into COS-7 cells, the fused P-450 protein expressed in the mitochondria exhibits steroid 18-hydroxylase or aldosterone synthase activity. These results provide the molecular genetic basis for the characteristic biochemical phenotype of GSH patients. PMID:1472060

Miyahara, K; Kawamoto, T; Mitsuuchi, Y; Toda, K; Imura, H; Gordon, R D; Shizuta, Y

1992-12-15

272

Nitric oxide synthase-dependent NADPH-diaphorase activity in the optic lobes of male and female Ceratitis capitata mutants  

Directory of Open Access Journals (Sweden)

Full Text Available Nitric oxide (NO is acknowledged as a messenger molecule in the nervous system with a pivotal role in the modulation of the chemosensory information. It has been shown to be present in the optic lobes of several insect species. In the present study, we used males and females from four different strains of the medfly Ceratitis capitata (Diptera, Tephritidae: or; or,wp (both orange eyed; w,M360 and w,Heraklion (both white eyed, as models to further clarify the involvement of NO in the mutants’ visual system and differences in its activity and localization in the sexes. Comparison of the localization pattern of NO synthase (NOS, through NADPH-diaphorase (NADPHd staining, in the optic lobes of the four strains, revealed a stronger reaction intensity in the retina and in the neuropile region lamina than in medulla and lobula. Interestingly, the intensity of NADPHd staining differs, at least in some strains, in the optic lobes of the two sexes; all the areas are generally strongly labelled in the males of the or and w,M360 strains, whereas the w,Heraklion and or,wp mutants do not show evident sexdependent NADPHd staining. Taken as a whole, our data point to NO as a likely transmitter candidate in the visual information processes in insects, with a possible correlation among NOS distribution, eye pigmentation and visual function in C. capitata males. Moreover, NO could influence behavioural differences linked to vision in the two sexes.

E Roda

2009-06-01

273

Distinct parts of leukotriene C{sub 4} synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein  

Energy Technology Data Exchange (ETDEWEB)

Leukotriene C{sub 4} is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C{sub 4} synthase (LTC{sub 4}S) participate in its biosynthesis. We report evidence that LTC{sub 4}S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC{sub 4}S. FLAP bound to the N-terminal part/first hydrophobic region of LTC{sub 4}S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC{sub 4}S. Fluorescent FLAP- and LTC{sub 4}S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC{sub 4}S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC{sub 4}S. Direct interaction of 5-LO and LTC{sub 4}S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC{sub 4}S.

Strid, Tobias; Svartz, Jesper; Franck, Niclas; Hallin, Elisabeth; Ingelsson, Bjoern; Soederstroem, Mats [Division of Cell biology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-58185 Linkoeping (Sweden); Hammarstroem, Sven, E-mail: sven.hammarstrom@liu.se [Division of Cell biology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-58185 Linkoeping (Sweden)

2009-04-17

274

Citrus Flavonoids Luteolin, Apigenin, and Quercetin Inhibit Glycogen Synthase Kinase-3? Enzymatic Activity by Lowering the Interaction Energy Within the Binding Cavity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Pancreatic cancer studies have shown that inhibition of glycogen synthase kinase-3? (GSK-3?) leads to decreased cancer cell proliferation and survival by abrogating nuclear factor ?B (NF?B) activity. In this investigation, various citrus compounds, including flavonoids, phenolic acids, and limonoids, were individually investigated for their inhibitory effects on GSK-3? by using a luminescence assay. Of the 22 citrus compounds tested, the flavonoids luteolin, apigenin, and quercetin had t...

Johnson, Jodee L.; Rupasinghe, Sanjeewa G.; Stefani, Felicia; Schuler, Mary A.; Mejia, Elvira Gonzalez

2011-01-01

275

Impact of Trans-Resveratrol-Sulfates and -Glucuronides on Endothelial Nitric Oxide Synthase Activity, Nitric Oxide Release and Intracellular Reactive Oxygen Species  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives...

Angela Ladurner; Daniel Schachner; Katharina Schueller; Marc Pignitter; Heiss, Elke H.; Veronika Somoza; Dirsch, Verena M.

2014-01-01

276

Beneficial effects of antioxidants and l-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by ...

Nigris, Filomena; Lerman, Lilach O.; Ignarro, Sharon Williams; Sica, Giacomo; Lerman, Amir; Palinski, Wulf; Ignarro, Louis J.; Napoli, Claudio

2003-01-01

277

Myocardial calcium-independent nitric oxide synthase activity is present in dilated cardiomyopathy, myocarditis, and postpartum cardiomyopathy but not in ischaemic or valvar heart disease.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

OBJECTIVE--To determine the activity of the calcium-dependent constitutive (cNOS) and calcium-independent inducible nitric oxide (iNOS) synthases in heart tissue from patients with different cardiac diseases. PATIENTS AND DESIGN--Endomyocardial biopsy specimens were obtained from patients with dilated hearts (by echocardiography and ventriculography) and normal coronary arteries (by selective angiography). Recognised clinical, radiological, and histopathological criteria were used to diagnose...

Belder, A. J.; Radomski, M. W.; Why, H. J.; Richardson, P. J.; Martin, J. F.

1995-01-01

278

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

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

279

Unleashing hydrogenase activity in carbon monoxide dehydrogenase/acetyl-CoA synthase and pyruvate:ferredoxin oxidoreductase.  

Science.gov (United States)

These results demonstrate that two well-studied metalloenzymes, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and pyruvate:ferredoxin oxidoreductase (PFOR), can reduce protons to H2 and, at much lower rates, oxidize H2 to protons and electrons. To our knowledge, this if the first time that PFOR has been shown to have hydrogenase activity. CODH/ACS and PFOR evolved H2 at maximum rates when CO and pyruvate were the electron donors, respectively, and when electron acceptors are absent; dithionite was a very poor substitute. PFOR, when purified to greater than 99% homogeneity, exhibited a specific activity for pyruvate-dependent H2 production of 135 nmol min-1 mg-1. The H2 evolution activity divided by the H2 uptake activity was 282:1; the highest ratio previously reported (22:1) was with the membrane-bound hydrogenase from Rhodospirillum rubrum [Fox, J.D., Kerby, R. L., Roberts, G. P., & Ludden, P. W. (1996) J. Bacteriol. 178, 1515-1524]. Highly purified samples of CODH/ACS (> 99% homogeneity) exhibited a specific activity of CO-dependent H2 evolution in the absence of electron carrier of 590 nmol min-1 mg-1. Equivalent rates of CO oxidation and H2 production were observed when determined in the absence of electron acceptor. This level of activity can account for the rate of H2 production that has been observed by growing cultures of Clostridium thermoaceticum and could solve the paradox that the highly CO-sensitive hydrogenases from acetogenic bacteria evolve H2 when grown on CO. The ratio of the rates of (H2 evolution):(H2 uptake) for purified CODH/ACS is between 20:1 and 30:1. H2 evolution and uptake by CODH/ACS were strongly inhibited by cyanide (ki = 1 microM), indicating that these reactions are catalyzed by cluster C, the site of CO oxidation. Our results extend earlier findings that the CODHs from Methanosarcina barkeri [Bhatnagar, L., Krzycki, J. A., & Zeikus, J. G. (1987) FEMS Microbiol. Lett. 41, 337-343] and Oligotropha carboxydovorans [Santiago, B., & Meyer, O. (1996) FEMS Microbiol. Lett. 136, 157-162] exhibit hydrogenase activity. Mechanistic implications of hydrogenase activity are discussed. Several physiological roles for proton reduction by CODH/ACS and PFOR are discussed, including the prevention of radical formation from reduced metal clusters when electron carriers (ferredoxin, flavodoxin, etc.) are limiting. PMID:8961945

Menon, S; Ragsdale, S W

1996-12-10

280

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

Science.gov (United States)

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. PMID:24060453

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

 
 
 
 
281

Inducible nitric oxide synthase activity does not contribute to the maintenance of peripheral vascular tone in patients with heart failure.  

Science.gov (United States)

Enhanced iNOS (inducible nitric oxide synthase) activity may contribute to vascular dysfunction in patients with heart failure. In the present study, we aimed to determine whether iNOS activity contributes to the maintenance of vascular tone in patients with symptomatic heart failure with the use of the highly selective iNOS inhibitor 1400W {N-[3-(aminomethyl)benzyl] acetamidine}. Bilateral forearm blood flow was measured using venous occlusion plethysmography in 12 patients with New York Heart Association class II-IV heart failure and eight matched healthy control subjects during intra-brachial infusion of 1400W (0.1-1 micromol/min), L-NMMA (N(G)-monomethyl-L-arginine; a non-selective NOS inhibitor; 2-8 micromol/min) and noradrenaline (control vasoconstrictor; 60-480 pmol/min). In both patients and controls, intra-brachial infusion of L-NMMA and noradrenaline caused a dose-dependent reduction in infused forearm blood flow (P<0.05 for both): peak reduction of 32+/-6% and 37+/-4% during L-NMMA and 52+/-6% and 49+/-5% during noradrenaline respectively (P values were not significant when patients were compared with controls). In contrast, 1400W had no effect on blood flow at 1 micromol/min [-3+/-4% in patients (95% confidence intervals, -11 to 5%) and 3+/-8% in controls; P value was not significant]. In conclusion, we have demonstrated that intrabrachial selective iNOS inhibition does not influence forearm blood flow in patients with heart failure. We conclude that iNOS activity does not contribute to peripheral vascular tone in patients with symptomatic heart failure. PMID:16803456

Dover, Anna R; Chia, Stanley; Ferguson, James W; Cruden, Nicholas L; Megson, Ian L; Fox, Keith A A; Newby, David E

2006-10-01

282

Calcium(II)3 (3,5-Diisopropylsalicylate)6(H2O)6 Activates Nitric Oxide Synthase: An Accounting for its Action in Decreasing Platelet Aggregation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Purposes of these studies were first; to determine whether or not Calcium(II)3 (3,5- diisopropylsalicylate)6(H2O)6 [Ca(II)3(3,5-DIPS)6], a lipophilic calcium complex, could decrease activated-platelet aggregation, and second; to determine whether or not it is plausible that Ca(II)3(3,5-DIPS)6 decreases activated-platelet aggregation by facilitating the synthesis of Nitric Oxide (NO) by Nitric Oxide Synthase (NOS). The influence of Ca(II)3(3,5-DIPS)6 on the initial rate of activ...

Donham, Douglas C.; Sorenson, John R. J.

2000-01-01

283

Phenylalanine ammonia-lyase, flavanone 3?-hydroxylase and flavonol synthase enzyme activity by a new in vitro assay method in berry fruits.  

Science.gov (United States)

An HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3?-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method considered kinetic studies to establish the incubation time to perform the assay. The method here described proved to be an interesting approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods. The enzyme activity method developed was applied to strawberry, raspberry, blackberry, redcurrant and blackcurrant fruits. PMID:24491710

Flores, Gema; De la Peña Moreno, Fernando; Blanch, Gracia Patricia; Del Castillo, Maria Luisa Ruiz

2014-06-15

284

A sesquiterpene, dehydrocostus lactone, inhibits the expression of inducible nitric oxide synthase and TNF-alpha in LPS-activated macrophages.  

Science.gov (United States)

Nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) are the major mediators produced in activated macrophages which contribute to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (dehydrocostus lactone) isolated from the medicinal plant, Saussurea lappa, inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW 264.7 cells by suppressing inducible nitric oxide synthase enzyme expression. This compound also decreased the TNF-alpha level in LPS-activated systems in vitro and in vivo. Thus, dehydrocostus lactone may be a possible candidate for the development of new drugs to treat endotoxemia accompanied by the overproduction of NO and TNF-alpha. PMID:10193198

Lee, H J; Kim, N Y; Jang, M K; Son, H J; Kim, K M; Sohn, D H; Lee, S H; Ryu, J H

1999-03-01

285

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase  

International Nuclear Information System (INIS)

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds o(by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15

286

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase  

Energy Technology Data Exchange (ETDEWEB)

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.

Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

1988-08-25

287

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.  

Science.gov (United States)

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved. PMID:18344340

Zelle, Rintze M; de Hulster, Erik; van Winden, Wouter A; de Waard, Pieter; Dijkema, Cor; Winkler, Aaron A; Geertman, Jan-Maarten A; van Dijken, Johannes P; Pronk, Jack T; van Maris, Antonius J A

2008-05-01

288

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  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese OBJETIVO: Um amplo corpo de evidência oriundo de estudos experimentais indica que a sepse se associa com um aumento da produção de espécies de oxigênio reativo, depleção de antioxidantes, e acúmulo de marcadores de estresse oxidativo. Além disto, a disfunção mitocondrial foi implicada na patogênese [...] da síndrome de disfunção de múltiplos órgãos. A citrato sintase é uma enzima que se localiza no interior das células, na matriz mitocondrial, sendo uma etapa importante do ciclo de Krebs; esta enzima foi utilizada como um marcador enzimático quantitativo da presença de mitocôndrias intactas. Assim, investigamos a atividade da citrato sintase no cérebro de ratos submetidos ao modelo sepse com de ligadura e punção do ceco. MÉTODOS: Em diferentes horários (3, 6, 12, 24 e 48 horas) após cirurgia de ligadura e punção do ceco, seis ratos foram sacrificados por decapitação, sendo seus cérebros removidos e dissecados o hipocampo, estriato, cerebelo, córtex cerebral e córtex pré-frontal, e utilizados para determinação da atividade de citrato sintase. RESULTADOS: Verificamos que a atividade de citrato sintase no córtex pré-frontal estava inibida após 12, 24 e 48 horas da ligadura e punção do ceco. No córtex cerebral, esta atividade estava inibida após 3, 12, 24 e 48 horas da ligadura e punção do ceco. Por outro lado a citrato sintase não foi afetada no hipocampo, estriato e cerebelo até 48 horas após a ligadura e punção do ceco. CONCLUSÃO: Considerando-se que é bem descrito o comprometimento da energia decorrente da disfunção mitocondrial na sepse, e que o estresse oxidativo desempenha um papel essencial no desenvolvimento da sepse, acreditamos que o comprometimento da energia pode também estar evolvido nestes processos. Se a inibição da citrato sintase também ocorre em um modelo de sepse, é tentador especular que a redução do metabolismo cerebral pode provavelmente estar relacionada com a fisiopatologia desta doença. Abstract in english 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 pathoge [...] nesis 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.

Giselli, Scaini; Natália, Rochi; Joana, Benedet; Gabriela Kozuchovski, Ferreira; Brena Pereira, Teodorak; Clarissa Martinelli, Comim; Larissa de Souza, Constantino; Francieli, Vuolo; Leandra Celso, Constantino; João, Quevedo; Emilio Luiz, Streck; Felipe, Dal-Pizzol.

2011-06-01

289

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.  

Science.gov (United States)

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME. PMID:1291764

Banu, M J; Nellaiappan, K; Dhandayuthapani, S

1992-06-01

290

Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Asparagus Spears During Storage  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We held asparagus (Asparagus officinalis L.) spears at 25?C for up to 5 days after harvest and examined changes in soluble carbohydrates and the activities of enzymes concerned with carbohydrate breakdown in both top and bottom portions of the spears. The acid invertase in soluble fraction showed a higher activity than that in cell wall bound fraction and the top portion of the spear showed a significantly higher soluble acid invertase activity than the bottom portion. But the activi...

Pankaj Kumar Bhowmik; Toshiyuki Matsui; Fabio Gimena Enriquez; Shameem Alam, A. K. M.; Kazuhide Kawada

2001-01-01

291

Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kB activation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug-resistant malaria. In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF-kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine-stimulated human astrocytoma T67 cells. These results suggest that artemisinin, in addition to its antiparasitic propertie...

Ghigo, Dario Antonio; Bosia, Amalia; Bergandi, Loredana; Aldieri, Elisabetta; Riganti, Chiara; Costamagna, Costanzo

2003-01-01

292

Pleofungins, novel inositol phosphorylceramide synthase inhibitors, from Phoma sp. SANK 13899. I. Taxonomy, fermentation, isolation, and biological activities.  

Science.gov (United States)

In the course of a screening for inositol phosphorylceramide (IPC) synthase inhibitors, the novel inhibitors pleofungins A, B, C, and D were found in a mycelial extract of a fungus, Phoma sp. SANK13899. Purification was performed by 50% methanol and ethyl acetate extraction, reversed phase open-column chromatography, and HPLC separations. Pleofungin A inhibited the IPC synthase of Saccharomyces cerevisiae and Aspergillus fumigatus at IC(50) values of 16 and 1.0 ng/ml, respectively. The inhibitor also suppressed the growth of Candida albicans, Cryptococcus neoformans, and A. fumigatus at MIC values of 2.0, 0.3, and 0.5 mug/ml, respectively. These biological properties indicate that pleofungins belong to a novel class of IPC synthase inhibitors efficacious against A. fumigatus. PMID:17420564

Yano, Tatsuya; Aoyagi, Azusa; Kozuma, Shiho; Kawamura, Yoko; Tanaka, Isshin; Suzuki, Yasuhiro; Takamatsu, Yasuyuki; Takatsu, Toshio; Inukai, Masatoshi

2007-02-01

293

Inhibition of p38 mitogen-activated protein kinase enhances c-Jun N-terminal kinase activity: Implication in inducible nitric oxide synthase expression  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Nitric oxide (NO is an inflammatory mediator, which acts as a cytotoxic agent and modulates immune responses and inflammation. p38 mitogen-activated protein kinase (MAPK signal transduction pathway is activated by chemical and physical stress and regulates immune responses. Previous studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli. The aim of the present study was to investigate the mechanisms involved in the regulation of inducible NO synthesis by p38 MAPK pathway. Results p38 MAPK inhibitors SB203580 and SB220025 stimulated lipopolysaccharide (LPS-induced inducible nitric oxide synthase (iNOS expression and NO production in J774.2 murine macrophages. Increased iNOS mRNA expression was associated with reduced degradation of iNOS mRNA. Treatment with SB220025 increased also LPS-induced c-Jun N-terminal kinase (JNK activity. Interestingly, JNK inhibitor SP600125 reversed the effect of SB220025 on LPS-induced iNOS mRNA expression and NO production. Conclusion The results suggest that inhibition of p38 MAPK by SB220025 results in increased JNK activity, which leads to stabilisation of iNOS mRNA, to enhanced iNOS expression and to increased NO production.

Kankaanranta Hannu

2006-02-01

294

PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement fo...

Mccool, Gabriel J.; Cannon, Maura C.

2001-01-01

295

Assembly and Activation of the Plasmodial Pyridoxal 5’-Phosphate Synthase Complex. Understanding the Structural Mechanism of PLP Biosynthesis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Biosynthesis of vitamin B6 is essential for all living cells. Most organisms use the pyridoxal 5’-phosphate (PLP) synthase complex to synthesize the cofactor form, PLP, from the three substrates ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P) and ammonia. PLP synthase complex is a glutamine amidotransferase (GATase) class I, consisting of 12 Pdx1 and 12 Pdx2 subunits. Pdx1 is responsible for the PLP synthesis and Pdx2 is the glutaminase that hydrolyses glutamine to produce ammoni...

Gue?dez Rodri?guez, Gabriela Liuvanova

2011-01-01

296

DksA-Dependent Resistance of Salmonella enterica Serovar Typhimurium against the Antimicrobial Activity of Inducible Nitric Oxide Synthase  

Science.gov (United States)

In coordination with the ppGpp alarmone, the RNA polymerase regulatory protein DksA controls the stringent response of eubacteria, negatively regulating transcription of translational machinery and directly activating amino acid promoters and de novo amino acid biosynthesis. Given the effects of nitric oxide (NO) on amino acid biosynthetic pathways and the intimate relationship of DksA with amino acid synthesis and transport, we tested whether DksA contributes to the resistance of Salmonella to reactive nitrogen species (RNS). Our studies show that the zinc finger predicted to position DksA in the secondary channel of the RNA polymerase is essential for the resistance of Salmonella enterica serovar Typhimurium to RNS in a murine model of systemic salmonellosis. Despite exhibiting auxotrophies for various amino acids, ?dksA mutant Salmonella strains regain virulence in mice lacking inducible NO synthase (iNOS). DksA is also important for growth of this intracellular pathogen in the presence of NO congeners generated by iNOS during the innate response of murine macrophages. Accordingly, dksA mutant Salmonella strains are hypersusceptible to chemically generated NO, a phenotype that can be prevented by adding amino acids. The DksA-dependent antinitrosative defenses do not rely on the Hmp flavohemoprotein that detoxifies NO to NO3? and appear to operate independently of the ppGpp alarmone. Our investigations are consistent with a model by which NO produced in the innate response to Salmonella exerts considerable pressure on amino acid biosynthesis. The cytotoxicity of NO against Salmonella amino acid biosynthetic pathways is antagonized in great part by the DksA-dependent regulation of amino acid biosynthesis and transport. PMID:22311927

Henard, Calvin A.

2012-01-01

297

Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Platelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture. Methods Fetal membranes were collected from Caesarean sections at term. RNA was extracted from membranes and subjected to a qualitative RT-PCR to assess the baseline expression of iNOS. Discs of fetal membranes were cultured for 24 hours in the presence of platelet-activating factor at a dose range of 0.1 nanomolar – 1 micomolar or 1 microgram/ml lipopolysaccharide. Nitric oxide production was measured via nitrite ions in the culture medium and mRNA for iNOS was detected by RT-PCR. Results Culturing the membrane discs in medium containing serum induced nitric oxide synthase expression and platelet-activating factor significantly stimulated the production of nitric oxide under these conditions. When cultured without serum inducible nitric oxide synthase expression was induced by lipopolysaccharide, but not by platelet-activating factor. Conclusion Platelet-activating factor may have a role in the initiation of labour, at term or preterm, via the increased local production of nitric oxide as an inflammatory mediator. In this model of intrauterine infection, lipopolysaccharide was found to induce iNOS expression by fetal membranes, and this mechanism could be involved in preterm labour.

Seyffarth Gunter

2004-06-01

298

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

299

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

International Nuclear Information System (INIS)

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)-?-stimulated monocytes to endothelial cells and suppressed the TNF-? induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-?-induced nuclear factor-?B activation, which was attenuated by pretreatment with NG-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: ? Puerarin induced the phosphorylation of eNOS and the production of NO. ? Puerarin activated eNOS through ER-dependent PI3-kinase and Ca2+-dependent AMPK. ? Puerarin-induced NO was involved in the inhibition of NF-kB activation. ? Puerarin may help for prevention of vascular dysfunction and diabetes.

300

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-gamma (200 U/ml) plus TNF (200 U/ml) or IL-1 beta (5 U/ml) increased NOS activity in HUVEC lysates, measured as conversion of [14C]L-arginine to [14C]L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-tre...

Rosenkranz-weiss, P.; Sessa, W. C.; Milstien, S.; Kaufman, S.; Watson, C. A.; Pober, J. S.

1994-01-01

 
 
 
 
301

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

OBJECTIVES: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targ...

Cai, S.; Alp, Nj; Mcdonald, D.; Smith, I.; Kay, J.; Canevari, L.; Heales, S.; Channon, Km

2002-01-01

302

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrul...

Ghigo, Dario Antonio; Trovati, Mariella; Bosia, Amalia; Fontana, Dario; Russo, Isabella; Doronzo, Gabriella; Mattiello, Luigi; Anfossi, Giovanni; Rolle, Luigi; Balbo, Alessandra

2002-01-01

303

Overexpression of a resveratrol synthase gene (PcRS) from Polygonum cuspidatum in transgenic Arabidopsis causes the accumulation of trans-piceid with antifungal activity.  

Science.gov (United States)

Although resveratrol-forming stilbene synthase (STS) genes have been well characterized in many plant species, there are only a few descriptions about STS genes from Polygonum cuspidatum Sieb. et Zucc, an important medicinal crop in Asian countries. To evaluate the biological functions of a Polygonum cuspidatum resveratrol synthase gene (PcRS), the PcRS gene was expressed in Arabidopsis under the control of Cauliflower mosaic virus (CaMV) 35S promoter. Integration and expression of transgene in the plant genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Transgenic plants accumulated a new compound in both the leaves and seeds, which was identified as trans-piceid by high-pressure liquid chromatography (HPLC) and electrospray mass spectrometry (HPLC-ESI-MS). Overexpression of PcRS in transgenic Arabidopsis caused restriction of Colletotrichum higginsianum colonization by inhibition of spore production, resulting in enhanced resistance against C. higginsianum. So, the PcRS gene could be deployed in other crop plants to significantly enhance resistance to fungal pathogens and improve the nutritional quality. In addition, altered seed coat pigmentation and significant reduction in anthocyanin levels were observed in transgenic Arabidopsis, while the expression of endogenous chalcone synthase (CHS) gene was not down-regulated. These results suggest that additional STS activities cause a lack of precursors for CHS which leads to the disturbance of the subsequent flavonoid biosynthesis steps in Arabidopsis. PMID:21717185

Liu, Zhongyu; Zhuang, Chuxiong; Sheng, Shujing; Shao, Li; Zhao, Wei; Zhao, Shujin

2011-11-01

304

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese 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 dose causando redução de 85,2% da atividade enzimática. A cinética variou de acordo com as concentrações de proteína e de arginina, sendo que as melhores leituras foram obtidas com 80 µg de proteína, 120 nM de arginina em 0,5 µCi de ³H arginina, em 60 minutos de incubação. A atividade da NOS na ausência de FAD (flavina adenina dinucleotídeo), FMN (flavina mononucleotídeo) e BH4 (tetrahidrobiopterina) foi de apenas 2,8% da atividade medida na presença destes três cofatores. A atividade da enzima foi completamente inibida pelo L-NAME (1 mM; 98,1 %), EGTA (5 mM; 98,8 %) e adição de trifluoperazina (TFP), nas concentrações de 200 µM e 500 µM, inibiu a atividade da enzima em 73,2% e 83,8 %, respectivamente. Em condições basais, o Km da NOS para Larginina foi de 0,84 ± 0,08 µM e o valor de Vmax foi de 0,122 ± 0,025 pmol.mg-1.min-1. A atividade média da NOS plaquetária humana foi de 0,020 ± 0,010 pmol.mg-1.min-1. Os resultados indicam que a eNOS em plaquetas humanas pode ser avaliada pelo método da conversão de [³H]-arginina em [³H]-citrulina, que em condições otimizadas, fornece resultados reprodutíveis e precisos com ótima sensibilidade para experimentos clínicos envolvendo doenças neurológicas e psiquiátricas. Abstract in english 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.

Elisa Mitiko, Kawamato; Isaias, Glezer; Carolina Demarchi, Munhoz; Cristiane, Bernardes; Cristoforo, Scavone; Tania, Marcourakis.

2002-09-01

305

Spectroscopic, thermal and structural studies on manganous malate crystals  

Science.gov (United States)

Prismatic crystals of manganous malate have been prepared by controlled ionic diffusion in hydrosilica gel. The structure was elucidated using single crystal X-ray diffraction. The crystals are orthorhombic with space group Pbca. Vibrations of the functional groups were identified by the FTIR spectrum. Thermogravimetric and differential thermal analyses (TG-DTA) were carried out to explore the thermal decomposition pattern of the material. Structural information derived from FTIR and TG-DTA studies is in conformity with the single crystal XRD data.

Thomas, J.; Lincy, A.; Mahalakshmi, V.; Saban, K. V.

2013-01-01

306

Spectroscopic, thermal and structural studies on manganous malate crystals  

Energy Technology Data Exchange (ETDEWEB)

Prismatic crystals of manganous malate have been prepared by controlled ionic diffusion in hydrosilica gel. The structure was elucidated using single crystal X-ray diffraction. The crystals are orthorhombic with space group Pbca. Vibrations of the functional groups were identified by the FTIR spectrum. Thermogravimetric and differential thermal analyses (TG-DTA) were carried out to explore the thermal decomposition pattern of the material. Structural information derived from FTIR and TG-DTA studies is in conformity with the single crystal XRD data.

Thomas, J., E-mail: smartlabindia@gmail.com; Lincy, A., E-mail: lincymaria@gmail.com; Mahalakshmi, V.; Saban, K. V. [Smart Materials Analytic Research and Technology (SMART), Department of Physics, St. Berchmans College (India)

2013-01-15

307

Spectroscopic, thermal and structural studies on manganous malate crystals  

International Nuclear Information System (INIS)

Prismatic crystals of manganous malate have been prepared by controlled ionic diffusion in hydrosilica gel. The structure was elucidated using single crystal X-ray diffraction. The crystals are orthorhombic with space group Pbca. Vibrations of the functional groups were identified by the FTIR spectrum. Thermogravimetric and differential thermal analyses (TG-DTA) were carried out to explore the thermal decomposition pattern of the material. Structural information derived from FTIR and TG-DTA studies is in conformity with the single crystal XRD data.

308

Fluvoxamine alters the activity of energy metabolism enzymes in the brain  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and cre [...] atine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg) for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

Gabriela K., Ferreira; Mariane R., Cardoso; Isabela C., Jeremias; Cinara L., Gonçalves; Karolina V., Freitas; Rafaela, Antonini; Giselli, Scaini; Gislaine T., Rezin; João, Quevedo; Emilio L., Streck.

2014-09-01

309

Fluvoxamine alters the activity of energy metabolism enzymes in the brain  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and cre [...] atine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg) for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

Gabriela K., Ferreira; Mariane R., Cardoso; Isabela C., Jeremias; Cinara L., Goncalves; Karolina V., Freitas; Rafaela, Antonini; Giselli, Scaini; Gislaine T., Rezin; Joao, Quevedo; Emilio L., Streck.

2014-03-17

310

The role of MALAT1 correlates with HPV in cervical cancer.  

Science.gov (United States)

Cervical cancer, the second most common type of cancer in women worldwide, is responsible for >275,100 mortalities each year and is associated with high-risk human papilloma virus (HR-HPV). HPVs have two important oncogenes, E6 and E7, which have crucial roles in malignant transformation in cervical cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA originally identified in non-small cell lung cancer. Previous studies have revealed that MALAT1 is expressed in numerous tissue types, and is significant in maintaining the normal function of the body. However, it also appeared to be notably upregulated in numerous carcinoma types compared with adjacent non-cancerous tissues. In the present study, it was identified that MALAT1 expression was upregulated in cervical cancer cell lines compared with normal cervical squamous cell samples. Further study into the effect of MALAT1 on cellular phenotype revealed that MALAT1 was able to promote cell migration and proliferation. Of note, it was revealed that the expression of MALAT1 was decreased with the knockdown of HPV16 E6/E7 in CaSki cells. Furthermore, the investigations in clinical samples also revealed that MALAT1 was expressed in HPV-positive cervical squamous cells, but not in HPV-negative normal cervical squamous cells. These results indicate that HPV correlates with MALAT1 deregulation in cervical cancer. PMID:24932303

Jiang, Yan; Li, Yuehui; Fang, Shujuan; Jiang, Binyuan; Qin, Changfei; Xie, Pingli; Zhou, Guohua; Li, Guancheng

2014-06-01

311

Mutation and Selection of Lactobacillus plantarum Strains That Do Not Produce Carbon Dioxide from Malate  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A differential medium was developed to distinguish between malate-decarboxylating (MDC+) and -non-decarboxylating (MDC?) strains of Lactobacillus plantarum. MDC? strains produced a visible acid reaction in the medium, whereas MDC+ strains did not. Use of the medium allowed for rapid screening and isolation of mutagenized cells that had lost the ability to produce CO2 from malate.

Daeschel, M. A.; Mcfeeters, R. F.; Fleming, H. P.; Klaenhammer, T. R.; Sanozky, R. B.

1984-01-01

312

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.  

Science.gov (United States)

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers. PMID:11502177

McGuire, K A; Siggaard-Andersen, M; Bangera, M G; Olsen, J G; von Wettstein-Knowles, P

2001-08-21

313

Cardiolipin synthase from Escherichia coli.  

Science.gov (United States)

Escherichia coli cardiolipin synthase catalyzes reversible phosphatidyl group transfer from one phosphatidylglycerol molecule to another to form cardiolipin (CL) and glycerol. The enzyme is specified by the cls gene, located at min 28.02 of the E. coli genetic map. Cells with mutations in cls have longer doubling times, tend to lose viability in the stationary phase, are more resistant to 3,4-dihydroxybutyl-1-phosphonate, and have an altered sensitivity to novobiocin. Although cls null mutants appear to lack CL synthase activity, they are still able to form trace quantities of CL. The enzyme appears to be regulated at both the genetic and enzymatic levels. CL synthase's molecular mass is 45-46 kDa, or about 8 kDa less than the polypeptide predicted by the gene sequence, suggesting that posttranslational processing occurs. CL synthase can use various polyols such as mannitol and arabitol to convert CL to the corresponding phosphatidylglycerol analog. When the amino acid sequences of four bacterial CL synthases are compared, three highly conserved regions are apparent. One of these regions contains a conserved pentapeptide sequence, RN(Q)HRK, and another has a conserved HXK sequence. These two sequences may be part of the active site. E. coli CL synthase has been studied by using a mixed micelle assay. The enzyme is inhibited by CL, the product of the reaction, and by phosphatidate. Phosphatidylethanolamine partially offsets inhibition caused by CL but not by phosphatidate. CDP-diacylglycerol does not appear to affect the activity of the purified enzyme but does stimulate the activity associated with crude membrane preparations. PMID:9370333

Tropp, B E

1997-09-01

314

Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Acetyl-CoA synthase from Clostridium thermoaceticum (ACSCt) is an ?2?2 tetramer containing two novel Ni-X-Fe4S4 active sites (the A and C clusters) and a standard Fe4S4 cluster (the B cluster). The acsA and acsB genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37oC under anaerobic conditions with Ni supplementation. The isolated recombinant His-tagged protein (AcsAB) exhibited characteristics essentially indist...

Loke, Huay-keng; Bennett, George N.; Lindahl, Paul A.

2000-01-01

315

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycero-P synthase) and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-...

Carman, G. M.; Wieczorek, D. S.

1980-01-01

316

Lipophilic 1,1-bisphosphonates are potent squalene synthase inhibitors and orally active cholesterol lowering agents in vivo.  

Science.gov (United States)

Squalene synthase catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene at the final branchpoint of the cholesterol biosynthetic pathway. We report herein that isoprenyl 1,1-bisphosphonates and related analogs are potent inhibitors of rat microsomal squalene synthase (I50 = 0.7-32 nM). In addition, members of this family are potent inhibitors of cholesterol biosynthesis in rats on intravenous and oral dosing, as well as cholesterol lowering agents in rats and hamsters. Significant inhibition of cholesterol biosynthesis in rats by lovastatin occurs with a concomitant inhibition of dolichol and coenzyme-Q9 synthesis. In contrast, bisphosphonate 4 has no effect on dolichol and coenzyme-Q9 biosynthesis in rats under conditions where cholesterol biosynthesis is > 90% inhibited. PMID:8227045

Ciosek, C P; Magnin, D R; Harrity, T W; Logan, J V; Dickson, J K; Gordon, E M; Hamilton, K A; Jolibois, K G; Kunselman, L K; Lawrence, R M

1993-11-25

317

Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface...

Chanrion, B.; Mannoury La Cour, C.; Bertaso, F.; Lerner-natoli, M.; Freissmuth, M.; Millan, M. J.; Bockaert, J.; Marin, P.

2007-01-01

318

Effect of the anorectic fatty acid synthase inhibitor C75 on neuronal activity in the hypothalamus and brainstem  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Intraperitoneal (i.p.) injection of C75, a fatty acid synthase inhibitor, causes a rapid (?2-h) and persistent (to at least 24-h) ?95% decrease in food intake. The persistent effect seems to be due to inhibition of the fasting-induced up-regulation of expression of hypothalamic orexigenic neuropeptides neuropeptide Y and agouti-related protein and down-regulation of expression of anorexigenic neuropeptides pro-opiomelanocortin/?-melanocyte-stimulating hormone and cocaine-amphetamine-rela...

Gao, Su; Lane, M. Daniel

2003-01-01

319

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys: IMPLICATIONS FOR ACTIVITY, ALLOSTERY, AND REGULATION*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemilumine...

Rosenfeld, Robin J.; Bonaventura, Joseph; Szymczyna, Blair R.; Maccoss, Michael J.; Arvai, Andrew S.; Yates, John R.; Tainer, John A.; Getzoff, Elizabeth D.

2010-01-01

320

Regulation of Cytosolic and Mitochondrial Oxidation via Malate-Aspartate Shuttle: An Observation Using Dynamic 13C NMR Spectroscopy  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The malate-aspartate (M-A) shuttle provides an important mechanism of metabolic communication between the cytosol and the mitochondria. In this study, dynamic 13C NMR spectroscopy was combined with a multi-domain model of cardiac metabolism for direct quantification of metabolic fluxes through the tricarboxylic acid (TCA) cycle (VTCA) and the M-A shuttle (VM?A) in intact heart. The sensitivity of this approach to altered M-A shuttle activity was examined at different cytosolic redox states....

Lu, Ming; Banerjee, Suhanti; Saidel, Gerald M.; Yu, Xin

2011-01-01

 
 
 
 
321

Measuring the activity of 1-deoxy-D-xylulose 5-phosphate synthase, the first enzyme in the MEP pathway, in plant extracts.  

Science.gov (United States)

The first enzyme in the methylerythritol phosphate (MEP) pathway is 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS). As such this enzyme is considered to be important in the control of plastidial isoprenoid production. Measuring the activity of DXS in plant extracts is therefore crucial to understanding the regulation of the MEP pathway. Due to the relatively low amounts of DXS, the activity of this enzyme can only be measured using highly sensitive analytical equipment. Here, a method is described to determine the DXS enzyme activity in a crude plant extract, by measuring DXP production directly using high performance liquid chromatography linked to a tandem triple quadrupole mass spectrometry detector (LC-MS/MS). PMID:24777787

Wright, Louwrance P; Phillips, Michael A

2014-01-01

322

Photofermentation of malate for biohydrogen production - A modeling approach  

Energy Technology Data Exchange (ETDEWEB)

A kinetic model for photofermentative biohydrogen production is developed in this study to predict the dynamics of the process. The proposed model contains 17 parameters to describe cell growth, substrate consumption, and hydrogen evolution as well as inhibition of the process by biomass, light intensity, and substrate. Batch experimental results from the literature were used to calibrate and validate the model with malic acid as a model substrate, using Rhodobacter sphaeroides as a model biomass. Temporal hydrogen evolution and cell growth predicted by the proposed model agreed well with the experimentally measured data obtained from four literature reports, with statistically significant correlation coefficients exceeding 0.9. Based on sensitivity analysis performed with the validated model, only six of the 17 parameters were found to be significant. Model simulations indicated that the range of optimal light intensity for maximum hydrogen yield from malate by R. sphaeroides was 150-250 W/m{sup 2}. (author)

Gadhamshetty, Venkataramana; Sukumaran, Anoop; Nirmalakhandan, Nagamany; Thein Myint, Maung [Civil Engineering Department, New Mexico State University, Las Cruces, NM 88003 (United States)

2008-05-15

323

Dinuclear nickel complexes modeling the structure and function of the acetyl CoA synthase active site  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A dinuclear nickel complex with methyl and thiolate ligands, Ni(dadtEt)Ni(Me)(SDmp) (2), has been synthesized as a dinuclear Nid–Nip-site model of acetyl-CoA synthase (ACS) (dadtEt is N,N?-diethyl-3,7-diazanonane-1,9-dithiolate; Dmp is 2,6-dimesitylphenyl). Complex 2 was prepared via 2 methods: (i) ligand substitution of a dinuclear Ni(II)–Ni(II) cation complex [Ni(dadtEt) Ni(tmtu)2] (OTf)2(1) with MeMgBr and KSDmp (tmtu is tetramethylthiourea), (ii) methyl transfer from methylcobaloxim...

Ito, Mikinao; Kotera, Mai; Matsumoto, Tsuyoshi; Tatsumi, Kazuyuki

2009-01-01

324

Post-translational Modification Regulates Prostaglandin D2 Synthase Apoptotic Activity: Characterization by Site-directed Mutagenesis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Lipocalin-type prostaglandin D2 synthase (L-PGDS) is a highly glycosylated protein found in several body fluids. Elevated L-PGDS levels have been observed in the serum of patients with renal impairment, diabetes mellitus, and hypertension. Recently, we demonstrated the ability of L-PGDS to induce apoptosis in a variety of cell types including epithelial cells, neuronal cells, and vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the effect several site-directed mu...

Ragolia, Louis; Hall, Christopher E.; Palaia, Thomas

2006-01-01

325

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)

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.

Dong Ju Son

2014-08-01

326

Effects of polyporus polysaccharide on activity and mRNA expression of inducible nitric oxide synthase in peritoneal macrophages of mice  

Directory of Open Access Journals (Sweden)

Full Text Available Objective: To study the mechanisms of the antitumor and immunoregulation functions of polyporus polysaccharide (PPS. Methods: The production of nitric oxide (NO, the activity and mRNA expression of inducible nitric oxide synthase (iNOS in peritoneal macrophages of mice administered with different dose of PPS were observed by Griess reaction, fluorimetry assay and RT-PCR, respectively. Results: PPS could elevate the iNOS activity with dose-dependence and stimulate the iNOS mRNA expression of peritoneal macrophages in mice. Conclusion: The regulation of PPS on the production of NO in peritoneal macrophages of mice may occur at transcriptional level of iNOS. This indicates that the mechanism of PPS's antitumor and immunoregulation functions may be related to increasing NO output of macrophages through stimulating iNOS's denovo synthesis.

HUANG Di-Nan

2004-09-01

327

Expression of Terpenoids 1, a glandular trichome-specific transcription factor from tomato that activates the terpene synthase 5 promoter.  

Science.gov (United States)

Terpene biosynthesis in tomato glandular trichomes has been well studied, with most if not all terpene synthases (TPSs) being identified. However, transcription factors (TFs) that regulate TPSs have not yet been discovered from tomato. In order to unravel the transcriptional regulation of the Solanum lycopersicum linalool synthase (SlMTS1, recently renamed SlTPS5) gene in glandular trichomes, we functionally dissected its promoter. A 207 bp fragment containing the minimal promoter and the 5'UTR appeared to be sufficient for trichome-specific expression in transgenic plants. Yeast-one-hybrid screens with this fragment identified a glandular trichome-specific transcription factor, designated Expression of Terpenoids 1 (SlEOT1). SlEOT1 is a member of a conserved family of TFs that includes the Arabidopsis Stylish 1 (AtSTY1) and Short Internode (AtSHI) genes. The EOT1 protein localized to the nucleus and specifically transactivated the SlTPS5 promoter in Nicotiana benthamiana leaves. PMID:24142382

Spyropoulou, Eleni A; Haring, Michel A; Schuurink, Robert C

2014-02-01

328

Chemiluminometric flow-injection method for determination of free l-malate in wine with co-immobilized malate dehydrogenase/NADH oxidase.  

Science.gov (United States)

A flow-injection system with a co-immobilized malate dehydrogenase/reduced nicotineamide adenine dinucleotide (NADH) oxidase reactor and a chemiluminometer is described for the determination of free l-malate in wine. Malate dehydrogenase and NADH oxidase were co-immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm x 4 mm i.d.). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 3 x 10(-7) M to 2.5 x 10(-4) M (the linear correlation coefficient was 0.9998); the detection limit (signal-to-noise ratio, 3) was 8 x 10(-8) M. The sample throughput was 30 h(-1) without carryover. The ractor was renewed every 2 weeks. PMID:18966411

Kiba, N; Inagaki, J; Furusawa, M

1995-11-01

329

Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells.  

Science.gov (United States)

In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of this study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time- and dose-response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance, but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor L-NAME blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin-endoperoxide synthase-2 mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin, and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for the LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands. PMID:24992832

Zamberlam, Gustavo; Sahmi, Fatiha; Price, Christopher A

2014-09-01

330

Systemic but not central nervous system nitric oxide synthase inhibition exacerbates the hypertensive effects of chronic melanocortin-3/4 receptor activation.  

Science.gov (United States)

We examined whether systemic or central nervous system (CNS) inhibition of nitric oxide synthase exacerbates the cardiovascular responses of chronic CNS melanocortin 3/4 receptor activation. Sprague-Dawley rats implanted with telemetry probes, venous catheters, and intracerebroventricular (ICV) cannulae were divided in 3 groups. After control measurements, the NO synthase inhibitor L-NAME was infused (10 ?g/kg/min intravenous) for 17 days and, starting on day 7 of L-NAME infusion, the melanocortin 3/4 receptor agonist melanotan II (MTII; 10 ng/hr; group 1) or saline vehicle (group 2) was infused ICV for 10 days. A third group not treated with L-NAME also received MTII ICV. Melanocortin 3/4 receptor activation caused a greater increase in mean arterial pressure (MAP) and heart rate in rats treated with intravenous L-NAME (35 ± 6 mm Hg and 56 ± 8 bpm) than L-NAME plus vehicle or MTII alone (22 ± 5 and 9 ± 2 mm Hg, and 26 ± 14 and 27 ± 5 bpm), despite a 58% and 50% reduction in food intake during the first 6 days of MTII infusion. To test if the amplified pressor response to MTII after L-NAME was attributable to a reduction in nitric oxide availability in the brain, we also infused L-NAME directly into the CNS alone or in combination with MTII. ICV infusion of L-NAME plus MTII caused only ? 10 mm Hg increase in MAP with no change in heart rate, similar to the effects of ICV infusion of MTII alone, whereas ICV infusion of L-NAME alone had no effect on MAP. These results suggest that reduction in peripheral, but not CNS, nitric oxide production augments MAP sensitivity to CNS melanocortin 3/4 receptor activation. PMID:21263126

do Carmo, Jussara M; Bassi, Mirian; da Silva, Alexandre A; Hall, John E

2011-03-01

331

Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities.  

Science.gov (United States)

Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we sought to investigate the potential neuroprotective effects of allicin against traumatic brain injury (TBI) in rats. We found that allicin treatment (10 and 50mg/kg, not 1mg/kg) significantly reduced brain edema and motor functional deficits, as well as apoptotic neuronal cell death in injured cortex. These protective effects could be observed even if the administration was delayed to 4h after injury. Moreover, allicin treatment decreased the expression levels of MDA and protein carbonyl, preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines. The results of Western blot analysis showed that allicin increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Blocking Akt/eNOS pathway activation by specific inhibitor LY294002 (10?L, 10mmol/L) or L-NIO (0.5mg/kg) partly reversed the protective effects of allicin and its anti-inflammatory activities. The allicin induced anti-oxidative activity was partly prevented by LY294002, but not L-NIO. In summary, our data strongly suggested that allicin treatment at an appropriate dose can exert protective effect against TBI through Akt/eNOS pathway-mediated anti-inflammatory and anti-oxidative activities. PMID:24530793

Chen, Wei; Qi, Jun; Feng, Feng; Wang, Mao-de; Bao, Gang; Wang, Tuo; Xiang, Mu; Xie, Wan-Fu

2014-03-01

332

Mechanistic and bioinformatic investigation of a conserved active site helix in ?-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.  

Science.gov (United States)

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily. PMID:24720347

Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

2014-05-13

333

High red blood cell nitric oxide synthase activation is not associated with improved vascular function and red blood cell deformability in sickle cell anaemia.  

Science.gov (United States)

Human red blood cells (RBC) express an active and functional endothelial-like nitric oxide (NO) synthase (RBC-NOS). We report studies on RBC-NOS activity in sickle cell anaemia (SCA), a genetic disease characterized by decreased RBC deformability and vascular dysfunction. Total RBC-NOS content was not significantly different in SCA patients compared to healthy controls; however, using phosphorylated RBC-NOS-Ser(1177) as a marker, RBC-NOS activation was higher in SCA patients as a consequence of the greater activation of Akt (phosphorylated Akt-Ser(473) ). The higher RBC-NOS activation in SCA led to higher levels of S-nitrosylated ?- and ?-spectrins, and greater RBC nitrite and nitrotyrosine levels compared to healthy controls. Plasma nitrite content was not different between the two groups. Laser Doppler flowmetric experiments demonstrated blunted microcirculatory NO-dependent response under hyperthermia in SCA patients. RBC deformability, measured by ektacytometry, was reduced in SCA in contrast to healthy individuals, and pre-shearing RBC in vitro did not improve deformability despite an increase of RBC-NOS activation. RBC-NOS activation is high in freshly drawn blood from SCA patients, resulting in high amounts of NO produced by RBC. However, this does not result in improved RBC deformability and vascular function: higher RBC-NO is not sufficient to counterbalance the enhanced oxidative stress in SCA. PMID:25316332

Grau, Marijke; Mozar, Anaïs; Charlot, Keyne; Lamarre, Yann; Weyel, Linda; Suhr, Frank; Collins, Bianca; Jumet, Stéphane; Hardy-Dessources, Marie-Dominique; Romana, Marc; Lemonne, Nathalie; Etienne-Julan, Maryse; Antoine-Jonville, Sophie; Bloch, Wilhelm; Connes, Philippe

2015-03-01

334

Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The mitochondrial tricarboxylic acid cycle enzyme malate dehydrogenase was purified from Saccharomyces cerevisiae, and an antibody to the purified enzyme was obtained in rabbits. Immunoscreening of a yeast genomic DNA library cloned into a lambda gt11 expression vector with anti-malate dehydrogenase immunoglobulin G resulted in identification of a lambda recombinant encoding an immunoreactive beta-galactosidase fusion protein. The yeast DNA portion of the coding region for the fusion protein ...

Mcalister-henn, L.; Thompson, L. M.

1987-01-01

335

NADP-Malate Dehydrogenase Gene Evolution in Andropogoneae (Poaceae): Gene Duplication Followed by Sub-functionalization  

Digital Repository Infrastructure Vision for European Research (DRIVER)

• Background and Aims Plastid NADP-dependent malate dehydrogenase (MDH) catalyses the conversion of oxaloacetate to malate. In C4 plants, it is involved in photosynthetic carbon assimilation. In Poaceae, one NADP-MDH gene has been identified in rice (C3; Erhartoideae) and maize (C4; Panicoideae), whereas two tandemly repeated genes have been identified in Sorghum (C4; Panicoideae). In the present study, the molecular evolution of the NADP-MDH multigene family was investigated in order to an...

Rondeau, P.; Rouch, C.; Besnard, G.

2005-01-01

336

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)

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 ?g 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 flavonoids in ginger can be increased by foliar application of SA in a controlled environment and that the anticancer activity in young ginger extracts could be improved.

Ehsan Karimi

2012-11-01

337

Processes of malate catabolism during the anaerobic metabolism of grape berries  

International Nuclear Information System (INIS)

In order to precise malate fate during the anaerobic metabolism of grape, malate-3-14C was injected into Carignan berries kept in darkness at 350C under carbon dioxide atmosphere. The injection of labelled malate was effected in presence or not of non-labelled oxalate which inhibits malic enzyme (EC I.I.I.40). The analyses of the samples fixed after 3 and 7 days anaerobiosis concerned the titration of various substrates, organic acids, amino-acids and glycolysis products, and the measuring of the NADP+-malic enzyme (EC I.I.I.40) and malate dehydrogenase (EC I.I.I.40). Radioactivity is mainly observed in ethanol, amino-butyrate the non-separated group glycerate-shikimate and succinate. Malic enzyme acts in the first sequence of a process leading from malate to ethanol. Alanin synthesis seems to be stimulated in presence of oxalate. The results obtained and some hypotheses presented in the literature induce to suggest a utilization scheme for malate in the anaerobic metabolism of grape

338

Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11.  

Science.gov (United States)

The genome sequence of the marine bacterium 'Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that 'Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 microM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 x 10(6) cells ml(-1) to 8.16 x 10(6) cells ml(-1) (R(2) = 0.992). Serine was capable of substituting for glycine at 1.5 microM. 'Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that 'Cand. P. ubique' is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade. PMID:19125817

Tripp, H James; Schwalbach, Michael S; Meyer, Michelle M; Kitner, Joshua B; Breaker, Ronald R; Giovannoni, Stephen J

2009-01-01

339

Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.  

Science.gov (United States)

Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state. PMID:11104686

Konno, H; Yodogawa, M; Stumpp, M T; Kroth, P; Strotmann, H; Motohashi, K; Amano, T; Hisabori, T

2000-01-01

340

Design, synthesis and antibacterial activities of vanillic acylhydrazone derivatives as potential ?-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors.  

Science.gov (United States)

Fatty acid biosynthesis is essential for bacterial survival. FabH, ?-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of acylhydrazone derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong broad-spectrum antibacterial activity. Compounds with potent antibacterial activities were tested for their Escherichia coli FabH inhibitory activity. Especially, compound E9 showed the most potent antibacterial activity with MIC values of 0.39-1.56 ?g/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC(50) of 2.5 ?M. Docking simulation was performed to position compound E9 into the E. coli FabH active site to determine the probable binding conformation. PMID:23124163

Wang, Xiao-Liang; Zhang, Yan-Bin; Tang, Jian-Feng; Yang, Yu-Shun; Chen, Ruo-Qi; Zhang, Fei; Zhu, Hai-Liang

2012-11-01

 
 
 
 
341

Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS.  

Science.gov (United States)

Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain. PMID:21944746

Bonnett, Shilah A; Rath, Christopher M; Shareef, Abdur-Rafay; Joels, Joanna R; Chemler, Joseph A; Håkansson, Kristina; Reynolds, Kevin; Sherman, David H

2011-09-23

342

Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes  

DEFF Research Database (Denmark)

AIMS/HYPOTHESIS: In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved insulin signalling to glycogen synthesis remains to be clarified. METHODS: Whole-body glucose metabolism was studied in 12 patients with type 2 diabetes, and 10 lean and 10 obese non-diabetic controls by means of indirect calorimetry and tracers during a euglycaemic-hyperinsulinaemic clamp. The diabetic patients underwent a second isoglycaemic-hyperinsulinaemic clamp maintaining fasting hyperglycaemia. Muscle biopsies from m. vastus lateralis were obtained before and after the clamp for examination of GS and relevant insulin signalling components. RESULTS: During euglycaemia, insulin-stimulated glucose disposal, glucose oxidation and non-oxidative glucose metabolism were reduced in the diabetic group compared with both control groups (p¿

Vind, B F; Birk, Jesper Bratz

2012-01-01

343

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

LENUS (Irish Health Repository)

Cystathionine ?-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 ?L 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.

Krijt, Jakub

2011-02-01

344

Vesicocutaneous fistula formation during treatment with sunitinib malate: Case report  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The oral multi-kinase inhibitor sunitinib malate improves the survival of patients with gastrointestinal stromal tumors (GIST after the disease progresses or intolerance to imatinib mesylate develops. Urinary fistulae arising during treatment with sunitinib for GIST have not been described. Case presentation We describe a 62-year-old female patient diagnosed with unresectable GIST that involved the abdominal wall, urinary bladder wall, bowel, mesentery and peritoneum in the pelvic cavity. Intestinocutaneous fistulae developed on a surgical lesion after orally administered imatinib was supplemented by an arterial infusion of 5-flurouracil. Sunitinib was started after the patient developed resistance to imatinib. On day 4 of the fourth course of sunitinib, a widely dilated cutaneous fistula discharged large amounts of fluid accompanied by severe abdominal pain. Urinary communication was indicated based on the results of an intravenous injection of indigo carmine. Computed tomography findings suggested a small opening on the anterior urinary bladder wall and fistulous communication between the bladder and abdominal walls bridged by a subcutaneous cavity. The fistula closed and the amount of discharge decreased when sunitinib was discontinued. Therefore, sunitinib might have been associated with the development of the vesicocutaneous fistula in our patient. Conclusion This is the first description of a vesicocutaneous fistula forming while under sunitinib treatment. Clinicians should be aware of the possible complication of vesicocutaneous fistula formation during treatment with molecular targeting agents in patients with extravesical invasion and peritoneal dissemination of GIST.

Sakashita Hiroyuki

2010-11-01

345

Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi.  

Science.gov (United States)

Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism. PMID:19690851

Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

2010-02-01

346

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.  

Science.gov (United States)

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa. PMID:136000

Valenta, M; Cepica, S; Slechta, V

1976-01-01

347

The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading  

DEFF Research Database (Denmark)

The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the alpha2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in alpha1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-beta and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that alpha2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken.

JØrgensen, Sebastian B; Nielsen, Jakob N.

2004-01-01

348

Sunitinib malate for gastrointestinal stromal tumour in imatinib mesylate–resistant patients: recommendations and evidence  

Science.gov (United States)

Question Is sunitinib malate—marketed as Sutent (Pfizer Canada, Kirkland, QC)—superior to placebo or other interventions for primary outcomes of interest in adult patients with gastrointestinal stromal tumour (gist) who have developed resistance or who exhibit intolerance to imatinib mesylate (im)? Background In patients with resectable disease, surgery is the mainstay of treatment for gist; in patients with unresectable or metastatic disease, the tyrosine kinase inhibitor im is the therapy of choice. However, some patients have primary resistance or intolerance to im, or they progress after optimal exposure (including an escalated dose). Here, we review the evidence for treating im-resistant gist with sunitinib malate. Methods Studies of sunitinib malate were identified through medline, embase, the Cochrane Library databases, and Web sites of guideline organizations. Outcomes of interest included time to progression, progression-free survival, overall survival, and toxicity. Results One phase iii randomized controlled trial, and one abstract and presentation describing that trial, served as the evidentiary base for this clinical practice guideline. Trial data confidently show that both time to progression and progression-free survival are highly statistically significant (p < 0.0001) in favour of sunitinib malate over placebo. Overall survival was improved with sunitinib malate (hazard ratio: 0.49; 95% confidence interval: 0.29 to 0.83; p = 0.007; absolute difference in weeks not reported). The most frequent of all adverse effects (experienced in greater proportion by patients on sunitinib malate) were grades 1 and 2 leucopenia (52% vs. 5% with placebo), neutropenia (43% vs. 4%), and thrombocytopenia (36% vs. 4%). Grade 3 hematologic adverse events were also reported more frequently in the sunitinib malate group, including leucopenia (4% vs. 0%), neutropenia (8% vs. 4%), lymphopenia (9% vs. 2%), and thrombocytopenia (4% vs. 0%). Toxicity comparisons did not include p values. The incidence of grades 1–3 fatigue was greater for the sunitinib malate group (34% vs. 22% with placebo). Other grade 3 nonhematologic treatment-related adverse events that occurred more frequently on sunitinib malate included hand–foot syndrome (4% vs. 0%), diarrhea (3% vs. 0%), and hypertension (3% vs. 0%). No grade 4 adverse events were observed. Conclusions In the target population, sunitinib malate is the recommended option for second-line therapy of metastatic gist. PMID:20697509

Younus, J.; Verma, S.; Franek, J.; Coakley, N.

2010-01-01

349

Interface Matters: The Stiffness Route to Stability of a Thermophilic Tetrameric Malate Dehydrogenase  

Science.gov (United States)

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate. PMID:25437494

Kalimeri, Maria; Girard, Eric; Madern, Dominique; Sterpone, Fabio

2014-01-01

350

A New Pathway for Salvaging the Coenzyme B12 Precursor Cobinamide in Archaea Requires Cobinamide-Phosphate Synthase (CbiB) Enzyme Activity  

Science.gov (United States)

The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5?-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica. A cbiP mutant strain of the archaeon Halobacterium sp. strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon. A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide. The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid. The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway. The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea. PMID:14645280

Woodson, Jesse D.; Zayas, Carmen L.; Escalante-Semerena, Jorge C.

2003-01-01

351

Impact of trans-resveratrol-sulfates and -glucuronides on endothelial nitric oxide synthase activity, nitric oxide release and intracellular reactive oxygen species.  

Science.gov (United States)

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings. PMID:25329867

Ladurner, Angela; Schachner, Daniel; Schueller, Katharina; Pignitter, Marc; Heiss, Elke H; Somoza, Veronika; Dirsch, Verena M

2014-01-01

352

Impact of Trans-Resveratrol-Sulfates and -Glucuronides on Endothelial Nitric Oxide Synthase Activity, Nitric Oxide Release and Intracellular Reactive Oxygen Species  

Directory of Open Access Journals (Sweden)

Full Text Available Resveratrol (3,5,4'-trihydroxy-trans-stilbene is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings.

Angela Ladurner

2014-10-01

353

Inhibition of endothelial nitric oxide synthase activity and suppression of endothelium-dependent vasorelaxation by 1,2-naphthoquinone, a component of diesel exhaust particles  

Energy Technology Data Exchange (ETDEWEB)

Diesel exhaust particles contain redox-active quinones, such as 9,10-phenanthraquinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which act as potent electron acceptors, thereby altering electron transfer on proteins. We have previously found that 9,10-PQ inhibits constitutive nitric oxide synthase (NOS) activity, by shunting electrons away from NADPH on the cytochrome P450 reductase domain of NOS, and thus suppresses acetylcholine (Ach)-induced vasorelaxation in the aortic ring. However, the effect of 1,2-NQ on endothelial NOS (eNOS) activity is still poorly understood. With the membrane fraction of cultured bovine aortic endothelial cells, we found that 1,2-NQ was a potent inhibitor of eNOS with an IC{sub 50} value of 1.4 {mu}M, whereas trans-1,2-dihydroxy-1,2-dihydronaphthalene (1,2-DDN), a redox-negative naphthalene analog of 1,2-NQ, did not show such an inhibitory action. Although 1,2-DDN (5 {mu}M) did not affect Ach-mediated vasorelaxation, 1,2-NQ caused a significant suppression of Ach-induced endothelium-dependent vasorelaxation in the aortic ring. However, 1,2-NQ did not affect sodium nitroprusside-induced endothelium-independent vasorelaxation. These results suggest that 1,2-NQ is an environmental quinone that inhibits eNOS activity, thereby disrupting NO-dependent vascular tone. (orig.)

Sun, Yang; Taguchi, Keiko; Sumi, Daigo [University of Tsukuba, Department of Environmental Medicine, Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Sciences, Ibaraki (Japan); Yamano, Shigeru [Fukuoka University, Faculty of Pharmaceutical Sciences, Fukuoka (Japan); Kumagai, Yoshito [University of Tsukuba, Department of Environmental Medicine, Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Sciences, Ibaraki (Japan); Southern California Particle Center and Supersite, Los Angeles, CA (United States)

2006-05-15

354

Reactive oxygen species-regulated glycogen synthase kinase-3? activation contributes to all-trans retinoic acid-induced apoptosis in granulocyte-differentiated HL60 cells.  

Science.gov (United States)

All-trans retionic acid (ATRA) treatment confers disease remission in acute promyelocytic leukemia (APL) patients by inducing granulocytic differentiation, which is followed by cell apoptosis. Although glycogen synthase kinase (GSK)-3? is known to be required for spontaneous cell death in neutrophils, the requirement of GSK-3? activation for the apoptotic effects remains unknown. This question is addressed in the present study using a model of ATRA-induced granulocytic differentiation and apoptosis in APL HL60 cells. ATRA at a therapeutic concentration (1 ?M) induced granulocytic differentiation, followed by apoptosis. ATRA treatment caused decreased Mcl-1, caspase-3 activation, and PARP cleavage following the inactivation of phosphatidylinositol 3-kinase/AKT and the activation of GSK-3?. Pharmacologically and genetically inhibiting GSK-3? effectively retarded ATRA-induced Mcl-1 degradation and apoptosis. Additional differentiation inducers, phorbol 12-myristate 13-acetate and dimethyl sulfoxide, also triggered GSK-3?-dependent apoptosis. Mechanistically, ATRA caused the generation of reactive oxygen species (ROS) through increased expression of NADPH oxidase subunits (p47(phox) and p67(phox)) to facilitate ATRA-induced GSK-3? activation and cell apoptosis. This study indicates that ROS initiate GSK-3?-dependent apoptosis in granulocyte-differentiated cells after long-term ATRA treatment. PMID:24406248

Wang, Chi-Yun; Yang, Tsan-Tzu; Chen, Chia-Ling; Lin, Wei-Chieh; Lin, Chiou-Feng

2014-03-01

355

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)

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 14C-labeled acetate to those supplied exogenously as 14C-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 fatty acids produced by FASN to those derived exogenously. • Cancer cells do not have a specific requirement for fatty acids produced by FASN. • Fatty acids produced by FASN are in excess of cell requirements and are excreted. • Increased FASN activity is not required to sustain elevations in glycolysis

356

Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity.  

Science.gov (United States)

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers. PMID:25454716

Hempel, Casper; Kohnke, Hannes; Maretty, Lasse; Jensen, Peter Ø; Staalsø, Trine; Kurtzhals, Jørgen A L

2014-11-01

357

Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity  

DEFF Research Database (Denmark)

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers.

Hempel, Casper; Kohnke, Hannes

2014-01-01

358

Homochiral Cu(II) and Ni(II) malates with tunable structural features  

International Nuclear Information System (INIS)

Four new homochiral metal–organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H2O (1), [Cu(mal)(bpe)]·2H2O (2), [Ni(mal)(bpy)]·1.3CH3OH (3) and [Ni(mal)(bpe)]·4H2O (4) (mal=S-malate, bpy=4,4?-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1–3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral (Ni(mal)) motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %. - Graphical abstract: Four new homochiral metal–organic frameworks are built from Ni2+ or Cu2+ cations, S-malate anions and N-donor linkers of different length, which controls the size of pores and guest accessible volume of the homochiral structure. Display Omitted - Highlights: • Four new homohiral metal–organic frameworks based on Ni2+ and Cu2+. • Cu(II)–malate layers and Ni(II)–malate chains are connected by N-donor linkers. • N-donor linkers of different length control the size of pores

359

Manipulation of Rumen Ecology by Yeast and Malate in Dairy Heifer  

Directory of Open Access Journals (Sweden)

Full Text Available Four, one-year old of dairy heifers were randomly assigned according to a 2x2 Factorial arrangement in a 4x4 Latin square design to study supplementation of malate level at 500 vs 1,000 g with yeast at 1,000 vs 2,000 g in concentrate. The treatments were as follows: T1 = supplementation of malate at 500 g + yeast at 1,000 g; T2 = supplementation of malate at 500 g + yeast at 2,000 g; T3 = supplementation of malate at 1,000 g + yeast at 1,000 g; T4 = supplementation of malate at 1,000 g + yeast at 2,000 g in concentrate, respectively. The cows were offered the treatment concentrate at 1 %BW and ruzi grass was fed ad libitum. The results have revealed that rumen fermentation and blood metabolites were similar for all treatments. However, the concentration of volatile fatty acid was significantly different especially the concentration of propionic acid was slightly higher in heifer receiving T4 than T3, T2 and T1 (24.4, 22.9, 22.4 and 19.7%, respectively. The populations of protozoa and fungal zoospores were significantly different as affected by malate level and yeast. In conclusion, the combined use of concentrate containing high level of cassava chip at 70% DM with malate at 1,000 g and yeast at 2,000 g in concentrate with ruzi grass as a roughage could improved rumen ecology in dairy heifers.

Sittisak Khampa

2009-01-01

360

Manipulation of Rumen Ecology by Malate and Cassava Hay in High-Quality Feed Block in Dairy Steers  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Four, dairy steers were randomly assigned according to a 2x2 Factorial arrangement in a 4x4 Latin square design to study supplementation of malate level at 500 and 1,000 g and cassava hay in high-quality feed block. The treatments were as follows: T1 = supplementation of high-quality feed block without cassava hay + malate at 500 g; T2 = supplementation of high-quality feed block without cassava hay + malate at 1,000 g; T3 = supplementation of high-quality feed block with cassava hay + malate...

Sittisak Khampa; Pala Chaowarat; Uthai Koatdoke; Rungson Singhalert; Metha Wanapat

2009-01-01

 
 
 
 
361

Identification of Pseudomonas fluorescens Chemotaxis Sensory Proteins for Malate, Succinate, and Fumarate, and Their Involvement in Root Colonization  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis protein (MCP) genes of P. fluorescens Pf0-1. To identify a MCP for l-malate, the plasmid library was screened using the PA2652 mutant of Pseudomonas aeruginosa PAO1, a mutant defective in chemotaxis to l-malate. The introduction of Pfl01_0728 and Pfl01_3768 genes restored the ability of the PA2652 mutant to respond to l-malate. ...

Oku, Shota; Komatsu, Ayaka; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

2014-01-01

362

Transfer of label from aspartate to malate by the cell-free extract of Sedum mexicanum leaves  

International Nuclear Information System (INIS)

The cell-free extract from the leaves of Sedum mexicanum, a typical CAM plant, formed 14C-malate from 14C-aspartate in the presence of NAD. No reduction of NAD was observed during the reaction. Analysis of this reaction revealed that the transfer of label from 14C-aspartate to malate took place by the action of malate dehydrogenase and aspartate aminotransferase, and the reaction was reversible in the model experiment with commercial enzymes. The pitfalls in assessing the data on dark 14CO2 fixation in CAM are discussed with reference to the transfer of label between malate and aspartate without actual synthesis. (Kaihara, S.)

363

Human cystathionine ?-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM.  

Science.gov (United States)

CBS (cystathionine ?-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies. PMID:22985361

Pey, Angel L; Majtan, Tomas; Sanchez-Ruiz, Jose M; Kraus, Jan P

2013-01-01

364

Constitutive activation of glycogen synthase kinase-3? correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Aberrant regulation of glycogen synthase kinase-3? (GSK-3? has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3? phosphorylated at Tyr216 (pGSK-3? and its relationship with other tumor-associated proteins in human gastric cancers. Methods Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3? inhibitor lithium chloride (LiCl for immunoblot analysis. Results We found that pGSK-3? was expressed in 129 (46% of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P P P P P Conclusions GSK-3? activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3? activation is a useful prognostic marker for the early-stage gastric cancer.

Cho Yu

2010-08-01

365

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)

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.

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

366

Apoptosis of Trypanosoma musculi co-cultured with LPS activated macrophages: enhanced expression of nitric oxide synthase INF-gamma and caspase.  

Science.gov (United States)

Trypanosoma musculi-macrophage co-cultures were studied to investigate the biological role of lipopolysaccharide (LPS) induced cytokines in controlling the proliferation of parasites in vitro. Macrophages, isolated by peritoneal lavage, sustained the growth and proliferation of the parasites. Macrophages activated with LPS were characterized by up-regulation of nitric oxide synthase (iNOS) and phagocytosis of fluorescent latex spheres. Activated macrophages showed marked inhibition of the association and proliferation of the parasites. The LPS treated macrophages produced cytokines, especially interferon gamma (INF-gamma), which was detected by Western blot. Trypanosomes, inhibited from association with macrophages, did not proliferate and instead formed clusters held together by their flagella. Cells in these clusters were apoptotic, as demonstrated by the Apoptag reaction and gel fragmentation assay. In addition, high levels of caspase 8 and caspase 3 were shown in floating trypanosome clusters. The results would suggest that INF-gamma and other cytokines released by activated macrophages, possibly functioning through the INF-gammaR1, Fas ligand, CD95 or other death ligands in the trypanosome plasma membrane initiates the apoptosis cascade in trypanosomes. PMID:16335591

Gugssa, A; Gebru, S; Lee, C M; Baccetti, B; Anderson, W

2005-08-01

367

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp. Atividade da sintetase da glutamina e sintase do glutamato em relação a fixação de nitrogênio em Lotus spp.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of no...

SUSANA GONNET; PEDRO DÍAZ

2000-01-01

368

Bacterial infection induces nitric oxide synthase in human neutrophils.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric o...

Wheeler, M. A.; Smith, S. D.; Garci?a-carden?a, G.; Nathan, C. F.; Weiss, R. M.; Sessa, W. C.

1997-01-01

369

Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution / Atividade de invertases e sacarose sintase em plantas de cafeeiro pulverizadas com solução de sacarose  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Uma prática cuja eficiência não foi ainda comprovada cientificamente, é a pulverização dos cafeeiros com solução diluída de sacarose, como fonte de carbono para as plantas. Este trabalho visou estudar o efeito da pulverização de açúcar via folha nos teores endógenos de carboidratos e na atividade da [...] s enzimas invertases e sacarose sintase em mudas de cafeeiros (Coffea arabica L.) com baixo (baixo) e alto (normal) nível de reservas de carbono. As pulverizações ocorreram nas concentrações de 0,5 e 1% de sacarose utilizando-se água como testemunha. A aplicação de sacarose a 1% aumentou a concentração de açúcares solúveis totais (AST) em plantas depauperadas, como também aumentou as atividades das enzimas invertase ácida da parede, invertase ácida do vacúolo, invertase neutra do citosol e sacarose sintase. Em plantas com níveis normais de carboidratos não foi observada nenhuma alteração nos teores dos AST, como também nas atividades de tais enzimas. Independentemente dos tratamentos aplicados e do estado fisiológico das plantas, não foram observadas diferenças na transpiração e na condutância estomática, mostrando, desta forma, o controle estomático da transpiração. A fotossíntese foi estimulada a 0,5% e 1% em plantas depauperadas, o que não aconteceu com plantas normais. A pulverização de sacarose em mudas de cafeeiros só é eficiente tratando-se de plantas depauperadas na concentração de 1%. Abstract in english 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 activ [...] ities 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%.

José Carlos da, Silva; José Donizeti, Alves; Amauri Alves de, Alvarenga; Marcelo Murad, Magalhães; Dárlan Einstein do, Livramento; Daniela Deitos, Fries.

370

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)

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 metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry. RESULTS: Reduced insulin-mediated glucose disposal (P < 0.05) was associated with a lower insulin-stimulated GS activity in PCOS patients (P < 0.05), compared with controls. This was, in part, explained by absent insulin-mediated dephosphorylation of GS at the NH2-terminal sites 2+2a, whereas dephosphorylation at the COOH-terminal sites 3a+3b was intact in PCOS subjects (P < 0.05). Consistently, multiple linear regression analysis showed that insulin activation of GS was dependent on dephosphorylation of sites 3a+3b in women with PCOS. 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 < 0.05) and restored the ability of insulin to dephosphorylate GS at sites 2 and 2a. CONCLUSIONS: Impaired insulin activation of GS including absent dephosphorylation at sites 2+2a contributes to insulin resistance in skeletal muscle in PCOS. The ability of pioglitazone to enhance insulin sensitivity, in part, involves improved insulin action on GS activity and dephosphorylation at NH2-terminal sites.

Glintborg, Dorte; HØjlund, Kurt

2008-01-01

371

Design, development and synthesis of ribitylaminopyrimidinedione derivatives as lumazine synthase mechanistic probes and trifluoromethylated pyrazoles as riboflavin synthase inhibitors  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inhibition of riboflavin synthase and lumazine synthase, the last two enzymes in the riboflavin biosynthetic pathway, provides a strategy for the development of therapeutically useful antibiotics.^ A homologous series of four derivatives of 5-amino-6-ribitylamino-1 H-pyrimidine-2,4-dione were designed and synthesiszed as potential enzyme inhibitors and mechanistic probes. Some of them displayed moderate inhibition activity on Mycobacterium tuberculosis lumazine synthase and riboflavin synth...

Zhao, Yujie

2009-01-01

372

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

Science.gov (United States)

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. PMID:24791414

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

2014-02-01

373

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

Science.gov (United States)

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 ?M, 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. PMID:23382512

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

374

Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase  

Energy Technology Data Exchange (ETDEWEB)

The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

2011-09-20

375

Higher plant cellulose synthases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The sole function of cellulose synthases, which are found in plants bacteria, fungi, and animals, is to produce the biopolymer cellulose. Although no crystal structure has yet been solved, a considerable amount is known about their structure, function and evolution.

Richmond, Todd

2000-01-01

376

Indomethacin inhibits activation of endothelial nitric oxide synthase in the rat kidney: possible role of this effect in the pathogenesis of indomethacin-induced renal damage.  

Science.gov (United States)

The clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is often associated with adverse effects in the kidney. Indomethacin, an NSAID that has been shown to induce oxidative stress in the kidney, was used to study the pathogenesis of renal damage induced by the drug in a rat model. Experimental animals were given indomethacin (20mg/kg) by oral gavage, sacrificed 1, 12 or 24h (h) later and the kidneys studied. Evidence of glomerular and tubular damage in the kidney was found in response to the drug. Renal tissue nitrite levels, a surrogate marker of nitric oxide (NO) synthesis, were significantly decreased at 12 and 24h. Indomethacin did not affect protein and mRNA levels of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS). However, it significantly reduced the ratio of dimeric (active) to monomeric (inactive) eNOS in the kidney, 12 and 24h after drug administration. This was associated with reductions in heme content, both in renal tissue and in NOS. Heme oxygenase 1 (HO-1) mRNA (at 1 and 12h), protein (at 12 and 24h) and activity (at 1, 12 and 24h) were elevated in response to indomethacin. Nuclear translocation of Nrf2 (at 12h) and p38 MAPK signaling (at 12h and 24h), both of which are known to induce HO-1, also occurred in response to the drug. In summary, our results show that indomethacin reduced levels of activated eNOS in the kidney. This effect is possibly mediated by heme depletion, secondary to HO-1 induction that occurred downstream of Nrf2 and p38 MAPK signaling. We postulate that reduced renal eNOS activity may result in decreased NO levels, and hence reduced renal perfusion, leading to glomerular and tubular injury with subsequent renal damage. PMID:25110317

Nagappan, Arumugam Suriyam; Varghese, Joe; Pranesh, Gautham Tumkur; Jeyaseelan, Visalakshi; Jacob, Molly

2014-09-25

377

Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages  

Energy Technology Data Exchange (ETDEWEB)

Inflammatory responses stimulated by bacterial endotoxin (lipopolysaccharide, LPS) involve calcium-mediated signaling, yet the cellular sensors that determine cell fate in response to LPS remain poorly understood. We report that exposure of RAW 264.7 macrophage-like cells to LPS induces a rapid increase in calmodulin (CaM) abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precedes nuclear localization of key transcription factors (i.e., NF?B p65 subunit, phospho-c-Jun, and Sp1) and subsequent increases in the pro-inflammatory cytokine tumor necrosis factor ? (TNF) and inducible nitric oxide synthase (iNOS). Cellular apoptosis following LPS challenge is blocked following inhibition of iNOS activity, whether accomplished using the pharmacological inhibitor 1400W, through gene silencing of TNF?, or by increasing the level of cellular CaM by stable transfection. Increasing CaM expression also results in reductions in the cellular release of TNF? and iNOS, and activation of their transcriptional regulators, indicating the level of available CaM plays a key role in determining the expression of the pro-inflammatory and pro-apoptotic cascade during cellular activation by LPS. These results indicate a previously unrecognized central role for CaM in maintaining cellular homeostasis in response to LPS, such that under resting conditions cellular concentrations of CaM are sufficient to inhibit the biosynthesis of proinflammatory mediators associated with macrophage activation. Although CaM and iNOS protein levels are coordinately increased as part of the oxidative burst, limiting cellular concentrations of CaM due to association with iNOS (and other high-affinity binders) commit the cell to an unchecked inflammatory cascade leading to apoptosis.

Weber, Thomas J.; Smallwood, Heather S.; Kathmann, Loel E.; Markillie, Lye MENG.; Squier, Thomas C.; Thrall, Brian D.

2006-01-18

378

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177  

Directory of Open Access Journals (Sweden)

Full Text Available NO production catalysed by eNOS (endothelial nitric-oxide synthase plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in HEK-293 (human embryonic kidney 293 cells with a series of deletion mutants in both the AIE (?594–604, ?605–612 and ?626–634 and the C-terminal tail (?14; deletion of 1164–1177. The expression of ?594–604 and ?605–612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, ?626–634 and ?1164–1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS. Intriguingly, mutant ?594–604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594–604 regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.

Pei?Rung Wu

2014-08-01

379

Defining the Potassium Binding Region in an Apple Terpene Synthase*S?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating...

Green, Sol; Squire, Christopher J.; Nieuwenhuizen, Niels J.; Baker, Edward N.; Laing, William

2009-01-01

380