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Sample records for malate synthase activity

  1. Biochemical characterization of malate synthase G of P. aeruginosa

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    Volckaert Guido

    2009-06-01

    Full Text Available Abstract Background Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA. In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen. Results Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5°C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 ?M, Km acetyl CoA (12 ?M and Vmax (16.5 ?mol/minutes/mg enzyme. In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source. Conclusion The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.

  2. ?-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

    Science.gov (United States)

    de Freitas, Carla S; Kato, Lucilia; de Oliveira, Cecília M A; Queiroz, Luiz H K; Santana, Mábio J; Schuquel, Ivânia T; Delprete, Piero G; da Silva, Roosevelt A; Quintino, Guilherme O; da Silva Neto, Benedito R; Soares, Célia M A; Pereira, Maristela

    2014-12-01

    As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase. PMID:25412318

  3. Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

    International Nuclear Information System (INIS)

    Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH4)SO4, glyoxylate and high concentration of MgCl2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

  4. Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Y.P; Randall, D.D. (Univ. of Missouri, Columbia (USA))

    1989-04-01

    Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH{sub 4})SO{sub 4}, glyoxylate and high concentration of MgCl{sub 2} (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by {sup 32}P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation.

  5. Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates

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    Hulya Leventerler

    2013-08-01

    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

  6. 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

  7. Evidence for a relationship between malate metabolism and activity of 1-sinapoylglucose: L-malate sinapoyltransferase in radish (Raphanus sativus L.) cotyledons.

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    Strack, D; Reinecke, J; Takeuchi, S

    1986-02-01

    The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl-?-D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO 3 (-) as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO 3 (-) also NH 4 (+) . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO 3 (-) nitrogen were subsequently supplied with NH 4 (+) nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO 3 (-) /NH 4 (+) -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO 3 (-) /NH 4 (+) -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid. PMID:24241853

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

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

    2014-03-01

    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

  9. Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints

    International Nuclear Information System (INIS)

    Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from ?4.5 to ?3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determinccuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes

  10. The malate synthase of Paracoccidioides brasiliensis Pb01 is required in the glyoxylate cycle and in the allantoin degradation pathway.

    Science.gov (United States)

    Zambuzzi-Carvalho, Patrícia Fernanda; Cruz, Aline Helena Da Silva; Santos-Silva, Ludier Kesser; Goes, Alfredo Miranda; Soares, Célia Maria De Almeida; Pereira, Maristela

    2009-11-01

    In the present study, we examined the characteristics of cDNA, the regulation of the gene expression of Paracoccidioides brasiliensis MLS (Pbmls), and the enzymatic activity of the protein P. brasiliensis MLS (PbMLS) from the P. brasiliensis Pb01 isolate. Pbmls cDNA contains 1617 bp, encoding a protein of 539 amino acids with a predicted molecular mass of 60 kDa. The protein presents the MLSs family signature, the catalytic residues essential for enzymatic activity and the peroxisomal/glyoxysomal targeting signal PTS1. The high level of Pbmls transcript observed in the presence of two-carbon (2C) sources suggests that in P. brasiliensis, the primary regulation of carbon flux into the glyoxylate cycle (GC) was at the level of the Pbmls transcript. The gene expression, protein level, and enzymatic activity of Pbmls were highly induced by oxalurate in the presence of glucose and by proline in the presence of acetate. In the presence of glucose, the gene expression, protein level, and enzymatic activity of Pbmls were mildly stimulated by proline. Our results suggested that PbMLS condenses acetyl-CoA from both 2C sources (GC) and nitrogen sources (from proline and purine metabolism) to produce malate. The regulation of Pbmls by carbon and nitrogen sources was reinforced by the presence of regulatory motifs CREA and UIS found in the promoter region of the gene. PMID:19888806

  11. MALATE DEHYDROGENASE ACTIVITY POST EXPOSURE RECOVERY FROM LEAD INTOXICATED FRESHWATER FISH ANABAS TESTUDINEUS

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    Afsar Shaikh

    2012-03-01

    Full Text Available Malate dehydrogenase activity are important amongst the several enzymes available in the cells, Carbohydrates play an important role in the cellular process  Under extreme stress conditions, carbohydrate enzyme such as Malate dehydrogenase  have been known to act as the energy supplier in metabolic pathways and biochemical reactions. In the present investigation fish  treated with an equitoxic dose of 10 ppm of lead nitrate and lead acetate intoxicated fish After a period of 15 days of exposure a batch from lead nitrate exposed fish and a batch from lead acetate exposed fish were transfered to lead-free water. Fishes were scarified on 1, 4, 8, 12 and 15 days for the analysis of of recovery pattern  in tissues viz. liver, muscle, kidney, gill and brain .It is found that lead toxicated fishes were recovered after 15 days depends upon physical condition of the fish.

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

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    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...

  13. SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth.

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    Yang, Hui; Zhou, Lisha; Shi, Qian; Zhao, Yuzheng; Lin, Huaipeng; Zhang, Mengli; Zhao, Shimin; Yang, Yi; Ling, Zhi-Qiang; Guan, Kun-Liang; Xiong, Yue; Ye, Dan

    2015-04-15

    The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection. PMID:25755250

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

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    Genowefa Kubik-Dorosz

    2014-02-01

    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.

  15. A Bacillus subtilis malate dehydrogenase gene.

    OpenAIRE

    Jin, S; De Jesús-Berríos, M; Sonenshein, A L

    1996-01-01

    A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.

  16. Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

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    TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

  17. Characterization of putative capsaicin synthase promoter activity.

    Science.gov (United States)

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

    2009-10-31

    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

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

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    Munir, Erman; Hattori, Takefumi; Shimada, Mikio

    2008-01-01

    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...

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

    Science.gov (United States)

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

    2013-06-01

    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

  20. 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.

  1. Malate transport and vacuolar ion channels in CAM plants.

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    Cheffings, C M; Pantoja, O; Ashcroft, F M; Smith, J A

    1997-03-01

    Malate is a ubiquitous vacuolar anion in terrestrial plants that plays an important role in carbon metabolism and ionic homeostasis. In plants showing crassulacean acid metabolism (CAM), malate is accumulated as a central intermediary in the process of photosynthetic carbon assimilation, and it is also one of the major charge-balancing anions present in the vacuole. During the CAM cycle, malic acid produced as a result of dark CO(2) fixation accumulates in the vacuole at night (2 H(+) per malate), and is remobilized from the vacuole in the following light period. CAM plants thus provide a good model for studying both the mechanism and control of malate transport across the tonoplast. Thermodynamic considerations suggest that malate(2-) (the anionic species transported out of the cytosol) is passively distributed across the tonoplast. Malic acid accumulation could thus be explained by malate(2-) transport into the vacuole occurring electrophoretically in response to the transmembrane electrical potential difference established by the tonoplast H(+)-ATPase and/or H(+)-PPase. Recent studies using the patch-clamp technique have provided evidence for the existence of a vacuolar malate-selective anion channel (VMAL) in both CAM species and C(3) species. The VMAL current has a number of distinctive properties that include strong rectification (opening only at cytosolicside negative membrane potentials that would favour malate uptake into the vacuole), lack of Ca(2+) dependence, and slow activation kinetics. The kinetics of VMAL activation can be resolved into three components, consisting of an instantaneous current and two slower components with voltage-independent time constants of 0.76 s and 5.3 s in Kalanchoë daigremontiana. These characteristics suggest that the VMAL channel represents the major pathway for malate transport into the vacuole, although the strong rectification of the channel means there may be a separate, still-to-be-identified, transport mechanism for malate efflux. PMID:21245236

  2. Epidermal growth factor stimulates glycogen synthase activity in cultured cells.

    OpenAIRE

    Chan, C. P.; Krebs, E. G.

    1985-01-01

    Addition of epidermal growth factor (EGF) to quiescent cultured cells was found to stimulate the activity of glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), an enzyme subjected to regulation by covalent modification. In Swiss mouse 3T3 cells, the activation by EGF paralleled the effect seen with insulin; the time course and dose-response curves of the two polypeptide factors were similar. Stimulation of enzyme activity ratio [(activity in the absence of glu...

  3. Structural basis for glucose-6-phosphate activation of glycogen synthase

    OpenAIRE

    Baskaran, Sulochanadevi; Roach, Peter J; DePaoli-Roach, Anna A; Hurley, Thomas D.

    2010-01-01

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to...

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

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal and 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

  5. Neural tube closure depends on nitric oxide synthase activity.

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    Nachmany, Amir; Gold, Veronica; Tsur, Asaf; Arad, Dan; Weil, Miguel

    2006-01-01

    Neural tube (NT) closure is a multifactorial process that involves yet unresolved molecular mechanisms. It had been shown previously that high levels of nitric oxide (NO) block the process of NT closure in the chick embryo by inhibiting methionine synthase (MS). The MS inhibition and its effect on NT closure could be alleviated by folic acid, suggesting the involvement of the folate-methionine pathway in the process. Here we test the hypothesis that endogenous nitric oxide synthase (NOS) activity regulates the MS activity required in the process of NT closure. The experiments described here reveal that NOS activity per se, is indeed critical for NT closure in the chick embryo. Inhibition of NOS activity with either 2,4-diamino-6-hydroxypyrimidine (DAHP), which blocks biosynthesis of the NOS co-factor tetrahydrobiopterin (BH4), or with calmidazolium, which blocks calcium-calmodulin binding to NOS, resulted in reduced MS activity and consequently ablated NT closure. Addition of BH4 or the calcium ionophore A23187 restored NOS and MS activities, resulting in NT closure. The results described here imply that NOS and MS activities can serve as functional markers in this developmental process as they are essential in the process of NT closure. PMID:16300634

  6. Long Noncoding RNA MALAT1 Controls Cell Cycle Progression by Regulating the Expression of Oncogenic Transcription Factor B-MYB

    OpenAIRE

    Tripathi, Vidisha; Shen, Zhen; Chakraborty, Arindam; Giri, Sumanprava; Freier, Susan M.; Wu, Xiaolin; Zhang, Yongqing; Gorospe, Myriam; Prasanth, Supriya G.; Lal, Ashish; Prasanth, Kannanganattu V.

    2013-01-01

    The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p...

  7. Malate-Aspartate Shuttle Inhibitor Aminooxyacetate Acid Induces Apoptosis and Impairs Energy Metabolism of Both Resting Microglia and LPS-Activated Microglia.

    Science.gov (United States)

    Chen, Heyu; Wang, Caixia; Wei, Xunbin; Ding, Xianting; Ying, Weihai

    2015-06-01

    NADH shuttles mediate the transfer of the reducing equivalents of cytosolic NADH into mitochondria. Cumulating evidence has suggested that malate-aspartate shuttle (MAS), one of the two types of NADH shuttles, plays significant roles in such biological processes as glutamate synthesis in neurons. However, there has been no information regarding the roles of NADH shuttle in the survival and energy metabolism of microglia. In current study, using microglial BV2 cells as a cellular model, we determined the roles of MAS in the survival and energy metabolism of microglia by using aminooxyacetate acid (AOAA)-a widely used MAS inhibitor. Our study has suggested that AOAA can effectively inhibit the MAS activity of the cells. We also found that AOAA can induce both early- and late-stage apoptosis of resting microglia and lipopolysaccharides (LPS)-activated microglia. AOAA also induced mitochondrial depolarization, increases in the cytosolic Ca(2+) concentrations, and decreases in the intracellular ATP levels. Moreover, our study has excluded the possibility that the major nonspecific effect of AOAA-inhibition of GABA transaminase-is involved in theses effects of AOAA. Collectively, our study has provided first information suggesting significant roles of MAS in the survival and energy metabolism in both resting microglia and LPS-activated microglia. PMID:25998884

  8. Nitric oxide synthase activity in the olfactory bulb of anuran and urodele amphibians

    OpenAIRE

    Porteros Herrero, A?ngel; Are?valo Are?valo, Rosario; Crespo, Carlos; Brin?o?n, Jesu?s G.; Weruaga Prieto, Eduardo; Aijo?n Noguera, Jose?; Alonso Pen?a, Jose? Ramo?n

    1996-01-01

    Nitric oxide synthase activity was studied by means of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the main and accessory olfactory bulbs of the frog Rana perezi and the newt Triturus marmoratus. In both species, NADPH-diaphorase staining was observed in all olfactory fibers. Vomeronasal fibers were NADPH-diaphorase labeled in Triturus but they were NADPH-diaphorase negative in Rana. Nitric oxide synthase immunoreactivity was not observed in the primary...

  9. Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo

    DEFF Research Database (Denmark)

    Volke, V; Wegener, Gregers

    1999-01-01

    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.

  10. Influence of Simvastatin Chronotherapy on Erythrocytes Nitric oxide Synthase Activity

    Directory of Open Access Journals (Sweden)

    Gamaleldin I. Harisa

    2015-01-01

    Full Text Available This study was undertaken to investigate the impact simvastatin chronotherapy on erythrocytes Nitric Oxide Synthase (NOS activity. In the present study we select 24 adult human subjects have plasma total cholesterol high borderline (200-230 mg dL-1. Subjects were administrated simvastatin (20 mg day time for 2 months. Afterward, they were recommended for washout period (2 weeks, then subjects were administered the same dose at night time for additional 2 months. The present results showed that both day time and night time simvastatin treatment regimen significantly decrease plasma atherogenic index, malondialdehyde and protein carbonyl levels. However, paraoxonase-1 activity and total thiol level were significantly increased. Moreover, simvastatin therapy improved nitrite (NO marker levels in both plasma and erythrocytes compared to baselines. As well, simvastatin day time and night time dosing significantly increased erythrocytes NOS activity (46 and 64%, respectively compared to baselines. Night time dosing induced marked increase of NOS activity (19% compared to day time. This study confirms that night time dosing boost hypocholesterolemic, antioxidant and NO modulating effects of simvastatin compared to day time.

  11. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B

    2014-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

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

  13. Autocatalytic activation of acetyl-CoA synthase.

    Science.gov (United States)

    Maynard, Ernest L; Tan, Xiangshi; Lindahl, Paul A

    2004-04-01

    Acetyl-CoA synthase (ACS identical with ACS/CODH identical with CODH/ACS) from Moorella thermoacetica catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group of a corrinoid-iron-sulfur protein (CoFeSP). A time lag prior to the onset of acetyl-CoA production, varying from 4 to 20 min, was observed in assay solutions lacking the low-potential electron-transfer agent methyl viologen (MV). No lag was observed when MV was included in the assay. The length of the lag depended on the concentrations of CO and ACS, with shorter lags found for higher [ACS] and sub-saturating [CO]. Lag length also depended on CoFeSP. Rate profiles of acetyl-CoA synthesis, including the lag phase, were numerically simulated assuming an autocatalytic mechanism. A similar reaction profile was monitored by UV-vis spectrophotometry, allowing the redox status of the CoFeSP to be evaluated during this process. At early stages in the lag phase, Co(2+)FeSP reduced to Co(+)FeSP, and this was rapidly methylated to afford CH(3)-Co(3+)FeSP. During steady-state synthesis of acetyl-CoA, CoFeSP was predominately in the CH(3)-Co(3+)FeSP state. As the synthesis rate declined and eventually ceased, the Co(+)FeSP state predominated. Three activation reductive reactions may be involved, including reduction of the A- and C-clusters within ACS and the reduction of the cobamide of CoFeSP. The B-, C-, and D-clusters in the beta subunit appear to be electronically isolated from the A-cluster in the connected alpha subunit, consistent with the ~70 A distance separating these clusters, suggesting the need for an in vivo reductant that activates ACS and/or CoFeSP. PMID:15015040

  14. 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)

    Bechard Matthew E.

    2003-01-01

    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.

  15. Salmonella typhimurium Mutants with Altered Glutamate Dehydrogenase and Glutamate Synthase Activities

    OpenAIRE

    Dendinger, Susan M.; Patil, Lalita G.; Brenchley, Jean E.

    1980-01-01

    Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH4)2SO4 as the nitrogen source (Asm?)...

  16. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    Science.gov (United States)

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg. PMID:23467831

  17. Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes.

    Science.gov (United States)

    Bromley, Pamela E; Li, Yuneng O; Murphy, Shawn M; Sumner, Catherine M; Lynch, Daniel V

    2003-09-15

    Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C(6) ceramide or [3H]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C(6) ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 microM (with half-maximum activity at approximately 200 microM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC(50) of 0.4-0.8 and 16-20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology. PMID:12941304

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

    Directory of Open Access Journals (Sweden)

    Rasmusson Allan G

    2009-03-01

    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.

  19. Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB.

    Science.gov (United States)

    Tripathi, Vidisha; Shen, Zhen; Chakraborty, Arindam; Giri, Sumanprava; Freier, Susan M; Wu, Xiaolin; Zhang, Yongqing; Gorospe, Myriam; Prasanth, Supriya G; Lal, Ashish; Prasanth, Kannanganattu V

    2013-03-01

    The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation. PMID:23555285

  20. Adipocyte Mineralocorticoid Receptor Activation Leads to Metabolic Syndrome and Induction of Prostaglandin D2 Synthase.

    Science.gov (United States)

    Urbanet, Riccardo; Nguyen Dinh Cat, Aurelie; Feraco, Alessandra; Venteclef, Nicolas; El Mogrhabi, Soumaya; Sierra-Ramos, Catalina; Alvarez de la Rosa, Diego; Adler, Gail K; Quilliot, Didier; Rossignol, Patrick; Fallo, Francesco; Touyz, Rhian M; Jaisser, Frédéric

    2015-07-01

    Metabolic syndrome is a major risk factor for the development of diabetes mellitus and cardiovascular diseases. Pharmacological antagonism of the mineralocorticoid receptor (MR), a ligand-activated transcription factor, limits metabolic syndrome in preclinical models, but mechanistic studies are lacking to delineate the role of MR activation in adipose tissue. In this study, we report that MR expression is increased in visceral adipose tissue in a preclinical mouse model of metabolic syndrome and in obese patients. In vivo conditional upregulation of MR in mouse adipocytes led to increased weight and fat mass, insulin resistance, and metabolic syndrome features without affecting blood pressure. We identified prostaglandin D2 synthase as a novel MR target gene in adipocytes and AT56, a specific inhibitor of prostaglandin D2 synthase enzymatic activity, blunted adipogenic aldosterone effects. Moreover, translational studies showed that expression of MR and prostaglandin D2 synthase is strongly correlated in adipose tissues from obese patients. PMID:25966493

  1. The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant

    Science.gov (United States)

    Fujisawa, Makoto; Fackelmayer, Oliver J.; Liu, Jun; Krulwich, Terry A.; Hicks, David B.

    2010-01-01

    A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and ?-subunit levels at least as high as wild-type. Purified mutant F1F0-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395–17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness. PMID:20716528

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

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

    Directory of Open Access Journals (Sweden)

    Rimma Sergeevna Yudina

    2012-08-01

    Full Text Available 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 malate dehydrogenase in diverse organs and tissues make this enzyme a reliable and convenient genetic marker, which can effectively be used in special, ecological and population genetics.

  5. Nitric oxide synthase activity in the olfactory bulb of anuran and urodele amphibians.

    Science.gov (United States)

    Porteros, A; Arévalo, R; Crespo, C; Briñón, J G; Weruaga, E; Aijón, J; Alonso, J R

    1996-06-10

    Nitric oxide synthase activity was studied by means of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the main and accessory olfactory bulbs of the frog Rana perezi and the newt Triturus marmoratus. In both species, NADPH-diaphorase staining was observed in all olfactory fibers. Vomeronasal fibers were NADPH-diaphorase-positive in Triturus but they were NADPH-diaphorase-negative in Rana. Nitric oxide synthase immunoreactivity was not observed in the primary afferents in any case. Granule cells were NADPH-diaphorase-positive and nitric oxide synthase-immunopositive in the main and accessory olfactory bulb of Rana, and in the main olfactory bulb of Triturus. The homogeneous NADPH-diaphorase staining of olfactory fibers is similar to what has been reported in teleosts, and it contrasts with the spatial segregation of NADPH-diaphorase-positive and -negative olfactory projections in rodents. These results confirm the interspecies variability of the NADPH-diaphorase/nitric oxide synthase distribution in the olfactory system of vertebrates. PMID:8816257

  6. Calcium-Dependent Nitric Oxide Synthase Activity in Rat Thymocytes

    OpenAIRE

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

    1998-01-01

    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...

  7. Castration increases and androgens decrease nitric oxide synthase activity in the brain: Physiologic implications

    OpenAIRE

    Singh, Rajan; Pervin, Shehla; Shryne, James; Gorski, Roger; Chaudhuri, Gautam

    2000-01-01

    Sex differences in nitric oxide synthase (NOS) activity in different regions of the rat brain and effects of testosterone and dihydrotestosterone (DHT) treatment in orchidectomized animals were investigated. Regional but no sex differences in NOS activity were detected in gonadectomized animals. Orchidectomy significantly increased NOS activity in the hypothalamus, “amygdala,” and cerebellum but not in the cortex. In the hypothalamus, the increase in NOS activity after castration and its ...

  8. 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)

    Pankaj Kumar Bhowmik

    2001-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-17

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

  10. Calcium (hydrogen-1-malate) hexahydrate on Echeveria gibbiflora leaves and its effect on sperm cells.

    Science.gov (United States)

    Reyes, R; Sánchez-Vázquez, M L; Merchant Larios, H; Ortega Hernández, A; Delgado, N M

    2005-01-01

    Echeveria gibbiflora is a plant widely used for its contraceptive activity in traditional Mexican medicine. Data on calcium crystals in plants are not outstanding. In the case of the Echeveria gibbiflora leaves, however, its quality, quantity, and salt type are quite surprising; one striking result of its X-ray crystallographic data shows the presence of calcium bis (hydrogen-1-malate) hexahydrate [2(C4H5O(5)1), Ca(1)2+, 6(H2O1)]. This highly soluble compound might explain the rapid shape changes of calcium crystals. Because SEM-EDS analysis shows that calcium malate crystals were obtained in a highly pure state and the immobilization and agglutination pattern that OBACE show on human and bull spermatozoa are not found even when high concentrations of calcium bis (hydrogen-1-malate) hexahydrate salt are present it is not feasible to involucrate molecules as calcium malate as part of the OBACE contraceptive activity. PMID:16214732

  11. Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

    OpenAIRE

    Alexander-caudle, C.; Latinwo, L. M.; Jackson, J. H.

    1990-01-01

    Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and e...

  12. Vacuolar malate uptake is mediated by an anion-selective inward rectifier.

    OpenAIRE

    Hafke, Jb; Hafke, Y.; Smith, Ja; Lu?ttge, U.; Thiel, G.

    2003-01-01

    Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic...

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

    Directory of Open Access Journals (Sweden)

    Ashok Kumar

    2009-01-01

    Full Text Available 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 the pod wall and seed in both cultivars. The pod wall reached its maximum dry weight 21 days after podding (DAP in ‘Kaniva’ and 28 and 35 DAP in ‘Sona’ with terminal drought and irrigation, respectively. The dry weight of the pod wall decreased during seed filling, particularly in the plants subjected to terminal drought. The increase in seed dry weight followed a sigmoid curve with a lag phase of 14 DAP and 21 DAP in ‘Kaniva’ and ‘Sona’, respectively, followed by a rapid almost-linear phase until 35 DAP when the dry weight leveled off and even decreased slightly near maturity. Sucrose synthase activity peaked at 32 DAP in ‘Kaniva’ and 35 DAP in ‘Sona’ and then decreased to near zero at maturity. A significant and positive association was observed between seed dry weight at maturity and peak sucrose synthase activity in both cultivars and both treatments. We suggest that sucrose synthase is a good physiological indicator for use in breeding for improved seed size in chickpea.

  14. Modification of glycogen synthase activity in isolated rat hepatocytes by tumor-promoting phorbol esters: evidence for differential regulation of glycogen synthase and phosphorylase.

    OpenAIRE

    Roach, P. J.; Goldman, M.

    1983-01-01

    Glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), in isolated rat hepatocytes, has been identified as a novel intracellular target for tumor-promoting phorbol esters such as phorbol 12-tetradecanoate 13-acetate (TPA). Exposure of hepatocytes to TPA resulted in a 50% decrease in the activity ratio of glycogen synthase without/with glucose 6-phosphate. The inactivation was dose dependent and was half-maximal at a TPA concentration of approximately 16 nM (10 ng/...

  15. Activation of peroxynitrite by inducible nitric-oxide synthase: a direct source of nitrative stress.

    OpenAIRE

    Mare?chal, A.; Mattioli, T. A.; Stuehr, D. J.; Santolini, J.

    2007-01-01

    In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite,...

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

    OpenAIRE

    DURDI QUJEQ

    1999-01-01

    A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyru...

  17. Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis.

    OpenAIRE

    Carpenter, Ep; Hawkins, Ar; Frost, Jw; Brown, Ka

    1998-01-01

    Dehydroquinate synthase (DHQS) has long been regarded as a catalytic marvel because of its ability to perform several consecutive chemical reactions in one active site. There has been considerable debate as to whether DHQS is actively involved in all these steps, or whether several steps occur spontaneously, making DHQS a spectator in its own mechanism. DHQS performs the second step in the shikimate pathway, which is required for the synthesis of aromatic compounds in bacteria, microbial euka...

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

    OpenAIRE

    Beller, Harry R.; Edwards, Elizabeth A.

    2000-01-01

    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...

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

    Directory of Open Access Journals (Sweden)

    T.M. Fonovich de Schroeder

    1998-05-01

    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.

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

    Scientific Electronic Library Online (English)

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

    1998-05-01

    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 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-04-02

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

  2. Glycogen Synthase Kinase3 Beta Phosphorylates Serine 33 of p53 and Activates p53's Transcriptional Activity

    OpenAIRE

    Price Brendan D; Turenne Gaetan A

    2001-01-01

    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. Resu...

  3. Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

    Science.gov (United States)

    Tilbrook, Kimberley; Poirier, Yves; Gebbie, Leigh; Schenk, Peer M; McQualter, Richard B; Brumbley, Stevens M

    2014-10-01

    Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the ?-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species. PMID:24944109

  4. Expression and Activity of Nitric Oxide Synthase Isoforms in Methamphetamine-Induced Striatal Dopamine Toxicity

    OpenAIRE

    Friend, Danielle M.; Son, Jong H.; Keefe, Kristen A.; Fricks-gleason, Ashley N.

    2013-01-01

    Nitric oxide is implicated in methamphetamine (METH)-induced neurotoxicity; however, the source of the nitric oxide has not been identified. Previous work has also revealed that animals with partial dopamine loss induced by a neurotoxic regimen of methamphetamine fail to exhibit further decreases in striatal dopamine when re-exposed to methamphetamine 7–30 days later. The current study examined nitric oxide synthase expression and activity and protein nitration in striata of animals adminis...

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

    OpenAIRE

    Ashok Kumar; Turner, Neil C.

    2009-01-01

    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...

  6. Interaction between Nitric Oxide Synthase Inhibitor Induced Oscillations and the Activation Flow Coupling Response

    OpenAIRE

    Ances, Beau M; Greenberg, Joel H.; Detre, John A

    2009-01-01

    The role of nitric oxide (NO) in the activation-flow coupling (AFC) response to periodic electrical forepaw stimulation was investigated using signal averaged laser Doppler (LD) flowmetry. LD measures of calculated cerebral blood flow (CBF) were obtained both prior and after intra-peritoneal administration of the non-selective nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA) (40 mg/kg). Characteristic baseline low frequency vasomotion oscillations (0.17 Hz) were observed aft...

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

    OpenAIRE

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

    2013-01-01

    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 co...

  8. Infectious and inflammatory stimuli decrease endothelial nitric oxide synthase activity in vitro.

    OpenAIRE

    Pescarmona, Gianpiero; Morra, Emanuella; Todros, Tullia; Silvagno, Maria Francesca; Cardaropoli, Simona

    2003-01-01

    BACKGROUND: Perturbation of iron metabolism, especially the increase of serum ferritin levels, is often associated with both inflammation and hypertension. Changes in iron availability can affect an important regulator of vascular tone, the endothelial nitric oxide synthase (eNOS), activated by a heme-dependent dimerization. OBJECTIVE: To study the regulation of the anti-hypertensive eNOS in human endothelial cells, in correlation with iron metabolism alterations and stimuli triggering them i...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-09

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

  10. 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.

  11. Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures.

    Science.gov (United States)

    Hezari, M; Ketchum, R E; Gibson, D M; Croteau, R

    1997-01-15

    The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated. To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T. canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T. brevifolia stem. Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system. PMID:9016812

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-12-15

    Here we demonstrate that human keratinocytes possess a Ca{sup 2+}/ calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm{sup 2}) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[{sup 3}H]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/cm{sup 2}) of particulate NO synthase. A 5-fold increase in superoxide (O{sub 2}{sup -}) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO{sup -}) production by UVB (20 mJ/cm{sup 2})-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm{sup 2}) 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 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{sup -} released by UVB-irradiated human keratinocytes in skin erythema and inflammation. (Author).

  13. 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)

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

    Science.gov (United States)

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

    2012-11-12

    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

  15. 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

  16. Aggregation states of mitochondrial malate dehydrogenase.

    OpenAIRE

    Sánchez, S. A.; Hazlett, T. L.; Brunet, J. E.; Jameson, D M

    1998-01-01

    The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate b...

  17. Functional copper at the acetyl-CoA synthase active site

    OpenAIRE

    Seravalli, Javier; Gu, Weiwei; Tam, Annie; Strauss, Erick; Begley, Tadhg P.; Cramer, Stephen P.; Ragsdale, Stephen W.

    2003-01-01

    The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO2 fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduct...

  18. Iron fortification of rice seeds through activation of the nicotianamine synthase gene

    OpenAIRE

    Lee, Sichul; Jeon, Un Sil; Lee, Seung Jin; Kim, Yoon-keun; Persson, Daniel Pergament; Husted, Søren; Schjørring, Jan K.; Kakei, Yusuke; Masuda, Hiroshi; Nishizawa, Naoko K.; An, Gynheung

    2009-01-01

    The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from...

  19. Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution.

    Science.gov (United States)

    Hall, M D; Levitt, D G; Banaszak, L J

    1992-08-01

    The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms. PMID:1507230

  20. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    International Nuclear Information System (INIS)

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differe enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

  1. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

    2014-08-01

    Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. PMID:24814295

  2. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase†‡

    OpenAIRE

    Vedula, L. Sangeetha; Cane, David E.; Christianson, David W.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with R- and S-azabisabolenes and PPi bind 3 Mg2+ ions and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change...

  3. Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length?

    OpenAIRE

    Grabin?ska, Kariona A.; Magnelli, Paula; Robbins, Phillips W.

    2006-01-01

    Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p...

  4. Endothelial nitric oxide synthase activation is critical for vascular leakage during acute inflammation in vivo

    OpenAIRE

    Bucci, Mariarosaria; Roviezzo, Fiorentina; Posadas, Inmaculada; Yu, Jun; Parente, Luca; Sessa, William C.; Ignarro, Louis J.; Cirino, Giuseppe

    2005-01-01

    The role of endothelium-derived nitric oxide (NO) in acute inflammation is not known. Here, we examine acute inflammation in congenic endothelial NO synthase-deficient (eNOS-/-) mice. Intraplantar injection of carrageenan induces a biphasic inflammatory response. The early phase (0-6 h) is largely eliminated, and the secondary phase (24-96 h) is markedly reduced in eNOS-/- but not WT mice. Inhibition of phosphatidylinositol 3-kinase or hsp90, pathways upstream of eNOS activation, also reduces...

  5. Complete tracking of transient proton flow through active chloroplast ATP synthase

    OpenAIRE

    1987-01-01

    Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of Pi and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typical...

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

    Directory of Open Access Journals (Sweden)

    Silva José Carlos da

    2003-01-01

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

  7. Evidence that Ferredoxin Interfaces with an Internal Redox Shuttle in Acetyl-CoA Synthase During Reductive Activation and Catalysis †

    OpenAIRE

    Bender, Gu?nes?; Ragsdale, Stephen W.

    2010-01-01

    Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-14C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends ...

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

    Science.gov (United States)

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

    2014-09-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual ??-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids. PMID:24990389

  9. Cyclic AMP-dependent and cyclic AMP-independent antagonism of insulin activation of cardiac glycogen synthase.

    Science.gov (United States)

    Ramachandran, C; Angelos, K L; Walsh, D A

    1982-02-10

    The hormonal regulation of glycogen synthase has been studied with isolated perfused hearts that were depleted of 85% of their endogenous glycogen. Glycogen depletion alone promoted a 3-fold activation of glycogen synthase and magnified by 3-fold the response to insulin. Glycogen depletion also facilitated the detection of epinephrine-promoted glycogen synthase inactivation. Hormonal effects on glycogen synthase have been correlated with changes in phosphorylase, phosphorylase kinase, and tissue cAMP levels. Insulin activation of glycogen synthase was observed within 90 s of hormone addition and was maximal by 4 min. A half-maximum effect was obtained at an insulin concentration of 100 microunits/ml. Insulin-dependent activation is reversed by beta-adrenergic agonists, alpha-adrenergic agonists, and glucagon. Each promote the same degree of inactivation and the maximum extent of inactivation produced by each is independent of whether or not the tissue has been stimulated with insulin. beta-Adrenergic agonists and glucagon act via cAMP, alpha-agonists most likely act via intracellular Ca2+ translocation, and insulin action would appear to be independent of either cAMP or Ca2+. The action of epinephrine on cardiac glycogen synthase is mediated by interaction with both alpha- and beta-receptors. As indicated by dose-response curves, receptor occupancy of each occurs to an almost equal extent at suboptimal epinephrine concentrations. Regulation of cardiac glycogen synthase by epinephrine thus is mediated by two second messenger systems which converge to produce the end physiological response. PMID:6276386

  10. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    Science.gov (United States)

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-01

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5?-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a ?-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the ?-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  11. Induction of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity by fungal elicitor in cultures of Petroselinum crispum.

    OpenAIRE

    Mccue, K. F.; Conn, E. E.

    1989-01-01

    The effects of a cell wall fraction of the fungus Phytophthora megasperma on the enzymatic activities of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) in extracts of cultured parsley cells (Petroselinum crispum) were examined. The specific activity of a plastidic form of DAHP synthase, designated DS-Mn by Ganson et al. [Ganson, R. J., d'Amato, T. A. & Jensen, R. A. (1986) Plant Physiol. 82, 203-210], was increased 2- to 3-fold in extracts of treated cells, with max...

  12. An evolutionarily conserved alternate metal ligand is important for activity in ?-isopropylmalate synthase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Frantom, Patrick A; Birman, Yuliya; Hays, Brittani N; Casey, Ashley K

    2014-10-01

    Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon-carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in ?-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to ?-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of ?-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for ?-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes. PMID:25064783

  13. Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Nassar Marwa

    2006-10-01

    Full Text Available Abstract Background Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. Results We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098 after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7, suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. Conclusion We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

  14. The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant: mutations in the critical alkaliphile-specific residue Lys-180 and other residues that support alkaliphile oxidative phosphorylation.

    Science.gov (United States)

    Fujisawa, Makoto; Fackelmayer, Oliver J; Liu, Jun; Krulwich, Terry A; Hicks, David B

    2010-10-15

    A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and ?-subunit levels at least as high as wild-type. Purified mutant F(1)F(0)-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395-17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness. PMID:20716528

  15. Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

    Science.gov (United States)

    Lakhal-Naouar, Ines; Jardim, Armando; Strasser, Rona; Luo, Shen; Kozakai, Yukiko; Nakhasi, Hira L.; Duncan, Robert C.

    2012-01-01

    Background Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. Methodology/Principal Findings Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. Conclusion/Significance Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target. PMID:23094117

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

    OpenAIRE

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

    2002-01-01

    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 wi...

  17. Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene[S

    Science.gov (United States)

    Burns, Tara Ann; Subathra, Marimuthu; Signorelli, Paola; Choi, Young; Yang, Xiaofeng; Wang, Yong; Villani, Maristella; Bhalla, Kapil; Zhou, Daohong; Luberto, Chiara

    2013-01-01

    Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells. PMID:23160178

  18. 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)

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

    2010-12-01

    Full Text Available PURPOSE: The etiology of obstructive bladder dysfunction includes free radical damage to mitochondria. Feeding rabbits a standardized grape suspension protects the ability of the bladder to contract and empty in part by preventing mitochondrial damage, thus maintaining smooth muscle and mucosal 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.

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

    Directory of Open Access Journals (Sweden)

    Vijay Venugopal

    2010-12-01

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

  20. ‘Dopamine-first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

    Science.gov (United States)

    Lichman, Benjamin R; Gershater, Markus C; Lamming, Eleanor D; Pesnot, Thomas; Sula, Altin; Keep, Nicholas H; Hailes, Helen C; Ward, John M

    2015-01-01

    Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity. PMID:25620686

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

    DEFF Research Database (Denmark)

    Willemoës, Martin; MØlgaard, Anne

    2005-01-01

    GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) 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

  2. Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

    Scientific Electronic Library Online (English)

    A.M., Herculano; M.E., Crespo-López; S.M.A., Lima; D.L.W., Picanço-Diniz; J.L.M. Do, Nascimento.

    2006-03-01

    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

  3. Increased nitric oxide synthase in the lung after ozone inhalation is associated with activation of NF-kappa B.

    OpenAIRE

    Laskin, D. L.; Sunil, V.; Guo, Y.; Heck, D. E.; Laskin, J. D.

    1998-01-01

    Acute inhalation of ozone is associated with a inflammatory response characterized by the accumulation of macrophages at sites of tissue injury. These cells, along with resident alveolar epithelial cells, become activated and release cytotoxic and proinflammatory mediators, such as nitric oxide (.NO), that we speculate contribute to toxicity. In these studies we analyzed mechanisms regulating increased .NO synthase activity in lung macrophages and type II cells after ozone inhalation. Brief e...

  4. Mechanism of influence of phosphorylation on serine 124 on a decrease of catalytic activity of human thymidylate synthase

    OpenAIRE

    Jarmu?a, Adam; Fr?czyk, Tomasz; Cieplak, Piotr; Rode, Wojciech

    2010-01-01

    Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine r...

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

    Science.gov (United States)

    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

    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

  6. Materials and methods for efficient succinate and malate production

    Science.gov (United States)

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

    2014-04-08

    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.

  7. UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis

    Science.gov (United States)

    Moon, Jong-Seok; Lee, Seonmin; Park, Mi-Ae; Siempos, Ilias I.; Haslip, Maria; Lee, Patty J.; Yun, Mijin; Kim, Chun K.; Howrylak, Judie; Ryter, Stefan W.; Nakahira, Kiichi; Choi, Augustine M.K.

    2015-01-01

    Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1? and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1? gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis. PMID:25574840

  8. Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala: Fabricius, 1794

    Directory of Open Access Journals (Sweden)

    A. C. Faraldo

    2007-08-01

    Full Text Available Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL, yeast-injected (YIL, and saline-injected (SIL larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

  9. 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

  10. Functional copper at the acetyl-CoA synthase active site.

    Science.gov (United States)

    Seravalli, Javier; Gu, Weiwei; Tam, Annie; Strauss, Erick; Begley, Tadhg P; Cramer, Stephen P; Ragsdale, Stephen W

    2003-04-01

    The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the "NiFeC" EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)-S(3) site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu-S bond length of 2.25 A and a metal neighbor at 2.65 A, consistent with the Cu-Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu-S(3)Se environment with a 2.4-A Se-Cu bond, strongly implicating a Cu-SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms. PMID:12589021

  11. Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase*

    OpenAIRE

    Irikura, Daisuke; Aritake, Kosuke; Nagata, Nanae; Maruyama, Toshihiko; Shimamoto, Shigeru; Urade, Yoshihiro

    2009-01-01

    We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 ?m)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a ...

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

    Directory of Open Access Journals (Sweden)

    Laurini Ricardo

    2003-06-01

    Full Text Available 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 Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of 14C-citrulline from 14C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS and neuronal (nNOS isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.

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

    Science.gov (United States)

    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

    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

  14. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.

    Science.gov (United States)

    Tripathi, Vidisha; Ellis, Jonathan D; Shen, Zhen; Song, David Y; Pan, Qun; Watt, Andrew T; Freier, Susan M; Bennett, C Frank; Sharma, Alok; Bubulya, Paula A; Blencowe, Benjamin J; Prasanth, Supriya G; Prasanth, Kannanganattu V

    2010-09-24

    Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression. PMID:20797886

  15. Tramadol anti-inflammatory activity is not related to a direct inhibitory action on prostaglandin endoperoxide synthases.

    Science.gov (United States)

    Buccellati, C; Sala, A; Ballerio, R; Bianchib, M

    2000-01-01

    The analgesic drug tramadol has been shown to relieve pain in inflammatory conditions, to inhibit the development of experimental inflammation, and to reduce prostaglandin (PG)E(2)concentrations in the inflammatory exudate. In this study, we evaluated the putative activity of tramadol to suppress prostaglandin endoperoxide synthase-1 (PGHS-1), and prostaglandin endoperoxide synthase-2 (PGHS-2) activities in human whole blood in vitro. Platelet thromboxane (Tx)B(2)production and monocyte PGE(2)production in LPS- stimulated blood were measured in samples incubated with different concentrations (300 ng/ml, 3 microg/ml, 30 microg/ml) of tramadol or its enantiomers. Neither tramadol nor the enantiomers inhibited the formation of arachidonic acid metabolites. Our results indicate that the anti-inflammatory effect of tramadol demonstrated in some models is not related to a direct inhibitory effect on the formation of prostanoids. PMID:11124014

  16. Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

    Directory of Open Access Journals (Sweden)

    A.M. Herculano

    2006-03-01

    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.

  17. Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

    OpenAIRE

    Notarnicola, Maria; Pisanti, Simona; Tutino, Valeria; Bocale, Domenica; Rotelli, Maria Teresa; Gentile, Antonio; Memeo, Vincenzo; Bifulco, Maurizio; Perri, Enzo; Caruso, Maria Gabriella

    2010-01-01

    Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growt...

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

    Science.gov (United States)

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

    2013-11-01

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNF?) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain. PMID:24147811

  19. Iridoid synthase activity is common among the plant progesterone 5?-reductase family.

    Science.gov (United States)

    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

    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

  20. Interaction between Nitric Oxide Synthase Inhibitor Induced Oscillations and the Activation Flow Coupling Response

    Science.gov (United States)

    Ances, Beau M.; Greenberg, Joel. H.; Detre, John A.

    2009-01-01

    The role of nitric oxide (NO) in the activation-flow coupling (AFC) response to periodic electrical forepaw stimulation was investigated using signal averaged laser Doppler (LD) flowmetry. LD measures of calculated cerebral blood flow (CBF) were obtained both prior and after intra-peritoneal administration of the non-selective nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA) (40 mg/kg). Characteristic baseline low frequency vasomotion oscillations (0.17 Hz) were observed after L-NNA administration. These LDCBF oscillations were synchronous within but not between hemispheres. L-NNA reduced the magnitude of the AFC response (p< 0.05) for longer stimuli (1 minute) with longer inter-stimulus intervals (2 minutes). In contrast, the magnitude of the AFC response for short duration stimuli (4 seconds) with short inter-stimulus intervals (20 seconds) was augmented (p < 0.05) after L-NNA. An interaction occurred between L-NNA induced vasomotion oscillations and the AFC response with the greatest increase occurring at the stimulus harmonic closest to the oscillatory frequency. Nitric oxide may therefore modulate the effects of other vasodilators involved in vasomotion oscillations and the AFC response. PMID:19900416

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

    DEFF Research Database (Denmark)

    Prats, Clara; Helge, JØrn W

    2009-01-01

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

  2. Iron fortification of rice seeds through activation of the nicotianamine synthase gene

    Science.gov (United States)

    Lee, Sichul; Jeon, Un Sil; Lee, Seung Jin; Kim, Yoon-Keun; Persson, Daniel Pergament; Husted, Søren; Schjørring, Jan K.; Kakei, Yusuke; Masuda, Hiroshi; Nishizawa, Naoko K.; An, Gynheung

    2009-01-01

    The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression. PMID:20080803

  3. Factors affecting lactate and malate utilization by Selenomonas ruminantium.

    Science.gov (United States)

    Evans, J D; Martin, S A

    1997-12-01

    Lactate utilization by Selenomonas ruminantium is stimulated in the presence of malate. Because little information is available describing lactate-plus-malate utilization by this organism, the objective of this study was to evaluate factors affecting utilization of these two organic acids by two strains of S. ruminantium. When S. ruminantium HD4 and H18 were grown in batch culture on DL-lactate and DL-malate, both strains coutilized both organic acids for the initial 20 to 24 h of incubation and acetate, propionate, and succinate accumulated. However, when malate and succinate concentrations reached 7 mM, malate utilization ceased, and with strain H18, there was a complete cessation of DL-lactate utilization. Malate utilization by both strains was also inhibited in the presence of glucose. S. ruminantium HD4 was unable to grow on 6 mM DL-lactate at extracellular pH 5.5 in continuous culture (dilution rate, 0.05 h-1) and washed out of the culture vessel. Addition of 8 mM DL-malate to the medium prevented washout on 6 mM DL-lactate at pH 5.5 and resulted in succinate accumulation. Addition of malate also increased bacterial protein, acetate, and propionate concentrations in continuous culture. These results suggest that 8 mM DL-malate enhances the ability of strain HD4 to grow on 6 mM DL-lactate at extracellular pH 5.5. PMID:9471965

  4. 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

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

    OpenAIRE

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

    2008-01-01

    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...

  6. Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs.

    Czech Academy of Sciences Publication Activity Database

    Lagana, G.; Bellocco, E.; Mannucci, C.; Leuzzi, U.; Tellone, E.; Kotyk, Arnošt; Galtieri, A.

    2006-01-01

    Ro?. 55, ?. 6 (2006), s. 675-688. ISSN 0862-8408 Institutional research plan: CEZ:AV0Z50110509 Keywords : elasmobranchs * lactate dehydrogenase * malate dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 2.093, year: 2006

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

    DEFF Research Database (Denmark)

    VigelsØ Hansen, Andreas; Andersen, Nynne Bjerre

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and 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

  8. Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

    Directory of Open Access Journals (Sweden)

    Olga N. Rozova

    2015-02-01

    Full Text Available We have expressed the l-malate dehydrogenase (MDH genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa enzyme displaying nearly equal reductive (malate formation and oxidative (oxaloacetate formation activities and higher affinity to malate (Km = 0.11 mM than to oxaloacetate (Km = 0.34 mM. The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa, two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM than to malate (Km = 1.28 mM. The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.

  9. N-Methyl-D-Aspartate Receptor-Dependent Denitrosylation of Neuronal Nitric Oxide Synthase Increase the Enzyme Activity

    OpenAIRE

    Qu, Zhong-wei; Miao, Wan-ying; Hu, Shu-qun; Li, Chong; Zhuo, Xing-li; Zong, Yan-yan; Wu, Yong-ping; Zhang, Guang-yi

    2012-01-01

    Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys331, one of the zinc-t...

  10. The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

    OpenAIRE

    Sordelli, Micaela S.; Beltrame, Jimena S.; Burdet, Juliana; Zotta, Elsa; Pardo, Romina; Cella, Maximiliano; Franchi, Ana M.; Ribeiro, Maria Laura

    2011-01-01

    Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot?1 h?1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dorma...

  11. 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

  12. Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.

    Science.gov (United States)

    Uttaro, A D; Opperdoes, F R

    1997-10-01

    Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg-1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+. PMID:9297700

  13. 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)

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

    1996-03-01

    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.

  14. Specificity of calreticulin transacetylase to acetoxy derivatives of benzofurans: effect on the activation of platelet nitric oxide synthase.

    Science.gov (United States)

    Gupta, Anjali; Priya, Nivedita; Jalal, Sarah; Singh, Prabhjot; Chand, Karam; Raj, Hanumantharao G; Parmar, Virinder S; DePass, Anthony L; Sharma, Sunil K

    2010-09-01

    Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we have studied the influence of differently substituted benzofurans on the CRTAase activity to study the effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl) on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase. Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin (DAMC). PMID:20600569

  15. Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

    OpenAIRE

    Bevan, Heather S.; Slater, Sadie C.; Clarke, Hayley; Cahill, Paul A.; Mathieson, Peter W.; Welsh, Gavin I.; Satchell, Simon C.

    2011-01-01

    Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10–20 dyn/cm2) in conditionally immortalized human GEnC. Laminar shear stress caused an orientatio...

  16. The effect of intermittent cryotherapy on the activities of citrate synthase and lactate dehydrogenase in regenerating skeletal muscle

    Scientific Electronic Library Online (English)

    Nuno Miguel Lopes de, Oliveira; João Luiz Quagliotti, Durigan; Flávia Simone, Munin; Maria Luiza Barcelos, Schwantes; Tania de Fátima, Salvini.

    2013-02-01

    Full Text Available This study examined the effect of three sessions of cryotherapy (three sessions of 30 minutes applied each 2 h) and muscle compression in the regenerating skeletal muscle of the rats. The middle belly of tibialis anterior muscle was injured by a frozen iron bar and received one of the following inte [...] rvention: injury + cryotherapy (treated with cryotherapy); injury + placebo (sand pack), and injury (I).The enzymatic activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the presence of 1mM or 10mM pyruvate. The ANOVA and Tukey's test (p

  17. Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus.

    OpenAIRE

    Currò, D.; Volpe, A R; Preziosi, P

    1996-01-01

    1. In the presence of atropine (1 microM) and guanethidine (5 microM), electrical field stimulation (EFS, 120 mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM)-precontracted longitudinal muscle strips from the rat gastric fundus. 2. L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adre...

  18. Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

    OpenAIRE

    Ricca, E.; Limauro, D.; Lago, C. T.; Felice, M.

    1988-01-01

    The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy aci...

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

    Directory of Open Access Journals (Sweden)

    Fu Y

    2014-06-01

    Full Text Available Yunfeng Fu,1 Xinyu Wang,1 Xiaodong Cheng,1 Feng Ye,2 Xing Xie,1,2 Weiguo Lu1,2 1Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, 2Women's Reproduction and Health Laboratory of Zhejiang Province, Hangzhou, People's Republic of China Background: Glycogen synthase kinase-3 (GSK-3 plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-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

  20. Characterization of the anti-inflammatory activity of enones based on the evaluation of their heme oxygenase-1 and inducible NO synthase activity

    OpenAIRE

    Ru?cker, Hannelore-maria

    2015-01-01

    To fight chronic inflammation in autoimmune diseases such as rheumatic arthritis and multiple sclerosis or cancer is an exigency. Therefore the inhibition of proinflammatory proteins such as inducible NO-synthase (iNOS) and the activation of anti-inflammatory proteins such as heme oxygenase-1 (HO-1) are important measures to be addressed. Many natural products and synthetic compounds with an ?,?-unsaturated carbonyl moiety reveal a variety of biological properties, including antioxidant, an...

  1. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs

    International Nuclear Information System (INIS)

    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)

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

    Science.gov (United States)

    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

    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

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

    DEFF Research Database (Denmark)

    Zerbe, Philipp; Chiang, Angela

    2014-01-01

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

  4. Determination of amino-acidic positions important for Ocimum basilicum geraniol synthase activity

    Directory of Open Access Journals (Sweden)

    Marc Bergdoll

    2013-02-01

    Full Text Available Terpenes are one of the largest and most diversified families of natural compounds. Although they have found numerous industrial applications, the molecular basis of their synthesis in plants has, until now, not been fully understood. Plant genomes have been shown to contain dozens of terpene synthase (TPS genes, however knowledge of their amino-acidic protein sequence in not sufficient to predict which terpene(s will be produced by a particular enzyme. In order to investigate the structural basis of a TPS specificity, we performed site directed mutations in the geraniol synthase from Ocimum basilicum. The results obtained suggest that a specific region on the catalytic site plays an important role in GPP transformation, either by stabilizing the GPP substrate on the catalytic site, or by enabling its transformation into a monoterpenol via an intermediate carbocation.

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

    OpenAIRE

    Fairman Robert; Kokona Bashkim; Forbes Siiri; Lighthall Rachel; Clauson Cheryl; Bollivar David W; Kundrat Lenka; Jaffe Eileen K

    2004-01-01

    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...

  6. Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

    OpenAIRE

    Giroud, Claire; Moreau, Magali; Mattioli, Tony A.; Balland, Ve?ronique; Boucher, Jean-luc; Xu-li, Yun; Stuehr, Dennis J.; Santolini, Je?ro?me

    2009-01-01

    Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate l-Arg first to N?-hydroxyl-l-arginine and then to l-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Alt...

  7. Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

    OpenAIRE

    Durbin, Mary L.; Denton, Amy L.; Clegg, Michael T.

    2001-01-01

    Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relati...

  8. Dual Regulation of Muscle Glycogen Synthase during Exercise by Activation and Compartmentalization*

    OpenAIRE

    Prats, Clara; Helge, Jørn W.; Nordby, Pernille; Qvortrup, Klaus; Ploug, Thorkil; Dela, Flemming; Wojtaszewski, Jørgen F. P.

    2009-01-01

    Glycogen synthase (GS) is considered the rate-limiting enzyme in glycogenesis but still today there is a lack of understanding on its regulation. We have previously shown phosphorylation-dependent GS intracellular redistribution at the start of glycogen re-synthesis in rabbit skeletal muscle (Prats, C., Cadefau, J. A., Cussó, R., Qvortrup, K., Nielsen, J. N., Wojtaszewki, J. F., Wojtaszewki, J. F., Hardie, D. G., Stewart, G., Hansen, B. F., and Ploug, T. (2005) J. Biol. Chem. 280, 23165–23...

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

    Directory of Open Access Journals (Sweden)

    Helena H. Chowdhury

    2014-10-01

    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.

  10. 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

  11. 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

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

    Science.gov (United States)

    Franch, J; Aslesen, R; Jensen, J

    1999-11-15

    We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen synthesis. Surprisingly, adrenaline did not inhibit glycogen synthesis stimulated by glycogen-depleting contractile activity. In agreement with this, the fractional activity of glycogen synthase was high when adrenaline was present after exercise, whereas adrenaline decreased the fractional activity of glycogen synthase to a low level during stimulation with insulin. Furthermore, adrenaline activated glycogen phosphorylase almost completely during stimulation with insulin, whereas a much lower activation of glycogen phosphorylase was observed after contractile activity. Thus adrenaline does not inhibit contraction-stimulated glycogen synthesis. PMID:10548555

  13. Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis.

    Science.gov (United States)

    Bender, Güne?; Ragsdale, Stephen W

    2011-01-18

    Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle. PMID:21141812

  14. Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure.

    OpenAIRE

    Tayeh, M A; Madigan, M. T.

    1987-01-01

    The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in ...

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

    OpenAIRE

    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

    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...

  16. Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

    OpenAIRE

    Lakhal-naouar, Ines; Jardim, Armando; Strasser, Rona; Luo, Shen; Kozakai, Yukiko; Nakhasi, Hira L.; Duncan, Robert C.

    2012-01-01

    Leishmaniasis is a neglected tropical disease that continues to pose a public health threat worldwide due to the absence of an effective vaccine, drug toxicity and parasite resistance. In an attempt to identify new potential drug targets, we focused our research on Leishmania donovani argininosuccinate synthase (LdASS), which is more highly expressed in the virulent form of the parasite. Using two cell lines that over expressed the wild type or a mutant form of LdASS, we demonstrated that LdA...

  17. Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

    OpenAIRE

    Strop, P.; Changchien, L.; Maley, F; Montfort, W. R.

    1997-01-01

    Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Es...

  18. The Cytoplasmic Tail of GM3 Synthase Defines Its Subcellular Localization, Stability, and In Vivo Activity

    OpenAIRE

    Uemura, Satoshi; Yoshida, Sayaka; Shishido, Fumi; Inokuchi, Jin-ichi

    2009-01-01

    GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH2-terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was e...

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

    OpenAIRE

    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

    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...

  20. The use of (E)- and (Z)-phosphoenol-3-fluoropyruvate as mechanistic probes reveals significant differences between the active sites of KDO8P and DAHP synthases.

    Science.gov (United States)

    Furdui, Cristina M; Sau, Apurba K; Yaniv, Orit; Belakhov, Valery; Woodard, Ronald W; Baasov, Timor; Anderson, Karen S

    2005-05-17

    The enzymes 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-2-heptulosonate-7-phosphate (DAHP) synthase catalyze a similar aldol-type condensation between phosphoenolpyruvate (PEP) and the corresponding aldose: arabinose 5-phosphate (A5P) and erythrose 4-phosphate (E4P), respectively. While KDO8P synthase is metal-dependent in one class of organisms and metal-independent in another, only a metal-dependent class of DAHP synthases has thus far been identified in nature. We have used catalytically active E and Z isomers of phosphoenol-3-fluoropyruvate [(E)- and (Z)-FPEP, respectively] as mechanistic probes to characterize the differences and/or the similarities between the metal-dependent and metal-independent KDO8P synthases as well as between the metal-dependent KDO8P synthase and DAHP synthase. The direct evidence of the overall stereochemistry of the metal-dependent Aquifex pyrophilus KDO8P synthase (ApKDO8PS) reaction was obtained by using (E)- and (Z)-FPEPs as alternative substrates and by subsequent (19)F NMR analysis of the products. The results reveal the si face addition of the PEP to the re face of the carbonyl of A5P, and establish that the stereochemistry of ApKDO8PS is identical to that of the metal-independent Escherichia coli KDO8P synthase enzyme (EcKDO8PS). In addition, both ApKDO8PS and EcKDO8PS enzymes exhibit high selectivity for (E)-FPEP versus (Z)-FPEP, the relative k(cat)/K(m) ratios being 100 and 33, respectively. In contrast, DAHP synthase does not discriminate between (E)- and (Z)-FPEP (the k(cat)/K(m) being approximately 7 x 10(-)(3) microM(-)(1) s(-)(1) for both compounds). The pre-steady-state burst experiments for EcKDO8PS showed that product release is rate-limiting for the reactions performed with either PEP, (E)-FPEP, or (Z)-FPEP, although the rate constants, for both product formation and product release, were lower for the fluorinated analogues than for PEP [125 and 2.3 s(-)(1) for PEP, 2.5 and 0.2 s(-)(1) for (E)-FPEP, and 9 and 0.1 s(-)(1) for (Z)-FPEP, respectively]. The observed data indicate substantial differences in the PEP subsites and open the opportunity for the design of selective inhibitors against these two families of enzymes. PMID:15882071

  1. 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

  2. A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.

    Science.gov (United States)

    Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Hisano, Tamao; Abe, Hideki; Tsuge, Takeharu

    2015-06-01

    Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation. PMID:25503319

  3. 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+

  4. Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry.

    Science.gov (United States)

    Jhee, K H; Yang, L H; Ahmed, S A; McPhie, P; Rowlett, R; Miles, E W

    1998-05-01

    To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates. PMID:9565551

  5. 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)

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

    2014-12-01

    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

  6. Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis.

    Science.gov (United States)

    Beckner, Marie E; Gobbel, Glenn T; Abounader, Roger; Burovic, Fatima; Agostino, Naomi R; Laterra, John; Pollack, Ian F

    2005-12-01

    Increased glycolysis is characteristic of malignancy. Previously, with a mitochondrial inhibitor, we demonstrated that glycolytic ATP production was sufficient to support migration of melanoma cells. Recently, we found that glycolytic enzymes were abundant and some were increased in pseudopodia formed by U87 glioma (astrocytoma) cells. In this study, we examined cell migration, adhesion (a step in migration), and Matrigel invasion of U87 and LN229 glioma cells when their mitochondria were inhibited with sodium azide or limited by 1% O(2). Cell migration, adhesion, and invasion were comparable, with and without mitochondrial inhibition. Upon discovering that glycolysis alone can support glioma cell migration, unique features of glucose metabolism in astrocytic cells were investigated. The ability of astrocytic cells to remove lactate, the inhibitor of glycolysis, via gluconeogenesis and incorporation into glycogen led to consideration of supportive genetic mutations. Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from constitutive inhibition by glycogen synthase kinase-3 (GSK3). We hypothesize that glycolysis in gliomas can support invasive migration, especially when aided by loss of PTEN's regulation on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway leading to inhibition of GSK3. Migration of PTEN-mutated U87 cells was studied for release of extracellular lactic acid and support by gluconeogenesis, loss of PTEN, and active PI3K. Lactic acid levels plateaued and phosphorylation changes confirmed activation of the PI3K/Akt pathway and glycogen synthase when cells relied only on glycolysis. Glycolytic U87 cell migration and phosphorylation of GSK3 were inhibited by PTEN transfection. Glycolytic migration was also suppressed by inhibiting PI3K and gluconeogenesis with wortmannin and metformin, respectively. These findings confirm that glycolytic glioma cells can migrate invasively and that the loss of PTEN is supportive, with activated glycogenic potential included among the relevant downstream effects. PMID:16170333

  7. Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

    Science.gov (United States)

    Díaz-Lobo, Mireia; Garcia-Amorós, Jaume; Fita, Ignacio; Velasco, Dolores; Guinovart, Joan J; Ferrer, Joan C

    2015-07-14

    Glycogen is a polymer of ?-1,4- and ?-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 ?M). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders. PMID:26055498

  8. [Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)].

    Science.gov (United States)

    Zhu, Rui-Juan; Rao, Xin-Feng; Wei, Deng-Bang; Wang, Duo-Wei; Wei, Lian; Sun, Sheng-Zhen

    2012-04-25

    To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P level of the zokor was significantly higher than that of the pika (P level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P level of plateau zokor was markedly higher than that of MDH2 (P 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen. PMID:22513468

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

    Directory of Open Access Journals (Sweden)

    Hildgund Schrempf

    2010-09-01

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

  10. 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)

    Vind, B F; Birk, J B

    2012-01-01

    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.

  11. Important differences in nitric oxide synthase activity and predominant isoform in reproductive tissues from human and rat

    Directory of Open Access Journals (Sweden)

    Al-Hijji J

    2003-02-01

    Full Text Available Abstract For the extrapolation of data obtained from experimental animals to the human situation, it is important to know the similarities and differences between human and animal species. Some important characteristics of nitric oxide synthase (NOS in myometrium and vagina from human and rat were compared. NOS-activity was measured by the formation of 14C-citrulline from 14C-arginine and the expression of NOS isoforms was examined by Western blotting. NOS activity in human uterus and vagina was significantly lower than in the tissues from rat. In contrast to the rat where NOS activity was predominantly found in the cytosolic fractions, NOS activity in particulate and cytosolic fractions from both human myometrium and vagina was similar. Data from Western blots confirmed that eNOS and nNOS isoforms were concentrated in the particulate and cytosolic fractions, respectively. Estrogen treatment of rats resulted in a down regulation of uterine cytosolic NOS activity. A down regulation of NOS in the cytosolic fraction was also seen in the human pregnant myometrium as compared with the nonpregnant myometrium. The vaginal NOS activity was considerably higher than the uterus in both species. In spite of some clear-cut qualitative and other differences between human and rat tissues, there are some interesting similarities. Downregulation in pregnancy of human uterine NOS is probably due to, at least in part, the influence of estrogen and progesterone.

  12. Suppression of pathogen-inducible NO synthase (iNOS) activity in tomato increases susceptibility to Pseudomonas syringae

    Science.gov (United States)

    Chandok, Meena R.; Ekengren, Sophia K.; Martin, Gregory B.; Klessig, Daniel F.

    2004-01-01

    Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and Arabidopsis plants. It was shown recently that a variant form of the Arabidopsis P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and susceptible tomato leaves upon inoculation with the Pseudomonas syringae pv. tomato strain DC3000. Virus-induced gene-silencing targeting LevarP, a putative tomato ortholog of AtvarP, led to complete suppression of DC3000-induced iNOS activation and an ?80% reduction in GDC activity; it also increased disease-symptom severity and DC3000 growth in both resistant and susceptible tomato. To determine whether enhanced susceptibility exhibited by LevarP-silenced, susceptible tomato was due to loss of (i) iNOS activity, (ii) GDC activity, or (iii) both, GDC activity was inhibited with or without concurrent suppression of iNOS. Treatment with methotrexate inhibited both iNOS and GDC activities and resulted in increased susceptibility, comparable with that observed in LevarP-silenced plants. When normal iNOS activity was maintained in the presence of methotrexate by the addition of tetrahydrobiopterin, there was no change in susceptibility, despite a dramatic reduction in GDC activity. Together, these results indicate that iNOS contributes to host defense response against DC3000. PMID:15146069

  13. Nitric oxide synthase inhibition blocks phencyclidine-induced behavioural effects on prepulse inhibition and locomotor activity in the rat.

    Science.gov (United States)

    Johansson, C; Jackson, D M; Svensson, L

    1997-05-01

    The ability of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), to block the behavioural effects of the potent psychotomimetic, phencyclidine, was tested in rats using two different behavioural models. L-NAME was found to block both phencyclidine-induced disruption of prepulse inhibition of acoustic startle and phencyclidine-induced stimulation of locomotor activity. A selective action of L-NAME on the effects of phencyclidine was indicated, since L-NAME did not alter the effects of amphetamine, another potent psychotomimetic, in these behavioural models. These observations suggest that a nitric oxide-dependent mechanism may be involved in the effects of phencyclidine in the central nervous system. PMID:9201805

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

    Directory of Open Access Journals (Sweden)

    Mohammad Taghi Mohammadi

    2010-03-01

    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.

  15. Characterization of AtALMT1 expression in aluminum inducible malate release and its role for rhizotoxic stress in Arabidopsis

    Science.gov (United States)

    Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here we further characterize AtALMT1, an Arabidopsis malate transporter, to clarify its specific role in malate release and Al stress responses. Malate ex...

  16. 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

  17. Exercise with calorie restriction improves insulin sensitivity and glycogen synthase activity in obese postmenopausal women with impaired glucose tolerance.

    Science.gov (United States)

    Ryan, Alice S; Ortmeyer, Heidi K; Sorkin, John D

    2012-01-01

    Our objective was to compare the effects of in vivo insulin on skeletal muscle glycogen synthase (GS) activity in normal (NGT) vs. impaired glucose-tolerant (IGT) obese postmenopausal women and to determine whether an increase in insulin activation of GS is associated with an improvement in insulin sensitivity (M) following calorie restriction (CR) and/or aerobic exercise plus calorie restriction (AEX + CR) in women with NGT and IGT. We did a longitudinal, clinical intervention study of CR compared with AEX + CR. Overweight and obese women, 49-76 yr old, completed 6 mo of CR (n = 46) or AEX + CR (n = 50) with Vo(2?max), body composition, and glucose tolerance testing. Hyperinsulinemic euglycemic (80 mU·m(-2)·min(-1)) clamps (n = 73) and skeletal muscle biopsies (before and during clamp) (n = 58) were performed before and after the interventions (n = 50). After 120 min of hyperinsulinemia during the clamp, GS fractional activity and insulin's effect to increase GS fractional activity (insulin - basal) were significantly lower in IGT vs. NGT (P level (insulin activation of GS) in older women at highest risk for diabetes are derived from a lifestyle intervention that includes exercise and diet. PMID:22008454

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

    International Nuclear Information System (INIS)

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [3H]-NaBH4, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

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

    Science.gov (United States)

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

    2015-06-01

    Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6?kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9?kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ?1.65?Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50?Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid-liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI). PMID:26057806

  20. Reconstitution of energy-linked activities of the solubilized F1F0 ATP synthase from Bacillus subtilis.

    OpenAIRE

    Hicks, D B; Cohen, D M; Krulwich, T A

    1994-01-01

    The F1F0 ATP synthases from wild-type Bacillus subtilis and an uncoupler-resistant mutant have comparable subunit structures. In accord with an earlier hypothesis, ATP hydrolysis and ATP-Pi exchange by the two synthases were equally stimulated and inhibited by protonophores, respectively, when reconstituted alone in either wild-type or mutant lipids.

  1. Pharmacological profile of FR260330, a novel orally active inducible nitric oxide synthase inhibitor.

    Science.gov (United States)

    Chida, Noboru; Hirasawa, Yoshimi; Ohkawa, Takehiko; Ishii, Yoshinori; Sudo, Yuji; Tamura, Kouichi; Mutoh, Seitaro

    2005-02-10

    In this study, we examined effects of a newly synthesized chemical compound, FR260330, (2E)-3-(4-chlorophenyl)-N-[(1S)-2-oxo-2-{[2-oxo-2-(4-{[6-(trifluoromethyl)-4-pyrimidinyl]oxy}-1-piperidinyl)ethyl]amino}-1-(2-pyridinylmethyl)ethyl]acrylamide on nitric oxide (NO) production in rat splenocytes and human colon cancer cell line, DLD-1 cells. FR260330 inhibited NOx production dose dependently in both cells. In lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treated murine macrophage cell line, RAW264.7, Western blot analysis with gel filtration chromatography revealed FR260330 might prevent dimerization of inducible nitric oxide synthase (iNOS), but had no effect on the expression of iNOS protein. Furthermore, oral administration of FR260330 reduced NOx production dose dependently in plasma from rats exposed to LPS (IC50=1.6 mg/kg). Meanwhile, higher dose (100 mg/kg) of oral administration of FR260330 did not change mean arterial blood pressure in rats. These results suggest that FR260330 might be a useful therapeutical approach to various inflammatory diseases, in which superoxide or peroxynitrite formed from iNOS-derived NO are involved. PMID:15713431

  2. 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)

    Mai Hazekawa

    2011-10-01

    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.

  3. Determination of delta-aminolaevulinic acid synthase activity in human bone marrow using high performance liquid chromatography

    International Nuclear Information System (INIS)

    A high performance liquid chromatographic (HPLC) method is described for the rapid and specific determination of the activity of the enzyme delta-aminolaevulinic acid synthase (ALA-S) in mitochondria prepared by sonication of human bone marrow cells. After incubation with 14C-?-ketoglutarate the 14C-delta-aminolaevulinic acid (ALA) formed is converted to a pyrrole derivative, 2-methyl-3-carbethoxy-4-(3-propionic acid) pyrrole. This is isolated by reversed-phase ion-pair chromatography on a Hypersil-SAS column with methanol-water (45:155, v/v) in the presence of 0.005 mol/l l-heptanesulphonic acid (PIC B-7) as the mobile phase. The radioactivity of the isolated pyrrole is determined by scintillation counting. The optimal substrate concentration and pH were 0.17 mmol/l ?-ketoglutarate and pH 7.4, with an optimal period of sonication of 18s. Under these conditions ALA production was proportional to the concentrations of erythroblasts in the initial sample and was linear with time up to 60 min. The addition of pyridoxal phosphate (PLP) did not affect ALA-S activity in normal subjects. The mean ALA-S activity in 10 haematologically normal control subjects was found to be 318.8 pmol.10-6 erythroblasts.h-1 (S.D. +- 125.8, range 193-444.6). (Auth.)

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

    DEFF Research Database (Denmark)

    Kolnes, Anders J; Birk, Jesper Bratz

    2015-01-01

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

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

    DEFF Research Database (Denmark)

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger

    2007-01-01

    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.

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

    Science.gov (United States)

    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

    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

  7. Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.

    Science.gov (United States)

    Hartwig, S; Frister, T; Alemdar, S; Li, Z; Krings, U; Berger, R G; Scheper, T; Beutel, S

    2014-05-01

    Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition. PMID:24576659

  8. The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

    Science.gov (United States)

    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

    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 Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) 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

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

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers

    2006-01-01

    Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method,we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC. Udgivelsesdato: null-null

  10. Estradiol regulation of lipocalin-type prostaglandin D synthase promoter activity: evidence for direct and indirect mechanisms.

    Science.gov (United States)

    Devidze, Nino; Fujimori, Ko; Urade, Yoshihiro; Pfaff, Donald W; Mong, Jessica A

    2010-04-19

    In the CNS, lipocalin-type prostaglandin D synthase (L-PGDS) is predominantly a non-neuronal enzyme responsible for the production of PGD(2), an endogenous sleep promoting substance. We have previously demonstrated that estradiol differentially regulates L-PGDS transcript levels in the rodent brain. In hypothalamic nuclei, estradiol increases L-PGDS transcript expression, whereas in the ventrolateral preoptic area L-PGDS gene expression is reduced after estradiol treatment. In the present study, we have used an immortalized glioma cell line transfected with a L-PGDS reporter construct and estrogen receptor (ER) alpha and ERbeta expression plasmids to further elucidate the mechanisms underlying estradiol regulation of L-PGDS gene expression. We found that physiologically relevant concentrations of estradiol evoked an inverted U response in cells expressing ERalpha. The most effective concentration of estradiol (10(-11)M) increased the promoter activity 3-fold over baseline. Expression of ERbeta did not increase activity over control and when ERbeta was co-expressed with ERalpha there was a significant attenuation of the promoter activity. While ERalpha significantly increased L-PGDS promoter activity, our previous in vivo studies demonstrate a greater magnitude of change in L-PGDS gene expression in the presences of estradiol. This led us to ask whether estradiol is signaling via a paracrine factor released by the neighboring neurons. Conditioned media from estradiol treated neurons applied to the glioma cell line resulted in a significant 7-fold increase in L-PGDS promoter activity supporting the possibility that neuronal-glial interactions are involved in estradiol regulation of L-PGDS. PMID:20193744

  11. Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase

    Science.gov (United States)

    Oyola-Robles, Delise; Gay, Darren C; Trujillo, Uldaeliz; Sánchez-Parés, John M; Bermúdez, Mei-Ling; Rivera-Díaz, Mónica; Carballeira, Néstor M; Baerga-Ortiz, Abel

    2013-01-01

    Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ?-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases. PMID:23696301

  12. ATP synthase.

    Science.gov (United States)

    Junge, Wolfgang; Nelson, Nathan

    2015-06-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  13. Observation of cytoplasmic and vacuolar malate in maize root tips by 13C-NMR spectroscopy

    International Nuclear Information System (INIS)

    The accumulation of malate by maize (Zea mays L.) root tips perfused with KH13CO3 was followed by 13C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ?5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH13CO3 treatment, 13C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ?6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment. (author)

  14. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Science.gov (United States)

    Sordelli, Micaela S; Beltrame, Jimena S; Burdet, Juliana; Zotta, Elsa; Pardo, Romina; Cella, Maximiliano; Franchi, Ana M; Ribeiro, Maria Laura

    2011-01-01

    Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies. PMID:21559512

  15. Effects of conditioned medium on activities of PAL, CHS, DAHP synthase (DS-Co and DS-Mn) and anthocyanin production in suspension cultures of Fragaria ananassa.

    Science.gov (United States)

    Mori, T; Sakurai, M; Sakuta, M

    2001-01-01

    A conditioned medium (CM) prepared from cell suspension cultures of strawberry stimulated anthocyanin synthesis. The effect was significantly (PDAHP) synthase (EC 4.1.2.15) (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) were monitored in the CM- and SM-cultured cells. PAL and CHS activities were found to increase significantly (P<0.05) in the CM-cultured cells. CHS transcript levels were higher in the CM-cultured cells compared to transcript abundance in SM-cultured cells. There was no significant difference in the DS-Mn and DS-Co activities of cells grown in conditioned or synthetic media. PMID:11164607

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

    Science.gov (United States)

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

    1994-01-01

    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.

  17. Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3 mutant on Cd-contaminated soil

    OpenAIRE

    Ku?hnlenz, Tanja; Schmidt, Holger; Uraguchi, Shimpei; Clemens, Stephan

    2014-01-01

    Experiments on Cd-contaminated soil demonstrated a contribution of phytochelatin synthesis to agriculturally relevant Cd accumulation and revealed constitutive activity of the hitherto functionally not understood Arabidopsis thaliana phytochelatin synthase2.

  18. Silencing Glycogen Synthase Kinase-3? Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1*

    OpenAIRE

    Shinohara, Mie; Ybanez, Maria D.; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2010-01-01

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3? (GSK-3?) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (?1 h). The silencing of GSK-3?, but not Akt-2 (protein kinase B) or glycogen syn...

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

    OpenAIRE

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

    2013-01-01

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

  20. Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

    OpenAIRE

    Baroja-Fernández, Edurne; Muñoz, Francisco José; LI, Jun; Bahaji, Abdellatif; Almagro, Goizeder; Montero, Manuel (O.F.M.); Etxeberria, Ed; Hidalgo, Maite; Sesma, María Teresa; Pozueta-Romero, Javier

    2011-01-01

    Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124–13129], who showed that (i) SUS activ...

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

    OpenAIRE

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

    2011-01-01

    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...

  2. Dysregulation of nitric oxide synthase activity and Bcl-2 and caspase-3 gene expressions in renal tissue of streptozotocin-induced diabetic rats

    OpenAIRE

    AKTU?, Hüseyin; ÇET?NTA?, Vildan BOZOK; Kosova, Buket; OLTULU, Fatih; DEM?RAY, ?irin BAKTI; ÇAVU?O?LU, Türker; Akarca, Saadet Özen; YAVA?O?LU, Altu?

    2012-01-01

    To evaluate the effects of diabetes on nitric oxide synthase (NOS) activity and on gene expressions of B-cell lymphoma protein 2 (Bcl-2) and caspase-3 in the renal tissues of streptozotocin (STZ)-induced rats. Materials and methods: Twenty rats were analyzed in 2 equally distributed groups of healthy controls and STZ-induced diabetic rats. Rats with blood glucose levels of >=250 mg/dL were considered diabetic. Renal tissue sections were analyzed histomorphologically and immunohistochemica...

  3. Tl(+) induces the permeability transition pore in Ca(2+)-loaded rat liver mitochondria energized by glutamate and malate.

    Science.gov (United States)

    Korotkov, Sergey M; Emelyanova, Larisa V; Konovalova, Svetlana A; Brailovskaya, Irina V

    2015-08-01

    It is known that Ca(2+) and heavy metals more actively induce MPTP opening in mitochondria, energized by the I complex substrates. Thus, a rise in a Tl(+)-induced MPTP was proposed in experiments on isolated rat liver mitochondria energized by the complex I substrate (glutamate and malate). Expose of the mitochondria to Ca(2+) into a medium containing TlNO3, glutamate, and malate as well as sucrose or KNO3 resulted in a decrease in state 3, state 4, or DNP-stimulated respiration as well as an increase of both mitochondrial swelling and ??mito dissipation. The MPTP inhibitors, CsA and ADP, almost completely eliminated the effect of Ca(2+), which was more pronounced in the presence of the complex I substrates than the complex II substrate (succinate) and rotenone (Korotkov and Saris, 2011). The present study concludes that Tl(+)-induced MPTP opening is more appreciable in mitochondria energized by glutamate and malate but not succinate in the presence of rotenone. We assume that the Tl(+)-induced MPTP opening along with followed swelling and possible structural deformations of the complex I in Ca(2+)-loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl(+) to Tl(3+) by mitochondrial oxygen reactive species is proposed for the mechanism. PMID:25910914

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

    Directory of Open Access Journals (Sweden)

    E Roda

    2009-06-01

    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.

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

    Science.gov (United States)

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

    1992-12-15

    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

  6. Manipulation of Rumen Ecology by Malate and Yeast in Native Cattle

    OpenAIRE

    Sittisak Khampa; Pala Chaowarat; Uthai Koatdoke; Rungson Singhalert; Metha Wanapat

    2009-01-01

    Four, one-year old of native cattle 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 with yeast (Saccharomyces cerevisiae) at 1,000 and 2,000 g in concentrate containing high levels of cassava chip. The treatments were as follows: T1 = supplementation of malate at 500 g with yeast at 1,000 g; T2 = supplementation of malate at 500 g with yeast at 2,000 g; T3 = supplementation of malate at 1,...

  7. Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase.

    Science.gov (United States)

    Irikura, Daisuke; Aritake, Kosuke; Nagata, Nanae; Maruyama, Toshihiko; Shimamoto, Shigeru; Urade, Yoshihiro

    2009-03-20

    We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (L-PGDS-transgenic mice. PMID:19131342

  8. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    Science.gov (United States)

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

    2008-10-24

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

  9. Observation of an Unusual Electronically Distorted Semiquinone Radical of PCB Metabolites in the Active Site of Prostaglandin H Synthase-2

    Science.gov (United States)

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

    2013-01-01

    The activation of the metabolites of airborne polychlorinated biphenyls (PCBs) into highly reactive radicals is of fundamental importance. We found that human recombinant prostaglandin H synthase-2 (hPGHS-2) biotransforms dihydroxy-PCBs, such as 4-chlorobiphenyl-2?,5?-hydroquinone (4-CB-2?,5?H2Q), into semiquinone radicals via one-electron oxidation. Using electron paramagnetic resonance (EPR) spectroscopy, we observed the formation of the symmetric quartet spectrum (1:3:3:1 by area) of 4-chlorobiphenyl-2?,5?-semiquinone radical (4-CB-2?,5?-SQ•?) from 4-CB-2?,5?H2Q. This spectrum changed to an asymmetric spectrum with time: the change can be explained as the overlap of two different semiquinone radical species. Hindered rotation of the 4-CB-2?,5?-SQ•? appears not to be a major factor for the change in lineshape because increasing the viscosity of the medium with glycerol produced no significant change in lineshape. Introduction of a fluorine, which increases the steric hindrance for rotation of the dihydroxy-PCB studied, also produced no significant changes. An in silico molecular docking model of 4-CB-2?,5?H2Q in the peroxidase site of hPGHS-2 together with ab initio quantum mechanical studies indicate that the close proximity of a negatively charged carboxylic acid in the peroxidase active site may be responsible for the observed perturbation in the spectrum. This study provides new insights into the formation of semiquinones from PCB metabolites and underscores the potential role of PGHS-2 in the metabolic activation of PCBs. PMID:20843536

  10. The tunnel of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase regulates delivery of CO to the active site.

    Science.gov (United States)

    Tan, Xiangshi; Loke, Huay-Keng; Fitch, Shawn; Lindahl, Paul A

    2005-04-27

    The effect of [CO] on acetyl-CoA synthesis activity of the isolated alpha subunit of acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica was determined. In contrast to the complete alpha(2)beta(2) enzyme where multiple CO molecules exhibit strong cooperative inhibition, alpha was weakly inhibited, apparently by a single CO with K(I) = 1.5 +/- 0.5 mM; other parameters include k(cat) = 11 +/- 1 min(-)(1) and K(M) = 30 +/- 10 microM. The alpha subunit lacked the previously described "majority" activity of the complete enzyme but possessed its "residual" activity. The site affording cooperative inhibition may be absent or inoperative in isolated alpha subunits. Ni-activated alpha rapidly and reversibly accepted a methyl group from CH(3)-Co(3+)FeSP affording the equilibrium constant K(MT) = 10 +/- 4, demonstrating the superior nucleophilicity of alpha(red) relative to Co(1+)FeSP. CO inhibited this reaction weakly (K(I) = 540 +/- 190 microM). NiFeC EPR intensity of alpha developed in accordance with an apparent K(d) = 30 microM, suggesting that the state exhibiting this signal is not responsible for inhibiting catalysis or methyl group transfer and that it may be a catalytic intermediate. At higher [CO], signal intensity declined slightly. Attenuation of catalysis, methyl group transfer, and the NiFeC signal might reflect the same weak CO binding process. Three mutant alpha(2)beta(2) proteins designed to block the tunnel between the A- and C-clusters exhibited little/no activity with CO(2) as a substrate and no evidence of cooperative CO inhibition. This suggests that the tunnel was blocked by these mutations and that cooperative CO inhibition is related to tunnel operation. Numerous CO molecules might bind cooperatively to some region associated with the tunnel and institute a conformational change that abolishes the majority activity. Alternatively, crowding of CO in the tunnel may control flow through the tunnel and deliver CO to the A-cluster at the appropriate step of catalysis. Residual activity may involve CO from the solvent binding directly to the A-cluster. PMID:15839681

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

    OpenAIRE

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

    2000-01-01

    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...

  12. 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)

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

    2014-06-15

    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

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

    Directory of Open Access Journals (Sweden)

    Giselli Scaini

    2011-06-01

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

  14. 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)

    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

    Full Text Available 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.

  15. Induction of nitric oxide synthase and activation of signaling proteins in Anopheles mosquitoes by the malaria pigment, hemozoin.

    Science.gov (United States)

    Akman-Anderson, Leyla; Olivier, Martin; Luckhart, Shirley

    2007-08-01

    Anopheles stephensi, a major vector for malaria parasite transmission, responds to Plasmodium infection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown that Plasmodium falciparum glycosylphosphatidylinositols (PfGPIs) can induce A. stephensi NO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In mosquito cells, signaling by PfGPIs and P. falciparum merozoites is mediated through Akt/protein kinase B (Akt/PKB), the mitogen-activated protein kinase kinase DSOR1, and extracellular signal-regulated kinase (ERK). In mammalian cells, a second parasite factor, malaria pigment or hemozoin (Hz), signals NOS induction through ERK- and nuclear factor kappa B-dependent pathways and has been demonstrated to be a novel proinflammatory ligand for Toll-like receptor 9. In this study, we demonstrate that Hz can also induce AsNOS gene expression in immortalized A. stephensi and Anopheles gambiae cell lines in vitro and in A. stephensi midgut tissue in vivo. In mosquito cells, Hz signaling is mediated through transforming growth factor beta-associated kinase 1, Akt/PKB, ERK, and atypical protein kinase C zeta/lambda. Our results show that Hz is a prominent parasite-derived signal for Anopheles and that signaling pathways activated by PfGPIs and Hz have both unique and shared components. Together with our previous findings, our data indicate that parasite signaling of innate immunity is conserved in mosquito and mammalian cells. PMID:17526741

  16. Fluvoxamine alters the activity of energy metabolism enzymes in the brain

    Scientific Electronic Library Online (English)

    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

    Full Text Available 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.

  17. Quininium Malates: partial chiral discrimination via diastereomeric salt formation

    Scientific Electronic Library Online (English)

    Nikoletta B., Báthori; Ayesha, Jacobs; Luigi R., Nassimbeni; Baganetsi K., Sebogisi.

    2014-01-01

    Full Text Available Quinine was employed as a resolving agent for racemic malic acid. The resultant product was a quininium salt containing 75 % of the D-malate anion. Quinine was also crystallized with pure L- and D-malic acids and the structures of the resulting diastereomeric salts were elucidated. The crystal packi [...] ngs were analyzed in terms of their non-bonded interactions and the conformation of the quinine, which was compared with other quinine structures recorded in the Cambridge Structural Database. The results indicate that the mechanism of enantiomeric resolution is reliant upon hydrogen bonded interactions.

  18. 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.

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

    OpenAIRE

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

    2003-01-01

    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...

  20. Activation of ceramide synthase 6 by celecoxib leads to a selective induction of C-ceramide

    OpenAIRE

    Schiffmann, Susanne; Ziebell, Simone; Sandner, Jessica; Birod, Kerstin; Deckmann, Klaus; Hartmann, Daniela; Rode, Sina; Schmidt, Helmut; Angioni, Carlo; Geisslinger, Gerd; Gro?sch, Sabine

    2010-01-01

    Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C-Cer-C-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50?M celecoxib leading to a signif...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-15

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

  2. 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.

  3. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  4. Malat1 is not an essential component of nuclear speckles in mice.

    Science.gov (United States)

    Nakagawa, Shinichi; Ip, Joanna Y; Shioi, Go; Tripathi, Vidisha; Zong, Xinying; Hirose, Tetsuro; Prasanth, Kannanganattu V

    2012-08-01

    Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine- and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA--Neat1--which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions. PMID:22718948

  5. In vitro effect of testosterone propionate on lactate dehydrogenase and malate dehydrogenase in brain and gonads of Channa punctatus.

    Science.gov (United States)

    Gupta, B L; Ansari, M A; Sinha, I P; Gupta, S P

    1993-01-01

    The activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and protein levels were measured in vitro incubation of tissue homogenate of brain and gonads with testosterone propionate (TP) in Channa punctatus (teleost). The activities of LDH and MDH were found to be highest in the ovarian tissues in comparison to the testis and the brain in control fish. TP (2 micrograms-4 micrograms/ml) stimulated testicular tissue with increases in LDH by 10-fold and MDH by 15-fold approximately, the ovary with significant increase in LDH and a decrease in MDH and the brain showed only an increase in MDH. PMID:8490571

  6. Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

    Science.gov (United States)

    Hutson, N J; Brumley, F T; Assimacopoulos, F D; Harper, S C; Exton, J H

    1976-09-10

    Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase... PMID:8456

  7. Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange.

    Science.gov (United States)

    Garg, Ashish; Xie, Xinqiang; Keatinge-Clay, Adrian; Khosla, Chaitan; Cane, David E

    2014-07-23

    Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains. PMID:25004372

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

    Directory of Open Access Journals (Sweden)

    Elisa Mitiko Kawamato

    2002-09-01

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

  9. 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)

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

    2002-09-01

    Full Text Available 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.

  10. AMP-activated Protein Kinase (AMPK) Activating Agents Cause Dephosphorylation of Akt and Glycogen Synthase Kinase-3

    OpenAIRE

    King, Taj D.; Song, Ling; Jope, Richard S.

    2006-01-01

    AMP-activated protein kinase (AMPK) is a key cellular sensor of reduced energy supply that is activated by increases in the cellular ratio of AMP/ATP. Phenformin and 5-aminoimida-zole-4-carboxamide riboside (AICAR) are two drugs widely used to activate AMPK experimentally. In both differentiated hippocampal neurons and neuroblastoma SH-SY5Y cells we found that these two agents not only activated AMPK, but conversely greatly reduced the activating Ser/Thr phosphorylation of Akt. This blockade ...

  11. Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum

    DEFF Research Database (Denmark)

    SØrensen, Jens Laurids; Hansen, Frederik Teilfeldt

    2012-01-01

    Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

  12. Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

    DEFF Research Database (Denmark)

    SØrensen, Jens Laurids; T. Hansen, Frederik

    2012-01-01

    Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F.?graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

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

    Czech Academy of Sciences Publication Activity Database

    Jendeková, L.; Kojšová, S.; Andriantsitohaina, R.; Pechá?ová, Olga

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

  15. Mechanism of phosphate solubilization and antifungal activity of Streptomyces spp. isolated from wheat roots and rhizosphere and their application in improving plant growth.

    Science.gov (United States)

    Jog, Rahul; Pandya, Maharshi; Nareshkumar, G; Rajkumar, Shalini

    2014-04-01

    The application of plant-growth-promoting rhizobacteria (PGPR) at field scale has been hindered by an inadequate understanding of the mechanisms that enhance plant growth, rhizosphere incompetence and the inability of bacterial strains to thrive in different soil types and environmental conditions. Actinobacteria with their sporulation, nutrient cycling, root colonization, bio-control and other plant-growth-promoting activities could be potential field bio-inoculants. We report the isolation of five rhizospheric and two root endophytic actinobacteria from Triticum aestivum (wheat) plants. The cultures exhibited plant-growth-promoting activities, namely phosphate solubilization (1916 mg l(-1)), phytase (0.68 U ml(-1)), chitinase (6.2 U ml(-1)), indole-3-acetic acid (136.5 mg l(-1)) and siderophore (47.4 mg l(-1)) production, as well as utilizing all the rhizospheric sugars under test. Malate (50-55 mmol l(-1)) was estimated in the culture supernatant of the highest phosphate solublizer, Streptomyces mhcr0816. The mechanism of malate overproduction was studied by gene expression and assays of key glyoxalate cycle enzymes - isocitrate dehydrogenase (IDH), isocitrate lyase (ICL) and malate synthase (MS). The significant increase in gene expression (ICL fourfold, MS sixfold) and enzyme activity (ICL fourfold, MS tenfold) of ICL and MS during stationary phase resulted in malate production as indicated by lowered pH (2.9) and HPLC analysis (retention time 13.1 min). Similarly, the secondary metabolites for chitinase-independent biocontrol activity of Streptomyces mhcr0817, as identified by GC-MS and (1)H-NMR spectra, were isoforms of pyrrole derivatives. The inoculation of actinobacterial isolate mhce0811 in T. aestivum (wheat) significantly improved plant growth, biomass (33%) and mineral (Fe, Mn, P) content in non-axenic conditions. Thus the actinobacterial isolates reported here were efficient PGPR possessing significant antifungal activity and may have potential field applications. PMID:24430493

  16. Malate metabolism and adaptation to chilling temperature storage by pretreatment with high CO2 levels in Annona cherimola fruit.

    Science.gov (United States)

    Maldonado, Roberto; Sanchez-Ballesta, M Teresa; Alique, Rafael; Escribano, M Isabel; Merodio, Carmen

    2004-07-28

    In this study we focused on the effect of a pretreatment with high (20%) CO2 levels on malic acid metabolism in cherimoya (Annona cherimola Mill) fruit stored at chilling temperature. We analyzed the activity of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), and the NADP-malic enzyme (NADP-ME), involved in the carboxylation/decarboxylation of malate. Our results show that CO2 treatment, which improves tolerance to prolonged storage at chilling temperature, was closely linked to considerably greater NADP-ME activity. These results, combined with lower PEPC activity, may explain the significantly lower amount of malic acid and titratable acidity quantified in CO2-treated fruit. Moreover, the high cytoplasmic MDH enzyme activity and the strong stimulation of NADP-ME activity exhibited by CO2-treated fruit could be contributing factors in the maintenance of fruit energy metabolism, pH stability, and the promotion of synthesis of defense compounds that prevent or repair damage caused by chilling temperature. PMID:15264911

  17. The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation

    OpenAIRE

    Tripathi, Vidisha; Ellis, Jonathan D.; Shen, Zhen; Song, David Y.; Pan, Qun; Watt, Andrew T.; Freier, Susan M.; Bennett, C. Frank; Sharma, Alok; Bubulya, Paula A.; Blencowe, Benjamin J.; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2010-01-01

    Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in A...

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

    OpenAIRE

    Hildgund Schrempf; Marta Kotasinska; Yury Herasimenka; Stefan Walter

    2010-01-01

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

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

    Science.gov (United States)

    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

    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

  20. Determination of amino-acidic positions important for Ocimum basilicum geraniol synthase activity

    OpenAIRE

    Marc Bergdoll; Philippe Hugueney; Damien Steyer; Patricia Claudel; Sophie Meyer; Fischer, Marc J. C.

    2013-01-01

    Terpenes are one of the largest and most diversified families of natural compounds. Although they have found numerous industrial applications, the molecular basis of their synthesis in plants has, until now, not been fully understood. Plant genomes have been shown to contain dozens of terpene synthase (TPS) genes, however knowledge of their amino-acidic protein sequence in not sufficient to predict which terpene(s) will be produced by a particular enzyme. In order to investigate the structur...

  1. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

    OpenAIRE

    Proudfoot, L; Nikolaev, A. V.; Feng, G J; Wei, W Q; Ferguson, M A; Brimacombe, J S; Liew, F. Y.

    1996-01-01

    Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant red...

  2. Estradiol Regulation of Lipocalin-Type Prostaglandin D Synthase Promoter Activity: Evidence for Direct and Indirect Mechanisms

    OpenAIRE

    Devidze, Nino; Fujimori, Ko; Urade, Yoshihiro; Donald W. Pfaff; Mong, Jessica A.

    2010-01-01

    In the CNS, lipocalin-type prostaglandin D synthase (L-PGDS) is predominantly a non-neuronal enzyme responsible for the production of PGD2, an endogenous sleep promoting substance. We have previously demonstrated that estradiol differentially regulates L-PGDS transcript levels in the rodent brain. In hypothalamic nuclei, estradiol increases L-PGDS transcript expression, whereas in the ventrolateral preoptic area L-PGDS gene expression is reduced after estradiol treatment. In the present study...

  3. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath

    OpenAIRE

    Lane, C.; Knight, D.; Burgess, S.; Franklin, P.; Horak, F.; Legg, J.; Moeller, A.; Stick, S.

    2004-01-01

    Background: The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled br...

  4. Induction of Nitric Oxide Synthase and Activation of Signaling Proteins in Anopheles Mosquitoes by the Malaria Pigment, Hemozoin?

    OpenAIRE

    Akman-Anderson, Leyla; Olivier, Martin; Luckhart, Shirley

    2007-01-01

    Anopheles stephensi, a major vector for malaria parasite transmission, responds to Plasmodium infection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown that Plasmodium falciparum glycosylphosphatidylinositols (PfGPIs) can induce A. stephensi NO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In...

  5. Inhibition of Glycogen Synthase Kinase-3? Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

    OpenAIRE

    Schu?tz, Stefanie V.; Schrader, Andres J.; Zengerling, Friedemann; Genze, Felicitas; Cronauer, Marcus V.; Schrader, Mark

    2011-01-01

    In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3? (GSK-3?) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us...

  6. Can photosynthesis-related parameters be used to establish the activity of acetolactate synthase-inhibiting herbicides on weeds?

    OpenAIRE

    Riethmuller-haage, I. C. P.; Bastiaans, L.; Kropff, M. J.; Harbinson, J.; Kempenaar, C.

    2006-01-01

    The application of the acetolactate synthase (ALS)¿inhibiting herbicide metsulfuron on greenhouse- and field-grown black nightshade and greenhouse-grown ladysthumb resulted in progressive inhibition of the level of carbon dioxide (CO2) fixation, the relative quantum efficiency of electron transport through photosystem I (PSI) and II (PSII), and the leaf chlorophyll content. Photosynthetic-related measurements, measured 2 to 4 d after treatment (DAT) at photon flux densities of 400 to 500 ¿m...

  7. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH?-terminal (sites 2 + 2a) phosphorylation

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Birk, Jesper Bratz

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr³?? on Akt (p-Akt-Thr³??), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr³?? (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser??³ or IRS-1-PI3K activity. Furthermore, p-Akt-Thr³?? and Akt2 activity were negatively associated with NH?-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P

  8. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2+2a) phosphorylation

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Birk, Jesper Bratz

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. We previously demonstrated that phosphorylation of Threonine-308 on Akt (pAkt-T308), Akt2 activity, and GS activity in muscle were positivity associated with insulin sensitivity. Now, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. 181 non-diabetic twins were examined with the euglycemic-hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing kinase activity assays and phospho-specific western blotting. The insulin-stimulated GS activity was positively associated with pAkt-T308 (P=0.01) and Akt2 activity (P=0.04), but not pAkt-S473 or IRS-1-PI3K activity. Furthermore, pAkt-T308 and Akt2 activity were negatively associated with NH(2)-terminal GS phosphorylation (P=0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P

  9. [Ureido-¹?N]citrulline UPLC-MS/MS nitric oxide synthase (NOS) activity assay: development, validation, and applications to assess NOS uncoupling and human platelets NOS activity.

    Science.gov (United States)

    Böhmer, Anke; Gambaryan, Stepan; Flentje, Markus; Jordan, Jens; Tsikas, Dimitrios

    2014-08-15

    In healthy human subjects, less than 0.2% of l-arginine is converted to l-citrulline and nitric oxide (NO) by NO synthases (NOS), a metabolic pathway present in all cell types. Assessment of NOS activity in vitro and in vivo by measuring l-citrulline or NO is difficult. l-citrulline is formed from l-arginine to a much higher extent by other pathways including the urea cycle. Furthermore, NO is a very short-lived gaseous molecule and is oxidized to nitrite and nitrate which are ubiquitous. In fact, nitrite and nitrate are also derived from food and air and are major laboratory contaminants. Further, NOS (in the uncoupled state) are also able to produce superoxide in addition and/or instead of l-citrulline and NO. The difficulties of NOS assays based on l-citrulline and NO measurement can only in part be overcome by sophisticated techniques including use of radio-labeled ((3)H or (14)C) and stable-isotope labeled ((15)N2 at the guanidine group) l-arginine analogs as substrates for NOS and measurement of radio-labeled l-citrulline and (15)N-labeled nitrite and nitrate, respectively. In the present work, we report on the development, validation and application of an UPLC-MS/MS method for the assessment of the activity of recombinant NOS enzymes by using [guanidino-(15)N2]-l-arginine (20 ?M for recombinant NOS, 5mM in cell systems) as the substrate and by measuring [ureido-(15)N]-l-citrulline as the reaction product (usually formed at concentrations below 1 ?M) using (2)H7-l-citrulline as the internal standard. The lower limit of detection of the method is about 80 fmol (2)H7-l-citrulline. In cell systems, exceeding [guanidino-(15)N2]-l-arginine is removed by strong cation exchanger solid-phase extraction. The method was cross-validated by a GC-MS assay that measures simultaneously (15)N-nitrite and (15)N-nitrate as pentafluorobenzyl derivatives, with unlabeled nitrite and nitrate serving as the internal standards. By means of this UPLC-MS/MS (15)N-citrulline assay, N(G)-nitro-arginine (100 ?M) was found to inhibit recombinant inducible NOS (iNOS) activity (by 38%), whereas nitrite and GSSG (each at 500 ?M) did not affect iNOS activity at all. Nitrite and GSSG at pathophysiological concentrations are unlikely to uncouple NOS. NOS activity was not detectable in platelets of healthy humans by the UPLC-MS/MS and GC-MS assays. PMID:25033468

  10. Specificity of starch synthase isoforms from potato.

    Science.gov (United States)

    Edwards, A; Borthakur, A; Bornemann, S; Venail, J; Denyer, K; Waite, D; Fulton, D; Smith, A; Martin, C

    1999-12-01

    In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo. PMID:10583366

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

    Directory of Open Access Journals (Sweden)

    Dong Ju Son

    2014-08-01

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

  12. The Gene Expression and Enzyme Activity of Plant 3-Deoxy-D-Manno-2-Octulosonic Acid-8-Phosphate Synthase Are Preferentially Associated with Cell Division in a Cell Cycle-Dependent Manner1

    Science.gov (United States)

    Delmas, Frédéric; Petit, Johann; Joubès, Jérôme; Séveno, Martial; Paccalet, Thomas; Hernould, Michel; Lerouge, Patrice; Mouras, Armand; Chevalier, Christian

    2003-01-01

    3-Deoxy-d-manno-2-octulosonic acid-8-phosphate (Kdo-8-P) synthase catalyzes the condensation of phosphoenolpyruvate with d-arabinose-5-phosphate to yield Kdo-8-P. Kdo-8-P is the phosphorylated precursor of Kdo, a rare sugar only found in the rhamnogalacturonan II pectic fraction of the primary cell walls of higher plants and of cell wall polysaccharides of some green algae. A cDNA named LekdsA (accession no. AJ294902) encoding tomato (Lycopersicon esculentum) Kdo-8-P synthase has been isolated. The recombinant protein rescued a kdsA thermosensitive mutant of Salmonella typhimurium impaired in the synthesis of a functional Kdo-8-P synthase. Using site-directed mutagenesis of LekdsA cDNA, the tomato Kdo-8-P synthase was shown to possess the same essential amino acids that form the active sites in the bacterial enzymes. The tomato kdsA gene expression and the relevant Kdo-8-P synthase activity were preferentially associated to dividing cells, in the course of the early development of tomato fruit and in meristematic tissues. Furthermore, the transcription of the kdsA gene was found to oscillate during the cell cycle in tobacco (Nicotiana tabacum) Bright-Yellow 2 synchronized cells with a maximum during mitosis. PMID:12970500

  13. Relationships among histidine and malate decarboxilation and the ADI pathway in a wine isolated Lactobacillus hilgardii

    OpenAIRE

    Giunta, Carlo; Purrotti, Micol; Fattori, Paolo; Pessione, Enrica; Mazzoli, Roberto; Lamberti, Cristina; Barello, Cristina; Pessione, Alessandro

    2008-01-01

    Lactobacillus hilgardii ISE5211 was isolated from an Italian red wine spontaneously undergoing malo-lactic fermentation. Besides performing malate decarboxylation, this strain is also able to produce histamine (4 g/L) and it has been proved that the genetic determinants for histidine decarboxylase (HDC) are plasmid-encoded . Since in lactic acid bacteria (LAB) malate and histidine decarboxylation contribute both to counteract acidic stress and to energy supply, the present study was inten...

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

    OpenAIRE

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

    2006-01-01

    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...

  15. Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

    OpenAIRE

    Dong, Yunzhou; WU, YONG; Wu, Mingyuan; Wang, Shuangxi; Zhang, Junhua; Xie, Zhonglin; Xu, Jian; SONG, Ping; Wilson, Kenneth; Zhao, Zhengxing; LYONS, TIMOTHY; Zou, Ming-Hui

    2009-01-01

    Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of ‘heavily oxidized’ glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eN...

  16. 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)

    HUANG Di-Nan

    2004-09-01

    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.

  17. Silencing glycogen synthase kinase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.

    Science.gov (United States)

    Shinohara, Mie; Ybanez, Maria D; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A; Han, Derick; Kaplowitz, Neil

    2010-03-12

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3beta (GSK-3beta) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury ( approximately 1 h). The silencing of GSK-3beta, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3alpha (GSK-3alpha), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3beta affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3beta decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3beta translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3beta reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3beta also resulted in an inhibition of the early phase (0-2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3beta is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver. PMID:20061376

  18. Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

    Science.gov (United States)

    Sheu, Der-Shyan; Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

    2012-05-01

    The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16?), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(?) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16?) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16?) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16?) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation. PMID:22408158

  19. 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)

    Chen, Wei; Qi, Jun; Feng, Feng; Wang, Mao-de; Bao, Gang; Wang, Tuo; Xiang, Mu; Xie, Wan-Fu

    2014-03-01

    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

  20. Citrulline malate supplementation increases muscle efficiency in rat skeletal muscle.

    Science.gov (United States)

    Giannesini, Benoît; Le Fur, Yann; Cozzone, Patrick J; Verleye, Marc; Le Guern, Marie-Emmanuelle; Bendahan, David

    2011-09-30

    Citrulline malate (CM; CAS 54940-97-5, Stimol®) is known to limit the deleterious effect of asthenic state on muscle function, but its effect under healthy condition remains poorly documented. The aim of this longitudinal double-blind study was to investigate the effect of oral ingestion of CM on muscle mechanical performance and bioenergetics in normal rat. Gastrocnemius muscle function was investigated strictly non-invasively using nuclear magnetic resonance techniques. A standardized rest-stimulation- (5.7 min of repeated isometric contractions electrically induced by transcutaneous stimulation at a frequency of 3.3 Hz) recovery-protocol was performed twice, i.e., before (t(0)-24 h) and after (t(0)+48 h) CM (3 g/kg/day) or vehicle treatment. CM supplementation did not affect PCr/ATP ratio, [PCr], [Pi], [ATP] and intracellular pH at rest. During the stimulation period, it lead to a 23% enhancement of specific force production that was associated to significant decrease in both PCr (28%) and oxidative (32%) costs of contraction, but had no effect on the time-courses of phosphorylated compounds and intracellular pH. Furthermore, both the rate of PCr resynthesis during the post-stimulation period (VPCr(rec)) and the oxidative ATP synthesis capacity (Q(max)) remained unaffected by CM treatment. These data demonstrate that CM supplementation under healthy condition has an ergogenic effect associated to an improvement of muscular contraction efficiency. PMID:21664351

  1. Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases.

    Science.gov (United States)

    Zhu, Guan; Keithly, Janet S

    2002-01-01

    We have cloned and sequenced a lactate dehydrogenase (LDH) gene from Cryptosporidium parvum (CpLDH1). With this addition, and that of four recently deposited alpha-proteobacterial malate dehydrogenase (MDH) genes, the phylogenetic relationships among apicomplexan LDH and bacterial MDH were re-examined. Consistent with previous studies, our maximum likelihood (ML) analysis using the quartet-puzzling method divided 105 LDH/MDH enzymes into five clades, and confirmed that mitochondrial MDH is a sister clade to those of y-proteobacteria, rather than to alpha-proteobacteria. In addition, a Cryptosporidium parvum MDH (CpMDH1) was identified from the ongoing Cryptosporidium genome project that appears to belong to a distinct clade (III) comprised of 22 sequences from one archaebacterium, numerous eubacteria, and several apicomplexans. Using the ML puzzling test and bootstrapping analysis with protein distance and parsimony methods, the resulting trees not only robustly confirmed the alpha-proteobacterial relationship of apicomplexan LDH/MDH, but also supported a monophyletic relationship of CpLDH1 with CpMDHI. These data suggest that, unlike most other eukaryotes, the Apicomplexa may be one of the few lineages retaining an alpha-proteobacterial-type MDH that could have been acquired from an ancestral alpha-proteobacterium through primary endosymbiosis giving rise to the mitochondria, or through an unknown lateral gene transfer (LGT) event. PMID:12120991

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

    Directory of Open Access Journals (Sweden)

    Ehsan Karimi

    2012-11-01

    Full Text Available The effect of foliar application of salicylic acid (SA at different concentrations (10?3 M and 10?5 M was investigated on the production of secondary metabolites (flavonoids, chalcone synthase (CHS activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231 in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS enzyme activity (involving flavonoid synthesis and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10?5 M SA treatment. As the SA concentration was decreased from 10?3 M to 10?5 M, the free radical scavenging power (FRAP increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 ?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.

  3. Effect of malate on digestibility and rumen microbial protein synthesis in growing lambs fed a high-concentrate diet

    OpenAIRE

    Carro Travieso, Mª Dolores; Ranilla, Mari?a Jose?; Gira?ldez, Francisco Javier; Manteco?n, A?ngel R.; Balcells, J.

    2003-01-01

    Twenty-four Merino lambs (initial weight 15.3+l .27 kg) were divided into three homogenous groups. Each group was randomly allocated to three malate (disodium malate:calcium malate; 0'16:0'84) levels: 0 (C), 4 g/kg concentrate (M4) and 8 g/kg concentrare (M8).Lambs were fed concentrate (based on barley, corn and soyabean meal) and barley straw ad líbitum...

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

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine ?-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.

  5. 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.)

  6. Supplementation of Malate and Yeast in Concentrate Containing High Cassava Chip on Rumen Ecology in Dairy Steers

    OpenAIRE

    Sittisak Khampa; Pala Chaowarat; Rungson Singhalert; Metha Wanapat

    2009-01-01

    Four, one-year old of 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 vs 1,000 g with yeast (Saccharomyces cerevisiae) at 1,000 vs 2,000 g in concentrate containing high levels of cassava chip. The treatments were as follows: T1 = supplementation of malate at 500 g with yeast at 1,000 g; T2 = supplementation of malate at 500 g with yeast at 2,000 g; T3 = supplementation of malate at 1,000...

  7. Manipulation of Rumen Ecology by Malate and Cassava Hay in High-Quality Feed Block in Dairy Steers

    OpenAIRE

    Sittisak Khampa; Pala Chaowarat; Uthai Koatdoke; Rungson Singhalert; Metha Wanapat

    2009-01-01

    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...

  8. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  9. Biochemistry: Acetohydroxyacid Synthase

    OpenAIRE

    Pham Ngoc Chien

    2010-01-01

    Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS) is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA) isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  10. 6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages.

    Science.gov (United States)

    Uto, Takuhiro; Fujii, Makoto; Hou, De-Xing

    2005-10-15

    6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is an active ingredient of Wasabi (Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. To clarify the cellular signaling mechanism underlying the anti-inflammatory action of 6-MITC, we investigated the effects of 6-MITC on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. 6-MITC showed a dose-dependent inhibition of LPS-induced nitric oxide (NO), iNOS mRNA and protein. LPS caused the c-Jun phosphorylation (a major component of AP-1) and IkappaB-alpha degradation. 6-MITC suppressed LPS-induced c-Jun phosphorylation, but did not inhibit IkappaB-alpha degradation. Cellular signaling analysis using MAPK-(U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) and Jak2-specific (AG490) inhibitors demonstrated that LPS stimulated iNOS expression via activating Jak2-mediated JNK, but not ERK and p38, pathway. 6-MITC suppressed iNOS expression through the inhibition of Jak2-mediated JNK signaling cascade with the attendant to AP-1 activation. In addition, the structure-activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings provide the molecular basis for the first time that 6-MITC is an effective agent to attenuate iNOS production. PMID:16139249

  11. 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)

    JØrgensen, Sebastian B; Nielsen, Jakob N.

    2004-01-01

    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.

  12. Beneficial effects of citrulline malate on skeletal muscle function in endotoxemic rat.

    Science.gov (United States)

    Giannesini, Benoît; Izquierdo, Marguerite; Le Fur, Yann; Cozzone, Patrick J; Verleye, Marc; Le Guern, Marie-Emmanuelle; Gillardin, Jean-Marie; Bendahan, David

    2009-01-01

    Although citrulline malate (CM; CAS 54940-97-5, Stimol) is used against fatigue states, its anti-asthenic effect remains poorly documented. The objective of this double-blind study was to evaluate the effect of oral ingestion of CM on a rat model of asthenia, using in situ (31)Phosphorus magnetic resonance spectroscopy ((31)P-MRS). Muscle weakness was induced by intraperitoneal injections of Klebsiella pneumoniae endotoxin (lipopolysaccharides at 3 mg/kg) at t(0) and t(0)+24 h. For each animal, muscle function was investigated strictly non-invasively before (t(0)-24 h) and during (t(0)+48 h) endotoxemia, through a standardized rest-stimulation-recovery protocol. The transcutaneous electrical stimulation protocol consisted of 5.7 min of repeated isometric contractions at a frequency of 3.3 Hz, and force production was measured with an ergometer. CM supplementation in endotoxemic animals prevented the basal phosphocreatine/ATP ratio reduction and normalized the intracellular pH (pH(i)) time-course during muscular activity as a sign of an effect at the muscle energetics level. In addition, CM treatment avoided the endotoxemia-induced decline in developed force. These results demonstrate the efficiency of CM for limiting skeletal muscle dysfunction in rats treated with bacterial endotoxin. PMID:19036344

  13. Developmental regulation and cellular distribution of human cytosolic malate dehydrogenase (MDH1).

    Science.gov (United States)

    Lo, Agnes Shuk-Yee; Liew, Choong-Tsek; Ngai, Sai-Ming; Tsui, Stephen Kwok-Wing; Fung, Kwok-Pui; Lee, Cheuk-Yu; Waye, Mary Miu-Yee

    2005-03-01

    Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across the mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and providing contractile function. Cellular localization studies indicate that MDH1 mRNA expression has a strong tissue-specific distribution, being expressed primarily in cardiac and skeletal muscle and in the brain, at intermediate levels in the spleen, kidney, intestine, liver, and testes and at low levels in lung and bone marrow. The observed MDH1 localizations reflect the role of NADH in the support of a variety of functions in different organs. These functions are primarily related to aerobic energy production for muscle contraction, neuronal signal transmission, absorption/resorption functions, collagen-supporting functions, phagocytosis of dead cells, and processes related to gas exchange and cell division. During neonatal development, MDH1 is expressed in human embryonic heart as early as the 3rd month and then is over-expressed from the 5th month until the birth. The expression of MDH1 is maintained in the adult heart but is not present in levels as high as in the fetus. Finally, over-expression of MDH1 is found in left ventricular cardiac muscle of dilated cardiomyopathy (DCM) patients when contrasted to the diseased non-DCM and normal heart muscle by in situ hybridization and Western blot. These observations are compatible with the activation of glucose oxidation in relatively hypoxic environments of fetal and hypertrophied myocardium. PMID:15565635

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

    International Nuclear Information System (INIS)

    Highlights: ? As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. ? RhoA activation increases PGE2 levels and its metabolic enzyme mPGES-1. ? RhoA-activated NF-?B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E2 (PGE2), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE2 levels and gene expression of the rate-limiting PGE2 producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1?-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1?-mediated phosphorylated nuclear factor-?B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE2 production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  15. Sucrose Synthase: Expanding Protein Function

    OpenAIRE

    SUBBAIAH, CHALIVENDRA C.; Huber, Steven C; Sachs, Martin M; Rhoads, David

    2007-01-01

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. This inference is based on the following observations: (a) tissue-specific, isoform-dependent and metabolically-regulated association of SUS with mitochondria and (b) isoform-specific and anoxia-resp...

  16. 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)

    Angela Ladurner

    2014-10-01

    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.

  17. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation

    DEFF Research Database (Denmark)

    Ramachandran, Roshni; Bhatt, Deepak Kumar

    2014-01-01

    BACKGROUND AND AIM: Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. MATERIALS AND METHODS: The femoral vein was catheterised in rats and GTN was infused (4 µg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP and Western blot for measuring nNOS protein expression. The effect of olcegepant, L-nitro-arginine methyl ester (L-NAME) and neurokinin (NK)-1 receptor antagonist L-733060 were analysed on Fos activation. RESULTS: GTN-treated rats showed a significant increase of nNOS and CGRP in dura mater and CGRP in the trigeminal nucleus caudalis (TNC). Upregulation of Fos was observed in TNC four hours after the infusion. This activation was inhibited by pre-treatment with olcegepant. Pre-treatment with L-NAME and L-733060 also significantly inhibited GTN induced Fos expression. CONCLUSION: The present study indicates that blockers of CGRP, NOS and NK-1 receptors all inhibit GTN induced Fos activation. These findings also predict that pre-treatment with olcegepant may be a better option than post-treatment to study its inhibitory effect in GTN migraine models.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-15

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

  19. 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

  20. Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes

    2014-01-01

    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.

  1. Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity.

    Science.gov (United States)

    Hempel, Casper; Kohnke, Hannes; Maretty, Lasse; Jensen, Peter Ø; Staalsø, Trine; Kurtzhals, Jørgen A L

    2014-11-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-02-15

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

  3. Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

    Science.gov (United States)

    Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

    2015-06-01

    Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling. PMID:25796222

  4. Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

    Science.gov (United States)

    Montori-Grau, Marta; Tarrats, Núria; Osorio-Conles, Oscar; Orozco, Anna; Serrano-Marco, Lucía; Vázquez-Carrera, Manuel; Gómez-Foix, Anna M

    2013-05-01

    Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3? activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3? activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3? (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3? (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3? or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose. PMID:23453973

  5. Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.

    OpenAIRE

    Silverman, S J; Sburlati, A; Slater, M L; Cabib, E.

    1988-01-01

    Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that th...

  6. Functional Analysis of the Elaeis oleifera Sesquiterpene SynthasePromoter Reveals Non-Specific Tissue Expression and Activation under Stress Condition

    Directory of Open Access Journals (Sweden)

    Nik Marzuki Sidik

    2009-01-01

    Full Text Available This research aimed to evaluate the specificity of sesquiterpene synthase promoter (SesqPro activity in the oil palm tissues and tomato hairy roots and to determine the functional region of the promoter. The effect of jasmonic acid (JA on the promoter activation and gene expression was also analyzed. A series of 5’ sequence deletions on the full-length SesqPro were generated and individually cloned into the pCAMBIA 1301 vector. Functional analysis was carried out on leaves, mesocarp slices and Immature Embryos (IE of oil palm and tomato hairy roots that had been transformed with full-length SesqPro (PSPr-VF6. GUS expression was found in all the tissues and a higher activity was detected in IE and mesocarp slices. All the constructed derivatives of SesqPro were transformed into IE and mesocarp slices in order to determine the promoter regions which are responsible for gene expression. The reduction of GUS activity was found to be related to the removal of DNA sequences within the promoter region. The promoter was induced by the elicitor molecule JA, thus suggesting the presence of JA responsive elements within the promoter. Incubation with 100 ?M of JA showed higher GUS activity in IE and mesocarp slices that had been transformed with PSPr-VF4 to PSPr-VF6. Nevertheless, the GUS activity was drastically reduced in IE and mesocarp slices containing the PSPr-VF3 promoter, suggesting that the presence of the G/A hybrid box located at -622 to -617 act as a specific element in response to elicitors. This study has shown that the action of SesqPro is non-specific and was influenced by JA induction.

  7. Activation of adipogenesis by lipocalin-type prostaglandin D synthase-generated ?¹²-PGJ? acting through PPAR?-dependent and independent pathways.

    Science.gov (United States)

    Fujimori, Ko; Maruyama, Toshihiko; Kamauchi, Shinya; Urade, Yoshihiro

    2012-08-15

    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS)-produced PGD(2) accelerates adipogenesis. In this study, we investigated the molecular mechanism of PGD(2)-mediated activation of adipogenesis in mouse adipocytic 3T3-L1 cells. LC/MS analysis showed that ?(12)-PGJ(2), one of the PGD(2) metabolites, was predominantly produced in the differentiated 3T3-L1 cells. ?(12)-PGJ(2) enhanced the expression of adipogenic genes in a ?(12)-PGJ(2)-concentration-dependent manner. Suppression of the expression of the adipogenic genes by L-PGDS siRNA or AT-56, an L-PGDS inhibitor, was cleared by the addition of ?(12)-PGJ(2). Moreover, the production of adiponectin and leptin was increased by treatment with ?(12)-PGJ(2). Furthermore, the results of a mammalian two-hybrid assay demonstrated that ?(12)-PGJ(2) enhanced the PPAR?-mediated transcription activity. However, ?(12)-PGJ(2)-activated expression of adipogenic genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase was inhibited only at 38% and 42%, respectively, by treatment with GW9662, a PPAR? antagonist in 3T3-L1 cells, although Troglitazone-mediated activation of the expression of these adipogenic genes was completely suppressed by GW9662, suggesting the existence of a PPAR?-independent mechanism for ?(12)-PGJ(2)-activated adipogenesis. These results, taken together, indicate that ?(12)-PGJ(2) is a dominant metabolite of L-PGDS-produced PGD(2) during adipogenesis and acts as an activator for adipogenesis through both PPAR?-dependent and -independent mechanisms in 3T3-L1 cells. PMID:22664386

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

    DEFF Research Database (Denmark)

    Glintborg, Dorte; HØjlund, Kurt

    2008-01-01

    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.

  9. Microbial enantioselective ester hydrolysis for the preparation of optically active 4,1-benzoxazepine-3-acetic acid derivatives as squalene synthase inhibitors.

    Science.gov (United States)

    Tarui, Naoki; Nakahama, Kazuo; Nagano, Yoichi; Izawa, Motowo; Matsumoto, Kiyoharu; Kori, Masakuni; Nagata, Toshiaki; Miki, Takashi; Yukimasa, Hidefumi

    2002-01-01

    Microbial enantioselective ester hydrolysis for the preparation of optically active (3R,5S)-(-)-5-phenyl-4,1-benzoxazepine-3-acetic acid derivatives as potent squalene synthase inhibitors was investigated. Pseudomonas diminuta and Pseudomonas taetrolens hydrolyzed the racemic ethyl ester of the 5-(2-chlorophenyl) analogue to yield the (-)-carboxylic acid with excellent enantiomeric excess (>99% ee). We found that the (-)-enantiomer was an active inhibitor. Bulkiness of the ester moiety did not affect the enantioselectivity but did affect reactivity. The racemic ethyl ester of the 5-(2-methoxyphenyl) analogue, 5-(2,3-dimethoxyphenyl) analogue and 5-(2,4-dimethoxyphenyl) analogue were also hydrolyzed with Pseudomonas taetrolens to afford enantiomerically pure (-)-carboxylic acids in large scale. As another route to (3R,5S)-(-)-7-chloro-5-(2,3-dimethoxyphenyl)-1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid [(-)-1c], the earlier intermediate (-)-2-amino-5-chloro-alpha-(2,3-dimethoxyphenyl)benzyl alcohol [(-)-12] was successfully obtained by asymmetric hydrolysis of (+/-)-5-chloro-alpha-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl acetate with Pseudomonas sp. S-13 with >99% ee in kilogram scale followed by alkaline treatment. The product (-)-12 was converted to (-)-1c without racemization. PMID:11824586

  10. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3? signaling.

    Science.gov (United States)

    Liu, Weiwei; Hu, Min; Wang, Yumei; Sun, Baozhen; Guo, Yu; Xu, Zhimin; Li, Jia; Han, Bing

    2015-03-01

    The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-? and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3? protein in CRL1623 cells, whereas the selective GSK-3? inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

  11. Cell surface F1Fo ATP synthase: a new paradigm?

    Science.gov (United States)

    Chi, Sulene L; Pizzo, Salvatore V

    2006-01-01

    The mitochondrial F1Fo adenosine triphosphate (ATP) synthase is one of the most thoroughly studied enzyme complexes known. Yet, a number of new observations suggesting that the enzyme is also located on the cell surface necessitate further investigation. While the mitochondrial synthase utilizes the proton gradient generated by oxidative phosphorylation to power ATP synthesis, the cell surface synthase has instead been implicated in numerous activities, including the mediation of intracellular pH, cellular response to antiangiogenic agents, and cholesterol homeostasis. Intriguingly, a common thread uniting these various models of cell surface ATP synthase functions is the apparently caveolar distribution of the enzyme. Recent studies concerning the cell surface ATP synthase manifest applications in the regulation of serum cholesterol levels, cellular proliferation and antitumor strategies. This review addresses the expression, interactions, functions, and consequences of inhibition of cell surface ATP synthase, an enzyme now displaying a shift in paradigm, as well as of location. PMID:17008306

  12. C-Reactive Protein Inhibits Insulin Activation of Endothelial Nitric Oxide Synthase via the Immunoreceptor Tyrosine-Based Inhibition Motif of Fc?RIIB and SHIP-1

    Science.gov (United States)

    Tanigaki, Keiji; Mineo, Chieko; Yuhanna, Ivan S.; Chambliss, Ken L.; Quon, Michael J.; Bonvini, Ezio; Shaul, Philip W.

    2009-01-01

    Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor Fc?RIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant Fc?RIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain–containing inositol 5?-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via Fc?RIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes. PMID:19423845

  13. Differential Activity of NO Synthase Inhibitors as Chemopreventive Agents in a Primary Rat Tracheal Epithelial Cell Transformation System

    Directory of Open Access Journals (Sweden)

    Sheela Sharma

    2002-01-01

    Full Text Available A model to study the effectiveness of potential chemopreventive agents that inhibit neoplastic process by different mechanisms has been used to test the efficacy of seven nitric oxide synthase (NOS inhibitors. Five selective inducible NOS (iNOS inhibitors: S-methyl isothiourea (S-MITU, S-2-aminoethyl isothiourea (S-2-AEITU, S-ethyl isothiourea (S-EITU, aminoguanidine (AG, 2-amino-4-methyl pyridine (2AMP, and two non selective general NOS inhibitors: L-N6-(1-iminoethyl lysine (IEL and N?-nitro-L-arginine (NNLA, were tested for efficacy against a carcinogen, benzo[a]pyrene (B[a]P-induced primary rat tracheal epithelial (RTE cell transformation assay. RTE cells were treated with B[a]P alone or with five nontoxic concentrations of an NOS inhibitor and the resulting foci at the end of 30 days were scored for inhibition of transformation. The results indicate that all three isothiourea compounds inhibited B[a]Pinduced RTE foci in a dose-dependent manner. SAEITU was the most effective inhibitor with an IC50 (the molar concentration that inhibits transformation by 50% of 9.1 ?M and 100% inhibition at the highest dose tested (30 ?M. However, both S-EITU and SMITU showed a maximum percent inhibition of 81% and 100% at 1 mM with an IC50 of 84 and 110 ?M, respectively. 2-AMP did not show any dose-dependent response, but was highly effective (57% inhibition at an intermediate dose of 30 ?M and an IC50 of 25 ?M. Similar to thiourea compounds, AG exhibited good dose-dependent inhibition with a maximum inhibition of 86% at 1 mM. NNLA and IEL were negative in this assay. Based on the IC50 values, NOS inhibitors were rated for efficacy from high to low as follows: S-2AEITU<2-AMP

  14. Enzyme activities of demersal fishes from the shelf to the abyssal plain

    Science.gov (United States)

    Drazen, Jeffrey C.; Friedman, Jason R.; Condon, Nicole E.; Aus, Erica J.; Gerringer, Mackenzie E.; Keller, Aimee A.; Elizabeth Clarke, M.

    2015-06-01

    The present study examined metabolic enzyme activities of 61 species of demersal fishes (331 individuals) trawled from a 3000 m depth range. Citrate synthase, lactate dehydrogenase, malate dehydrogenase, and pyruvate kinase activities were measured as proxies for aerobic and anaerobic activity and metabolic rate. Fishes were classified according to locomotory mode, either benthic or benthopelagic. Fishes with these two locomotory modes were found to exhibit differences in metabolic enzyme activity. This was particularly clear in the overall activity of citrate synthase, which had higher activity in benthopelagic fishes. Confirming earlier, less comprehensive studies, enzyme activities declined with depth in benthopelagic fishes. For the first time, patterns in benthic species could be explored and these fishes also exhibited depth-related declines in enzyme activity, contrary to expectations of the visual interactions hypothesis. Trends were significant when using depth parameters taken from the literature as well as from the present trawl information, suggesting a robust pattern regardless of the depth metric used. Potential explanations for the depth trends are discussed, but clearly metabolic rate does not vary simply as a function of mass and habitat temperature in fishes as shown by the substantial depth-related changes in enzymatic activities.

  15. Nitric oxide synthase inhibition prevents activity-induced calcineurin-NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions.

    Science.gov (United States)

    Martins, Karen J B; St-Louis, Mathieu; Murdoch, Gordon K; MacLean, Ian M; McDonald, Pamela; Dixon, Walter T; Putman, Charles T; Michel, Robin N

    2012-03-15

    The calcineurin–NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin–NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(?1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(?1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3? phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3?-induced facilitation of calcineurin–NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling. PMID:22219342

  16. 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)

    Pei?Rung Wu

    2014-08-01

    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.

  17. Role of AKT-Glycogen Synthase Kinase axis in Monocyte Activation in Humans with and without Type 2 Diabetes

    OpenAIRE

    Nandy, Debashis; Asmann, Yan W.; Mukhopadhyay, Debabrata; Basu, Ananda

    2009-01-01

    Monocyte activation by chemokines is a vital trigger for initiation of atherosclerotic process. Circulating levels of Platelet Activating Factor (PAF), a recognized chemokine, is known to be increased in type 2 diabetes that is linked to accelerated atherosclerosis. To explore the molecular basis we examined the signaling pathways involved in PAF induced monocyte activation. PAF increased migration in monocytes obtained from THP-1 cells, nondiabetic and diabetic subjects that was blocked by A...

  18. Homochiral Cu(II) and Ni(II) malates with tunable structural features

    Energy Technology Data Exchange (ETDEWEB)

    Zavakhina, Marina S. [Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Lavrentiev av., 630090 Novosibirsk (Russian Federation); Samsonenko, Denis G. [Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Lavrentiev av., 630090 Novosibirsk (Russian Federation); Novosibirsk State University, 2 Pirogova st., 630090 Novosibirsk (Russian Federation); Virovets, Alexander V. [Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Lavrentiev av., 630090 Novosibirsk (Russian Federation); Dybtsev, Danil N. [Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Lavrentiev av., 630090 Novosibirsk (Russian Federation); Division of the Advanced Materials Science, POSTECH, San 31, Hyojadong, 790-784 Pohang (Korea, Republic of); Fedin, Vladimir P., E-mail: cluster@niic.nsc.ru [Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Lavrentiev av., 630090 Novosibirsk (Russian Federation); Novosibirsk State University, 2 Pirogova st., 630090 Novosibirsk (Russian Federation)

    2014-02-15

    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)]·H{sub 2}O (1), [Cu(mal)(bpe)]·2H{sub 2}O (2), [Ni(mal)(bpy)]·1.3CH{sub 3}OH (3) and [Ni(mal)(bpe)]·4H{sub 2}O (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 Ni{sup 2+} or Cu{sup 2+} 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 Ni{sup 2+} and Cu{sup 2+}. • 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.

  19. Induction of ATP synthase ? by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells.

    Science.gov (United States)

    Kim, Hye-Eun; Lee, Seong-Gene

    2013-07-01

    Hydrogen peroxide (H2O2) production due to oxidative stress is associated with apoptosis and melanogenesis in melanocytes. Here, we analyzed the effects of H2O2 on melanogenesis by measuring the melanin content and analyzing the expression of melanogenesis-related proteins, including cAMP-responsive element binding protein (CREB), microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and phenylalanine hydroxylase (PAH). Treatment with 1mM H2O2 increased the cellular melanin content; the expression of PAH, TYR, and MITF; and the phosphorylation of CREB in B16F10 and SK-Mel-2 cells. In addition, H2O2 increased the expression of ATP synthase ? (ATP5B), a mitochondrial F1 complex, and increased intracellular ATP levels. Studies using the ATP5B inhibitor oligomycin (OM) showed that the induction of cAMP resulted from an increase in ATP caused by the induction of ATP5B. OM treatment increased H2O2-mediated apoptosis via accelerated ATP depletion and apoptosis-related gene expressions. In summary, H2O2 may induce melanogenesis via the upregulation of PAH and activation of cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through the induction of ATP5B. PMID:23523934

  20. Maintained activity of glycogen synthase kinase-3? despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    International Nuclear Information System (INIS)

    Glycogen synthase kinase-3? (GSK3?) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3?. However, the inactive form of GSK3? which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3? substrates, such as ?-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3? at serine-9 and other substrates including tau, ?-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3? inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3? may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3? inhibitors could be a valuable drug candidate in AD.

  1. Glycogen synthase kinase 3? dictates podocyte motility and focal adhesion turnover by modulating paxillin activity: implications for the protective effect of low-dose lithium in podocytopathy.

    Science.gov (United States)

    Xu, Weiwei; Ge, Yan; Liu, Zhihong; Gong, Rujun

    2014-10-01

    Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3? (GSK3?), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3? in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3? inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3? overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion-associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3? highly mimicked, whereas ectopic expression of a constitutively active GSK3? mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3?. Furthermore, paxillin interacted with GSK3? and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3? overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3?-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy. PMID:25239564

  2. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: dhkim@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

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

    Science.gov (United States)

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

    2008-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J

    2012-01-01

    Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling was carried out to reveal the co-localization of neurotransmitters. VIP-immunoreactive (-ir) neurons as well as fibers were frequently found in human SPG. Many, homogenously stained NOS-ir cells were found, but no positive fibers. In addition, PACAP-ir was observed in some of the neurons and in fibers. Co-localization was found between VIP and NOS. In rat VIP-, NOS-, and PACAP-ir were found in many neurons and fibers. Co-localization of PACAP and NOS was observed in neurons. PACAP and GS double staining revealed that the PACAP-ir was localized in/close to the cell membrane, but not in the satellite glial cells. PAC1 and VPAC1 immunoreactivity was found in the satellite glial cells of both human and rat. Western blot revealed protein expression of PAC1, VPAC1, and VPAC2 in rat SPG. The trigeminal-autonomic reflex may be active in migraine attacks. We hypothesized that VIP, PACAP, NOS, PAC1, VPAC1, and VPAC2 play a role in the activation of parasympathetic cranial outflow during migraine attacks.

  6. Long noncoding RNA MALAT1 promotes brain metastasis by inducing epithelial-mesenchymal transition in lung cancer.

    Science.gov (United States)

    Shen, Liqin; Chen, Lei; Wang, Yongsheng; Jiang, Xiaochun; Xia, Hongping; Zhuang, Zhixiang

    2015-01-01

    Brain metastasis often has a poor prognosis in patients with advanced non-small cell lung cancer (NSCLC). Therefore, it is urgent to identify factors associated with lung cancer brain metastasis. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) also known as noncoding nuclear-enriched abundant transcript 2 is a long noncoding RNA, which is highly conserved amongst mammals. It has been shown to be increased in a variety of tumors including NSCLC and regulate the expression of metastasis-associated genes. However, the role of MALAT1 in lung cancer brain metastasis has not been investigated. In this study, we examined the level of MALAT1 in 78 cases of NSCLC samples with 19 brain metastasis and 59 non-brain metastasis by qRT-PCR. We observed that the level of MALAT1 was significantly higher in brain metastasis than that of non brain metastasis samples (P cell subline using the brain metastasis lung cancer cell H1915. We found that MALAT1 is increased in highly invasive subline of brain metastasis lung cancer cells. Further functional studies indicate that silencing MALAT1 inhibits highly invasive subline of brain metastasis lung cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). Therefore, increased level of long noncoding RNA MALAT1 promotes lung cancer brain metastasis by inducing EMT, which may be a promising prognosis factor and therapeutic target to treat lung cancer brain metastasis in future. PMID:25217850

  7. A chalcone synthase/stilbene synthase DNA probe for conifers.

    Science.gov (United States)

    Baker, S M; White, E E

    1996-05-01

    A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them. PMID:24166547

  8. Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not.

    Science.gov (United States)

    Seravalli, Javier; Xiao, Yuming; Gu, Weiwei; Cramer, Stephen P; Antholine, William E; Krymov, Vladimir; Gerfen, Gary J; Ragsdale, Stephen W

    2004-04-01

    The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. One structure of the Moorella thermoacetica enzyme revealed that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bridged to a binuclear CuNi center with Cu at the proximal metal site (M(p)) and Ni at the distal metal site (M(d)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity and Cu content, we had proposed that the Cu-containing enzyme is active [Seravalli, J., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3689-3694]. Here we have reexamined this proposal. Enzyme preparations with a wider range of Ni (1.6-2.8) and Cu (0.2-1.1) stoichiometries per dimer were studied to reexamine the correlation, if any, between the Ni and Cu content and ACS activity. In addition, the effects of o-phenanthroline (which removes Ni but not Cu) and neocuproine (which removes Cu but not Ni) on ACS activity were determined. EXAFS results indicate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examined to investigate the electronic state of this proposed intermediate in the ACS reaction mechanism. The combined results strongly indicate that the CuNi enzyme is inactive, that the NiNi enzyme is active, and that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](2+) (net S = 0) cluster bridged to a Ni(1+) (S = (1)/(2)) at M(p) that is bridged to planar four-coordinate Ni(2+) (S = 0) at M(d), with the spin predominantly on the Ni(1+). Furthermore, these studies suggest that M(p) is inserted during cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieved with NiFeC signals of approximately 0.8 spin/mol. PMID:15049702

  9. Laser microdissection of conifer stem tissues: Isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca

    Directory of Open Access Journals (Sweden)

    Hamberger Björn

    2010-06-01

    Full Text Available Abstract Background Laser microdissection (LMD has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in regular patterns with xylem, phloem and cortex development controlled by the activity of the cambial zone (CZ. Defense responses of conifer stems against insects and pathogens involve increased accumulation of terpenoids in cortical resin ducts (CRDs and de novo formation of traumatic resin ducts from CZ initials. These tissues are difficult to isolate for tissue-specific molecular and biochemical characterization and are thus good targets for application of LMD. Results We describe robust methods for isolation of individual tissue-types from white spruce (Picea glauca stems for analysis of RNA, enzyme activity and metabolites. A tangential cryosectioning approach was important for obtaining large quantities of CRD and CZ tissues using LMD. We report differential expression of genes involved in terpenoid metabolism between CRD and CZ tissues and in response to methyl jasmonate (MeJA. Transcript levels of ?-pinene synthase and levopimaradiene/abietadiene synthase were constitutively higher in CRDs, but induction was stronger in CZ in response to MeJA. 3-Carene synthase was more strongly induced in CRDs compared to CZ. A differential induction pattern was observed for 1-deoxyxyulose-5-phosphate synthase, which was up-regulated in CRDs and down-regulated in CZ. We identified terpene synthase enzyme activity in CZ protein extracts and terpenoid metabolites in both CRD and CZ tissues. Conclusions Methods are described that allow for analysis of RNA, enzyme activity and terpenoid metabolites in individual tissues isolated by LMD from woody conifer stems. Patterns of gene expression are demonstrated in specific tissues that may be masked in analysis of heterogenous samples. Combined analysis of transcripts, proteins and metabolites of individual tissues will facilitate future characterization of complex processes of woody plant development, including periodic stem growth and dormancy, cell specialization, and defense and may be applied widely to other plant species.

  10. Active site labeling of the shikimate pathway enzyme, dehydroquinase. Evidence for a common substrate binding site within dehydroquinase and dehydroquinate synthase.

    Science.gov (United States)

    Kleanthous, C; Campbell, D G; Coggins, J R

    1990-07-01

    Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes. PMID:2193027

  11. The SH2B1 obesity locus is associated with myocardial infarction in diabetic patients and with NO synthase activity in endothelial cells

    Science.gov (United States)

    Prudente, Sabrina; Morini, Eleonora; Larmon, Jay; Andreozzi, Francesco; Pietro, Natalia Di; Nigro, Angela; Gervino, Ernest V; Mannino, Gaia Chiara; Bacci, Simonetta; Hauser, Thomas H; Bellacchio, Emanuele; Formoso, Gloria; Pellegrini, Fabio; Proto, Vittoria; Menzaghi, Claudia; Frittitta, Lucia; Pandolfi, Assunta; Sesti, Giorgio; Doria, Alessandro; Trischitta, Vincenzo

    2013-01-01

    Objective Obesity and cardiovascular disease recognize a common metabolic soil and may therefore share part of their genetic background. Genome-wide association studies have identified variability at the SH2B1 locus as a predictor of obesity. We investigated whether SNP rs4788102, which captures the entire SH2B1 variability, is associated with coronary artery disease (CAD) and/or myocardial infarction (MI) in patients with type 2 diabetes mellitus (T2DM). Design and Setting SNP rs4788102 was typed in 2,015 White subjects with T2DM from three CAD case-control studies [n=740 from the Gargano Hearth Study (GHS, Italy); n=818 from the Joslin Hearth Study (JHS, Boston); n=457 from the University of Catanzaro (CZ, Italy)]. Results SNP rs4788102 (G/A) was not associated with CAD (overall allelic OR=1.06, 95% CI=0.93-1.21; p=0.37). On the contrary, it was associated with MI in GHS (1.42, 1.12-1.81; p=0.004) and in the three samples analyzed together (1.21, 1.04-1.41; p=0.016). Insulin stimulated nitric oxide synthase (NOS) activity in human vein endothelial cells from G/G (n=4, p=0.03) but not the G/A (n=5, p=0.83) genotype. Of the SNPs in perfect LD with rs4788102, one (rs7498665) affects amino acid polarity (Ala484Thr) and falls into a highly conserved protein segment of SH2B1 containing a class II SH3 domain binding site. Conclusions Variability at the SH2B1 obesity locus is associated with MI in diabetic patients and with reduced insulin-stimulated NOS activity in human endothelial cells. Further studies are needed to replicate this association and dissect the biology underlying this finding. PMID:21907990

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

    Science.gov (United States)

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

    2012-10-01

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

  13. Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism.

    OpenAIRE

    Leibovich, S. J.; Polverini, P J; Fong, T W; Harlow, L A; Koch, A E

    1994-01-01

    Human monocytes (M phi) require stimulation with substances such as bacterial endotoxin [LPS (lipopolysaccharide)] to produce angiogenic activity. In this study, we report that stimulation of M phi with LPS (5 micrograms/ml) in the absence of L-arginine greatly reduced their production of angiogenic activity, as assessed in vivo in rat corneas and in vitro by chemotaxis of human umbilical vein endothelial cells (HU-VECs). D-Arginine did not substitute for L-arginine in the production of angio...

  14. Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

    Science.gov (United States)

    Wang, Xiao Ming; Soetaert, Karine; Peirs, Priska; Kalai, Michaël; Fontaine, Véronique; Dehaye, Jean Paul; Lefèvre, Philippe

    2015-01-01

    PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions. PMID:25860441

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

    Directory of Open Access Journals (Sweden)

    Guidon Gérard

    2006-12-01

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

  16. Specificities of calreticulin transacetylase to acetoxy derivatives of 3-alkyl-4-methylcoumarins: effect on the activation of nitric oxide synthase.

    Science.gov (United States)

    Kathuria, Abha; Gupta, Anjali; Priya, Nivedita; Singh, Prabhjot; Raj, Hanumantharao G; Prasad, Ashok K; Parmar, Virinder S; Sharma, Sunil K

    2009-02-15

    Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and modulates their biological activities. CRTAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (DAMC). We have studied earlier, the influence of acetoxy groups on the benzenoid ring, the effect of reduction of double bond at C-3 and C-4 position, the effect of methyl/phenyl group at C-4, and the influence of position of carbonyl group with respect to oxygen heteroatom in the benzopyran nucleus, for the catalytic activity of CRTAase. In this communication, we have extended our previous work; wherein we studied the influence of an alkyl group (ethyl, hexyl and decyl) at the C-3 position of the acetoxy coumarins on the CRTAase activity. The substitution at C-3 position of coumarin nucleus resulted in the reduction of CRTAase activity and related effects. Accordingly the formation of NO in platelets by C-3 alkyl substituted acetoxy coumarins was found to be much less compared to the unsubstituted analogs. In addition the alkyl substitution at C-3 position exhibited the tendency to form radicals other than NO. PMID:19181531

  17. Activation of ceramide synthase 6 by celecoxib leads to a selective induction of C16:0-ceramide

    OpenAIRE

    2010-01-01

    Abstract Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C14:0-Cer–C26:0-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50?M...

  18. Integrin ?9?1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase

    OpenAIRE

    Gupta, Shiv K.; Vlahakis, Nicholas E

    2009-01-01

    Integrins are important mediators of cell adhesion and migration, which in turn are essential for diverse biological functions, including wound healing and cancer metastasis. The integrin ?9?1 is expressed on numerous mammalian tissues and can mediate accelerated cell migration. As the molecular signaling mechanisms that transduce this effect are poorly defined, we investigated the pathways by which activated integrin ?9?1 signals migration. We found for the first ...

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

    Directory of Open Access Journals (Sweden)

    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be context or cell line dependent, but could also be explained on the basis that although NF-kappaB is an important mediator of pancreatic cancer cell survival, it plays a minor role in gemcitabine resistance. Further work is needed to understand the mechanisms of this effect, including the potential for rational combination of GSK3 inhibitors with other targeted agents for the treatment of pancreatic cancer.

  20. Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage ?-glucan

    Science.gov (United States)

    Fujita, Naoko; Toyosawa, Yoshiko; Utsumi, Yoshinori

    2012-01-01

    The relationship between the solubility, crystallinity, and length of the unit chains of plant storage ?-glucan was investigated by manipulating the chain length of ?-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa I, coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total ?-glucan in the isa1 mutant, whereas it was only 35–70% in the transgenic SSIIa I /isa1 lines. Thus, the ?-glucans from the SSIIa I /isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the ?-glucans of the insoluble fraction, while no crystallinity was confirmed for ?-glucans in isa1. Concerning the degree of polymerization (DP) ?30, the chain lengths of these ?-glucans differed significantly in the order of SSIIa I /isa1 insoluble > SSIIa I /isa1 soluble > ?-glucans in isa1. The amount of long chains with DP ?33 was higher in the insoluble fraction ?-glucans than in the other two ?-glucans. No difference was observed in the chain length distributions of the ?-amylase limit dextrins among these ?-glucans. These results suggest that in the SSIIa I /isa1 transgenic lines, the unit chains of ?-glucans were elongated by SSIIaI, whereas the expression of SSIIaI did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIaI. PMID:23048127

  1. Destabilization of the homotetrameric assembly of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from the hyperthermophile Pyrococcus furiosus enhances enzymatic activity.

    Science.gov (United States)

    Nazmi, Ali Reza; Schofield, Linley R; Dobson, Renwick C J; Jameson, Geoffrey B; Parker, Emily J

    2014-02-01

    Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 ?M. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment. PMID:24239948

  2. Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria.

    Science.gov (United States)

    Bustad, Helene J; Vorland, Marta; Rønneseth, Eva; Sandberg, Sverre; Martinez, Aurora; Toska, Karen

    2013-01-01

    The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype-phenotype relations for AIP. PMID:23815679

  3. Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria

    Directory of Open Access Journals (Sweden)

    Aurora Martinez

    2013-08-01

    Full Text Available The autosomal dominantly inherited disease AIP (acute intermittent porphyria is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W, enzyme kinetics (R167W or both (R173W. A combination of native PAGE, CD, DSF (differential scanning fluorimetry and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18{_}V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 {angstrom} resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3{beta} (HsGSK-3{beta}) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  5. Glycogen synthase kinase 3 ? activity is required for hBora/Aurora A-mediated mitotic entry

    OpenAIRE

    Lee, Yu-cheng; Liao, Po-chi; Liou, Yih-cherng; Hsiao, Michael; Huang, Chi-ying; Lu, Pei-jung

    2013-01-01

    The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G2 phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3? can interact with hBora and phosphorylate hBora at Ser274 and ...

  6. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-03-02

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

  7. Effects of supplemental citrulline malate ingestion during repeated bouts of lower-body exercise in advanced weightlifters.

    Science.gov (United States)

    Wax, Benjamin; Kavazis, Andreas N; Weldon, Kevin; Sperlak, Joseph

    2015-03-01

    The purpose of this investigation was to test the efficacy of citrulline malate supplementation on exercise performance, blood lactate, heart rate, and blood pressure during lower-body dynamic resistance exercise. We hypothesized that citrulline malate ingestion before performing submaximal repeated bouts of multiple lower-body resistance exercises would improve performance. Twelve advanced resistance-trained male subjects participated in a randomized, counterbalanced, double-blind study. Subjects were randomly assigned to placebo (PL) or citrulline malate (8 g) groups and then performed repeated bouts of multiple lower-body resistance exercise. Specifically, subjects performed 5 sequential sets (60% 1 repetition maximum) to failure on the leg press, hack squat, and leg extension machines. Blood lactate, heart rate, systolic blood pressure, and diastolic blood pressure were determined before and after exercise. The exercise protocol resulted in sequential significant (p ? 0.05) decrease in the number of repetitions in all 3 exercises. However, subjects in the citrulline malate group performed significantly (p ? 0.05) higher number of repetitions during all 3 exercises compared with PL group. Blood lactate and heart rate were significantly increased (p ? 0.05) after exercise compared with before exercise but were not significantly different between citrulline malate and PL (p > 0.05). No significant (p > 0.05) differences were detected for blood pressure measurements. In conclusion, our results suggest that citrulline malate supplementation may be beneficial in improving exercise performance during lower-body multiple-bout resistance exercise in advanced resistance-trained men. PMID:25226311

  8. Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo.

    OpenAIRE

    Gaughran, J P; Lai, M.H.; Kirsch, D R; Silverman, S J

    1994-01-01

    Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.

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

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

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

  10. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-05

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

  11. Metal-Catalyzed Oxidation of Phenylalanine-Sensitive 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Escherichia coli: Inactivation and Destabilization by Oxidation of Active-Site Cysteines

    OpenAIRE

    Park, Ohkmae K.; Bauerle, Ronald

    1999-01-01

    The in vitro instability of the phenylalanine-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and d-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life...

  12. Soybean seed galactinol synthase activity as determined by a novel colorimetric assay / Atividade de galactinol sintase de semente de soja determinada por um novo ensaio calorimétrico

    Scientific Electronic Library Online (English)

    MARLUCI, RIBEIRO; CARLOS R., FELIX; SILENE DE PAULINO, LOZZI.

    Full Text Available Galactinol sintase (GS) é a enzima-chave para a biossíntese de oligosacarídeos de rafinose (RO), que são os fatores antinutricionais causadores de flatulência, os quais estão presentes em sementes de soja e em outros legumes. A GS catalisa a formação de galactinol e UDP a partir de UDP-gal e mioinos [...] itol. A atividade dessa enzima é determinada atualmente pelo método radioisotópico que, apesar de adequado tecnicamente, apresenta vários inconvenientes, tais como a necessidade de substrato de alto custo, bem como de cuidados adicionais e serviços especializados para descarte dos resíduos radioativos. Assim, desenvolveu-se um método colorimétrico alternativo ao método radioisotópico, baseado na determinação colorimétrica indireta do UDP formado pela hidrólise enzimática (apirase) desse nucleotídeo e determinação do Pi resultante pelo método de Fiske & SubbaRow, com modificações. A cor desenvolvida é estável e o método é sensível para detecção de quantidades nanomolares de Pi. Os perfis de atividade da GS em sementes de soja em diferentes fases de desenvolvimento, determinados pelos métodos colorimétrico e radioisotópico, são semelhantes. Adicionalmente, a GS de sementes de soja foi purificada (46-vezes) por tratamento do extrato das sementes com MnCl2, e uma seqüência de cromatografias em colunas de DEAE-Sepharose, Phenyl-Sepharose CL-4B e Q-Sepharose. As atividades de GS no extrato bruto e na amostra parcialmente purificada foram máximas em pH 7.0 e 50 ºC. Ditiotreitol e MnCl2 aumentaram consideravelmente a atividade da enzima parcialmente purificada. Enquanto UDP-glc pode ser hidrolisado pela enzima com uma atividade relativa correspondendo a 49% da atividade contra UDP-gal, UDP-man e sacarose foram completamente ineficazes como substratos alternativos. Os valores de K M para conversão de UDP-gal e mio-inositol foram de 2,0 mM e 2,93 mM, respectivamente, determinados pelo método de Lineaweaver-Burk. Abstract in english Galactinol synthase (GS) is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO) which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses forma [...] tion of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity. The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold) by treatment of seed extract with MnCl2, sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates.

  13. Soybean seed galactinol synthase activity as determined by a novel colorimetric assay Atividade de galactinol sintase de semente de soja determinada por um novo ensaio calorimétrico

    Directory of Open Access Journals (Sweden)

    MARLUCI RIBEIRO

    2000-01-01

    Full Text Available Galactinol synthase (GS is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses formation of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity. The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold by treatment of seed extract with MnCl2, sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates.Galactinol sintase (GS é a enzima-chave para a biossíntese de oligosacarídeos de rafinose (RO, que são os fatores antinutricionais causadores de flatulência, os quais estão presentes em sementes de soja e em outros legumes. A GS catalisa a formação de galactinol e UDP a partir de UDP-gal e mioinositol. A atividade dessa enzima é determinada atualmente pelo método radioisotópico que, apesar de adequado tecnicamente, apresenta vários inconvenientes, tais como a necessidade de substrato de alto custo, bem como de cuidados adicionais e serviços especializados para descarte dos resíduos radioativos. Assim, desenvolveu-se um método colorimétrico alternativo ao método radioisotópico, baseado na determinação colorimétrica indireta do UDP formado pela hidrólise enzimática (apirase desse nucleotídeo e determinação do Pi resultante pelo método de Fiske & SubbaRow, com modificações. A cor desenvolvida é estável e o método é sensível para detecção de quantidades nanomolares de Pi. Os perfis de atividade da GS em sementes de soja em diferentes fases de desenvolvimento, determinados pelos métodos colorimétrico e radioisotópico, são semelhantes. Adicionalmente, a GS de sementes de soja foi purificada (46-vezes por tratamento do extrato das sementes com MnCl2, e uma seqüência de cromatografias em colunas de DEAE-Sepharose, Phenyl-Sepharose CL-4B e Q-Sepharose. As atividades de GS no extrato bruto e na amostra parcialmente purificada foram máximas em pH 7.0 e 50 ºC. Ditiotreitol e MnCl2 aumentaram consideravelmente a atividade da enzima parcialmente purificada. Enquanto UDP-glc pode ser hidrolisado pela enzima com uma atividade relativa correspondendo a 49% da atividade contra UDP-gal, UDP-man e sacarose foram completamente ineficazes como substratos alternativos. Os valores de K M para conversão de UDP-gal e mio-inositol foram de 2,0 mM e 2,93 mM, respectivamente, determinados pelo método de Lineaweaver-Burk.

  14. Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

    OpenAIRE

    Schaefer, A. L.; Val, D L; Hanzelka, B L; J.E. Cronan; Greenberg, E. P.

    1996-01-01

    Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synt...

  15. Active Stabilization of Human Endothelial Nitric Oxide Synthase mRNA by hnRNP E1 Protects against Antisense RNA and MicroRNAs

    OpenAIRE

    Ho, J. J. David; Robb, G. Brett; Tai, Sharon C.; Turgeon, Paul J.; Mawji, Imtiaz A.; Man, H. S. Jeffrey; Marsden, Philip A.

    2013-01-01

    Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3? untranslated region (3? UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and...

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

    OpenAIRE

    Prosser, I.; Altug, I. G.; Philips, A. L.; Ko?nig, W. A.; Bouwmeester, H. J.; Beale, M.

    2004-01-01

    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 function...

  17. Glucocorticoid protects rodent hearts from ischemia/reperfusion injury by activating lipocalin-type prostaglandin D synthase–derived PGD2 biosynthesis

    OpenAIRE

    Tokudome, Satori; Sano, Motoaki; Shinmura, Ken; Matsuhashi, Tomohiro; Morizane, Shintaro; Moriyama, Hidenori; Tamaki, Kayoko; Hayashida, Kentaro; Nakanishi, Hiroki; Yoshikawa, Noritada; Shimizu, Noriaki; Endo, Jin; Katayama, Takaharu; Murata, Mitsushige; Yuasa, Shinsuke

    2009-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD2 biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. In the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types,...

  18. Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis.

    OpenAIRE

    Silverman, P. M.; Eoyang, L.

    1987-01-01

    Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a ...

  19. The Acute Physiological Effects of the Vaso-Active Drug, L-NNA, a Nitric Oxide Synthase Inhibitor, on Renal and Tumour Perfusion in Human Subjects

    OpenAIRE

    Kent Yip; Vicky Goh; Jane Gregory; Ian Simcock; James Stirling, J.; Jane Taylor, N.; Robert Kozarski; Andrew Mitchell; Sam Bosopem; Gavin Halbert; Roberto Alonzi; David Miles; Peter Hoskin

    2013-01-01

    Purpose: To assess the baseline variation in global renal and tumour blood flow, blood volume and extraction fraction, and changes in these parameters related to the acute physiological effects of a single dose of a non selective inhibitor of nitric oxide synthase, L-NNA. Materials & Methods: Ethical approval and informed consent were obtained for this Phase I clinical study. Patients with advanced solid tumours refractory to conventional therapy were recruited and give...

  20. Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations

    OpenAIRE

    Anna Dowiercia?; Piotr Wilk; Wojciech Rypniewski; Wojciech Rode; Adam Jarmu?a

    2014-01-01

    The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands...

  1. Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria

    OpenAIRE

    Aurora Martinez; Karen Toska; Helene J. Bustad; Marta Vorland; Eva Rønneseth; Sverre Sandberg

    2013-01-01

    The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated ...

  2. Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit.

    Science.gov (United States)

    Ahmed, S A; Ruvinov, S B; Kayastha, A M; Miles, E W

    1991-11-15

    The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association. PMID:1939184

  3. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-?-dependent pathway in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Highlights: ? Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. ? Adiponectin also increases the phosphorylation of AMPK. ? A pharmacological activator of AMPK increases mRNA levels of PPAR? and HAS2. ? Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR? antagonist. ? Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR?-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1?-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-? (PPAR?), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR? antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR?-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  4. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan); Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  5. Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin ?-Ketoacyl-Acyl Carrier Protein Synthase

    OpenAIRE

    He, Min(Department of Applied Physics, Nanjing University of Science and Technology, Nanjing, 210094, China); Varoglu, Mustafa; Sherman, David H.

    2000-01-01

    A three-dimensional model of the Streptomyces coelicolor actinorhodin ?-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme ?-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions...

  6. Monoterpene synthases from common sage (Salvia officinalis)

    Science.gov (United States)

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

    1999-01-01

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

  7. Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-?-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

    Science.gov (United States)

    Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba

    2014-09-15

    Recent data show that colon cancer cells selectively overexpress cystathionine-?-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects. PMID:24667534

  8. Isolations and characterization of highly water-soluble dimeric lanthanide citrate and malate with ethylenediaminetetraacetate.

    Science.gov (United States)

    Chen, Mao-Long; Gao, Song; Zhou, Zhao-Hui

    2012-01-28

    Highly water-soluble lanthanum and cerium citrates or malates with ethylenediaminetetraacetate (NH(4))(8)[Ln(2)(Hcit)(2)(EDTA)(2)]·9H(2)O [Ln = La, 1; Ce, 2], K(8)[La(2)(Hcit)(2)(EDTA)(2)]·16H(2)O (3) and K(6)[Ln(2)(Hmal)(2)(EDTA)(2)]·14H(2)O [Ln = La, 4; Ce, 5] (H(4)cit = citric acid, H(3)mal = malic acid, and H(4)EDTA = ethylenediaminetetracetic acid) were prepared from the reactions of lanthanide ethylenediaminetetraacetate trihydrates with citric or malic acid at pH 5.0-6.5. These compounds were characterized by elemental analyses, IR, TG-DTG, solution (13)C{(1)H} NMR, solid state (13)C NMR spectra and X-ray structural analyses. The main structural feature of the compounds consists of a dinuclear unit deca-coordinated by EDTA and citrate or malate. The ?-hydroxy and ?-carboxy groups of citrate and malate chelate in five-membered ring with one lanthanide ion, while one of the ?-carboxy group coordinates with the other lanthanide ion, forming a dimeric structure. The other pendent ?-carboxy groups in 1-3 form very strong intramolecular hydrogen bond with ?-hydroxy groups [O1O7 2.594(4), 2.587(8) and 2.57(1) Å for 1-3 respectively]. (13)C NMR spectra of the lanthanum compounds show obvious downfield shifts based on solid and solution NMR measurements, indicating the coordinations of mixed-ligand in lanthanum complexes, while highfield shifts are observed in cerium complexes. PMID:22116197

  9. Amide hydrogen exchange shows that malate dehydrogenase is a folded monomer at pH 5

    OpenAIRE

    Chen, Jiwen; Smith, David L.

    2001-01-01

    Although there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH-induced changes in solvent accessibility, MDH was exposed to D2O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the in...

  10. Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

    Science.gov (United States)

    Mazzoli, R; Lamberti, C; Coisson, J D; Purrotti, M; Arlorio, M; Giuffrida, M G; Giunta, C; Pessione, E

    2009-01-01

    Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion. PMID:18283413

  11. Nitric oxide synthase in the pineal gland

    OpenAIRE

    Lopez-figueroa, M. O.; Moller, M.

    1996-01-01

    The recent discovery of nitric oxide (NO) as a biological messenger molecule with unique characteristics has opened a new field in pineal research. This free radical gas is synthesized by the enzyme nitric oxide synthase (NOS) from L-arginine. The activation of adrenoreceptors in the membrane of the pinealocytes mediates the increase in NO through a mechanism that involves G proteins. In the pinealocyte, NO stimulates guanylyl cyclase resulting in an increased ...

  12. Thermotoga maritima 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase: the ancestral eubacterial DAHP synthase?

    Science.gov (United States)

    Wu, Jing; Howe, David L; Woodard, Ronald W

    2003-07-25

    The gene encoding the 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase from the thermophilic microorganism Thermotoga maritima was cloned, and the enzyme was overexpressed in Escherichia coli. The purified DAHP synthase displays a homotetrameric structure and exhibits maximal activity at 90 degrees C. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for approximately 5 h at 80 degrees C, 21 h at 70 degrees C, and 86 h at 60 degrees C. The enzyme appears to follow Michaelis-Menten kinetics with Km for phosphoenolpyruvate = 9.5-13 microm, Km for d-erythrose 4-phosphate = 57.3-350.1 microm, and kcat = 2.3-7.6 s-1 between 50 degrees C and 70 degrees C. Metal analysis indicates that DAHP synthase as isolated contains Zn2+, and the enzyme is inactivated by treatment with EDTA. The apo-enzyme is partially reactivated by a variety of divalent metals including Zn2+, Cd2+, Mn2+, Cu2+, Co2+, and Ni2+. These observations suggest that T. maritima DAHP synthase is a metalloenzyme. The activity of T. maritima DAHP synthase is inhibited by two of the three aromatic amino acids (l-Phe and l-Tyr) formed in the Shikimate pathway. This report is the first description of a thermophilic eubacterial DAHP synthase. PMID:12743122

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

    OpenAIRE

    Choi, Won-il; Jeon, Bu-nam; Park, Hyejin; Yoo, Jung-yoon; Kim, Yeon-sook; Koh, Dong-in; Kim, Myung-hwa; Kim, Yu-ri; Lee, Choong-eun; Kim, Kyung-sup; Osborne, Timothy F.; Hur, Man-wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator o...

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

    Czech Academy of Sciences Publication Activity Database

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

    2005-01-01

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

  15. Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

    OpenAIRE

    Sheu, Der-Shyan; Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

    2012-01-01

    The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaCCsp) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly ...

  16. An active triple-catalytic hybrid enzyme engineered by linking COX-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

    OpenAIRE

    Ruan, Ke-he; So, Shui-ping; Cervantes, Vanessa; Wu, Hanjing; Wijaya, Cori; Jentzen, Rebecca R.

    2008-01-01

    It remains a challenge to create the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme (Trip-cat enzyme-2, [Ruan, KH., et al. (2006) Biochemistry 45, 14003 – 14011]), which links cyclooxygenase-2 (COX-2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s triple-catalytic (Trip-cat) functions. The stable up-regulation of the biosynthesis of the vascular protector, PGI2, in cells is...

  17. Evolutionary and mechanistic insights from the reconstruction of ?-humulene synthases from a modern (+)-germacrene A synthase.

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% ?-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases. PMID:25230152

  18. Resveratrol Inhibits Invasion and Metastasis of Colorectal Cancer Cells via MALAT1 Mediated Wnt/?-Catenin Signal Pathway

    OpenAIRE

    Ji, Qing; Liu, Xuan; Fu, Xiaoling; Zhang, Long; Sui, Hua; Zhou, Lihong; Sun, Jian; Cai, Jianfeng; Qin, Jianmin; Ren, Jianlin; Li, Qi

    2013-01-01

    Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC), in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1) also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/?-catenin signaling, th...

  19. A continuous spectrophotometric assay for human cystathionine beta-synthase.

    Science.gov (United States)

    Shen, Weijun; McGath, Molly K; Evande, Ruby; Berkowitz, David B

    2005-07-01

    We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-1) for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Km(app) = 2.2+/-0.5 mM for L-serine and 6.6+/-2.2 for cysteamine, with kcat = 2.5+/-0.4 s(-1). These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine. PMID:15958186

  20. Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation

    International Nuclear Information System (INIS)

    The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NaCl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively. 24 refs.; 7 figs.; 4 tabs

  1. Upregulation of long non-coding RNA MALAT1 correlates with tumor progression and poor prognosis in clear cell renal cell carcinoma.

    Science.gov (United States)

    Zhang, Hai-Min; Yang, Feng-Qiang; Chen, Shao-Jun; Che, Jianping; Zheng, Jun-Hua

    2015-04-01

    Long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, pancreatic cancer, and cervical cancer. However, the role of lncRNA MALAT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. Expression levels of lncRNA MALAT1 in ccRCC tissues and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR), and its association with overall survival of patients was analyzed by statistical analysis. Small interfering RNA (siRNA) was used to suppress MALAT1 expression in renal cancer cells. In vitro assays were conducted to further explore its role in tumor progression. The expression level of MALAT1 was higher in ccRCC tissues and renal cancer cells compared to adjacent non-tumor tissues and normal human proximal tubule epithelial cells HK-2. The ccRCC patients with higher MALAT1 expression had an advanced clinical features and a shorter overall survival time than those with lower MALAT1 expression. And multivariate analysis showed that the status of MALAT1 expression was an independent predictor of overall survival in ccRCC. Additionally, our data indicated that knockdown expression of MALAT1 decreased renal cancer cell proliferation, migration, and invasion. Our data suggested that lncRNA MALAT1 was a novel molecule involved in ccRCC progression, which provided a potential prognostic biomarker and therapeutic target. PMID:25480417

  2. Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

    OpenAIRE

    Chang, Ying-ying; Eichel, Johannes; Cronan, John E.

    2000-01-01

    Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation ...

  3. Identification of avian wax synthases

    Directory of Open Access Journals (Sweden)

    Biester Eva-Maria

    2012-02-01

    Full Text Available Abstract Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.

  4. Neem leaf glycoprotein is superior than cisplatin and sunitinib malate in restricting melanoma growth by normalization of tumor microenvironment.

    Science.gov (United States)

    Barik, Subhasis; Bhuniya, Avishek; Banerjee, Saptak; Das, Arnab; Sarkar, Madhurima; Paul, Tanmoy; Ghosh, Tithi; Ghosh, Sarbari; Roy, Soumyabrata; Pal, Smarajit; Bose, Anamika; Baral, Rathindranath

    2013-09-01

    We have observed earlier that therapeutic treatment with neem leaf glycoprotein (NLGP) inhibits murine B16-melanoma growth in vivo and improves survivability of treated mice. Anti-tumor effect of NLGP is directly associated with enhanced CD8(+) T cell activity and downregulation of suppressive cellular functions. Objective of this present study is to know the efficacy of NLGP in comparison to two popular drugs, Cisplatin and Sunitinib malate (Sutent) in relation to the modulation of tumor microenvironment (TME). Analysis of cytokine milieu within TME revealed IL-10, TGF?, IL-6 rich type 2 characters was significantly switched to type 1 microenvironment with dominance of IFN? and IL-2 within NLGP-TME, which was not found in other cases; however Cisplatin-TME appeared better in type 2 to type 1 conversion than Sutent-TME as evidenced by RT-PCR, ELISA and immunohistochemical analysis. NLGP-TME educated CD8(+) T cells exhibited greater cytotoxicity to B16 Melanoma cells in vitro and these cells showed comparatively higher expression of cytotoxicity related molecules, perforin and granzyme B than Cisplatin-TME and Sutent-TME educated T cells. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of melanoma in vivo. Such tumor growth inhibition was in significantly lower extent when therapeutic CD8(+) T cells were exposed to either Cisplatin-TME or Sutent-TME or control-TME. Accumulated evidences strongly suggest that non toxic NLGP normalized TME allows T cells to perform optimally than other TMEs under study to inhibit the melanoma growth. PMID:23747315

  5. The lncRNA Malat1 Is Dispensable for Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2012-07-01

    Full Text Available Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1 is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3?-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

  6. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    Aquino-Silva M. R.

    2003-01-01

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  7. Synthesis, crystal growth, structural, optical, thermal and mechanical properties of novel organic NLO material: Ammonium malate

    Science.gov (United States)

    Anandha babu, G.; Bhagavannarayana, G.; Ramasamy, P.

    2008-03-01

    Ammonium malate (AM), an organic nonlinear optical material, has been synthesized and single crystals, with dimensions up to 38×35×27 mm 3, have been grown from aqueous solution. The crystal structure of AM has been determined, and it belongs to the non-centro symmetric space group Cc. The structural perfection of the grown crystals has been analysed by high-resolution X-ray diffraction (HRXRD) rocking curve measurements. Fourier transform infrared (FTIR) spectroscopic studies were also performed for the identification of different modes present in the compound. The UV-vis transmission spectrum has been recorded in the range 200-1100 nm. The second harmonic conversion efficiency of AM was determined using the Kurtz powder technique; it was observed to be greater than that of KDP. Thermal behaviour of AM was studied by TG and DTA. The dielectric and mechanical behaviour of the specimen was also studied.

  8. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

    OpenAIRE

    Houghton, P.J.; Germain, G S; Hazelton, B J; Pennington, J W; Houghton, J A

    1989-01-01

    GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphat...

  9. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    International Nuclear Information System (INIS)

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 ?g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  10. Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

    Science.gov (United States)

    Heyes, Logan C; Reichau, Sebastian; Cross, Penelope J; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4?M. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated. PMID:25245459

  11. Energy transduction in ATP synthase.

    Science.gov (United States)

    Elston, T; Wang, H; Oster, G

    1998-01-29

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit. This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model. PMID:9461222

  12. Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and beta-adrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms.

    Science.gov (United States)

    Moule, S K; Welsh, G I; Edgell, N J; Foulstone, E J; Proud, C G; Denton, R M

    1997-03-21

    Previous studies using L6 myotubes have suggested that glycogen synthase kinase-3 (GSK-3) is phosphorylated and inactivated in response to insulin by protein kinase B (PKB, also known as Akt or RAC) (Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and Hemmings, B. A. (1995) Nature 378, 785-789). In the present study, marked increases in the activity of PKB have been shown to occur in insulin-treated rat epididymal fat cells with a time course compatible with the observed decrease in GSK-3 activity. Isoproterenol, acting primarily through beta3-adrenoreceptors, was found to decrease GSK-3 activity to a similar extent (approximately 50%) to insulin. However, unlike the effect of insulin, the inhibition of GSK by isoproterenol was not found to be sensitive to inhibition by the phosphatidylinositol 3'-kinase inhibitors, wortmannin or LY 294002. The change in GSK-3 activity brought about by isoproterenol could not be mimicked by the addition of permeant cyclic AMP analogues or forskolin to the cells, although at the concentrations used, these agents were able to stimulate lipolysis. Isoproterenol, but again not the cyclic AMP analogues, was found to increase the activity of PKB, although to a lesser extent than insulin. While wortmannin abolished the stimulation of PKB activity by insulin, it was without effect on the activation seen in response to isoproterenol. The activation of PKB by isoproterenol was not accompanied by any detectable change in the electrophoretic mobility of the protein on SDS-polyacrylamide gel electrophoresis. It would therefore appear that distinct mechanisms exist for the stimulation of PKB by insulin and isoproterenol in rat fat cells. PMID:9065430

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

    International Nuclear Information System (INIS)

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

  14. Structural classification and properties of ketoacyl synthases

    OpenAIRE

    Chen, Yingfei; Kelly, Erin E; Masluk, Ryan P; Nelson, Charles L.; Cantu, David C; Reilly, Peter J

    2011-01-01

    Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and t...

  15. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile. PMID:19454282

  16. Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines.

    Science.gov (United States)

    Park, O K; Bauerle, R

    1999-03-01

    The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines. PMID:10049398

  17. Glucocorticoid protects rodent hearts from ischemia/reperfusion injury by activating lipocalin-type prostaglandin D synthase-derived PGD2 biosynthesis.

    Science.gov (United States)

    Tokudome, Satori; Sano, Motoaki; Shinmura, Ken; Matsuhashi, Tomohiro; Morizane, Shintaro; Moriyama, Hidenori; Tamaki, Kayoko; Hayashida, Kentaro; Nakanishi, Hiroki; Yoshikawa, Noritada; Shimizu, Noriaki; Endo, Jin; Katayama, Takaharu; Murata, Mitsushige; Yuasa, Shinsuke; Kaneda, Ruri; Tomita, Kengo; Eguchi, Naomi; Urade, Yoshihiro; Asano, Koichiro; Utsunomiya, Yasunori; Suzuki, Takeshi; Taguchi, Ryo; Tanaka, Hirotoshi; Fukuda, Keiichi

    2009-06-01

    Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD2 biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. In the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types, upregulated the expression of L-PGDS together with cytosolic calcium-dependent phospholipase A2 and COX2 via the glucocorticoid receptor (GR) in rat cardiomyocytes. Accordingly, PGD2 was the most prominently induced prostaglandin in vivo in mouse hearts and in vitro in cultured rat cardiomyocytes after exposure to GR-selective agonists. In isolated Langendorff-perfused mouse hearts, dexamethasone alleviated ischemia/reperfusion injury. This cardioprotective effect was completely abrogated by either pharmacologic inhibition of COX2 or disruption of the gene encoding L-PGDS. In in vivo ischemia/reperfusion experiments, dexamethasone reduced infarct size in wild-type mice. This cardioprotective effect of dexamethasone was markedly reduced in L-PGDS-deficient mice. In cultured rat cardiomyocytes, PGD2 protected against cell death induced by anoxia/reoxygenation via the D-type prostanoid receptor and the ERK1/2-mediated pathway. Taken together, these results suggest what we believe to be a novel interaction between glucocorticoid-GR signaling and the cardiomyocyte survival pathway mediated by the arachidonic acid cascade. PMID:19451694

  18. microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus

    DEFF Research Database (Denmark)

    Leucci, Eleonora; Patella, Francesca

    2013-01-01

    microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

  19. Type 2 Diabetic Rats on Diet Supplemented With Chromium Malate Show Improved Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism

    Science.gov (United States)

    Feng, Weiwei; Zhao, Ting; Mao, Guanghua; Wang, Wei; Feng, Yun; Li, Fang; Zheng, Daheng; Wu, Huiyu; Jin, Dun; Yang, Liuqing; Wu, Xiangyang

    2015-01-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPK?1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes. PMID:25942313

  20. Type 2 diabetic rats on diet supplemented with chromium malate show improved glycometabolism, glycometabolism-related enzyme levels and lipid metabolism.

    Science.gov (United States)

    Feng, Weiwei; Zhao, Ting; Mao, Guanghua; Wang, Wei; Feng, Yun; Li, Fang; Zheng, Daheng; Wu, Huiyu; Jin, Dun; Yang, Liuqing; Wu, Xiangyang

    2015-01-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPK?1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes. PMID:25942313

  1. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... synthase deficiency tend to be resistant (refractory) to treatment with antiseizure medications. GM3 synthase deficiency profoundly disrupts brain development. Most affected children have severe intellectual disability and do not develop skills such as reaching ...

  2. Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine

    OpenAIRE

    Giunta, Carlo; Pessione, Enrica; Lamberti, Cristina; Mazzoli, Roberto; Purrotti, Micol

    2009-01-01

    Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ...

  3. Unique rotary ATP synthase and its biological diversity.

    Science.gov (United States)

    von Ballmoos, Christoph; Cook, Gregory M; Dimroth, Peter

    2008-01-01

    F1F0 ATP synthases convert energy stored in an electrochemical gradient of H+ or Na+ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore under diverse environmental conditions. The ATP synthase of each particular cell is confronted with specific challenges, imposed by the specific environment, and thus by necessity must adapt to these conditions for optimal operation. Examples of these adaptations include diverse mechanisms for regulating the ATP hydrolysis activity of the enzyme, the utilization of different coupling ions with distinct ion binding characteristics, different ion-to-ATP ratios reflected by variations in the size of the rotor c ring, the mode of ion delivery to the binding sites, and the different contributions of the electrical and chemical gradients to the driving force. PMID:18573072

  4. Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

    Science.gov (United States)

    Souza, Cleverson D

    2015-03-15

    This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP. PMID:25700780

  5. Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons : I. Molecular properties of the purified isoenzymes.

    Science.gov (United States)

    Walk, R A; Michaeli, S; Hock, B

    1977-01-01

    Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD(+) oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: ?1/2 at 40°C=3.0 min; mMDH: stable at 40°C; ?1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3-9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition. PMID:24420393

  6. [3H]Tetrahydrocerulenin, a specific reagent for radio-labelling fatty acid synthases and related enzymes

    International Nuclear Information System (INIS)

    The authors have synthesised [3H]tetrahydrocerulenin, a radiolabelled derivative of cerulenin, an antibiotic from Cephalosporium caerulens which specifically inhibits the condensing enzyme of fatty acid synthases and polyketide synthases from various sources. [3H]Tetrahydrocerulenin binds to fatty acid synthase from pig liver with the same specificity as cerulenin. They also used [3H]tetrahydrocerulenin to monitor condensing enzyme activity in cell-free extracts of the erythromycin-producing organism Streptomyces erythreus. (Auth.)

  7. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs. PMID:17109150

  8. Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats

    Scientific Electronic Library Online (English)

    Artur, Guimarães Filho; Rodrigo Maranguape Silva da, Cunha; Paulo Roberto Leitão de, Vasconcelos; Sergio Botelho, Guimarães.

    2014-06-01

    Full Text Available PURPOSE: To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. METHODS: One-hundred and eight male Wistar rats were ra [...] ndomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2-??C T method using the threshold cycle (CT) value for normalization. RESULTS: MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. CONCLUSION: Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.

  9. Pancreatoprotection of Malate-Containing Infusion Solutions in Experimental Hemorrhagic Shock

    Directory of Open Access Journals (Sweden)

    ?.Y. Yakovlev

    2013-06-01

    Full Text Available The aim of the investigation was to study in experiment the effect of Sterofundin isotonic on the development of post-hemorrhagic damage of the pancreas.Materials and Methods. The experiments were carried out on 36 male Wistar rats weighing 230–250 g. Hemorrhagic shock was stimulated by acute massive hemorrhage to the extent of 2.5 mL/100 g at rate of 2 mL/min. The blood loss an hour later was followed by the replacement of hypovolemia within 60 min by 200% of the lost volume: in control group — by Ringer’s solution, in test group — by Sterofundin isotonic. Then there was performed reinfusion in the volume of 70% of blood loss. On day 1 and 3 after hemorrhagic shock we assessed the laboratory findings and morphological changes of the pancreas.Results. There was found pancreatoprotective action of isotonic Sterofundin, a malate-containing blood substitute in infusion therapy of experimental hemorrhagic shock.

  10. High Performance Liquid Chromatographic Analysis of Almotriptan Malate in Bulk and Tablets

    Directory of Open Access Journals (Sweden)

    Chandra Bala Sekaran

    2013-02-01

    Full Text Available Purpose: A simple RP-HPLC method has been developed and validated for the determination of almotriptan malate (ATM in bulk and tablets. Methods: Chromatographic separation of ATM was achieved by using a Thermo Scientific C18 column. A Mobile phase containing a mixture of methanol, water and acetic acid (4:8:0.1 v/v was pumped at the flow rate of 1 mL/min. Detection was performed at 227 nm. According to ICH guidelines, the method was validated. Results: The calibration curve was linear in the concentration range 5–60 ?g/mL for the ATM with regression coefficient 0.9999. The method was precise with RSD <1.2%. Excellent recoveries of 99.60 - 100.80% proved the accuracy of the method. The limits of detection and quantification were found to be 0.025 and 0.075 ?g/mL, respectively. Conclusion: The method was successfully applied for the quantification of ATM in tablets with acceptable accuracy and precision.

  11. Synthesis and crystal structures of two nickel coordination polymers generated from asymmetric malate ligand

    International Nuclear Information System (INIS)

    Two nickel coordination polymers [Ni(H2O)(C4H4O5)].H2O 1 and [Ni(H2O)(mal)(phen)] 2, have been hydrothermally synthesized and structurally characterized by single crystal X-ray diffraction. Crystal data for 1: C4H8O7Ni, monoclinic Cc, a=13.156(3)A, b=7.5436(15)A, c=9.6982(19)A, ?=130.96(3)o, Z=4. Crystal data for 2: C16H14N2O6Ni, orthorhombic Pna21, a=9.6113(19)A, b=19.691(4)A, c=8.0944(16)A, Z=4. Compound 1 is constructed from [Ni(H2O)(C4H4O5)] sheets pillared through ?-carboxylate groups into a 3D framework, which exhibits a diamond-like network. Compound 2 exhibits a 3D supramolecular network. To our knowledge, compound 1 represents the first diamond-like topology in the system of metal-malate. Other characterizations by elemental analysis, IR and TG are also described. The magnetic behavior of compound 1 has been studied

  12. Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

    International Nuclear Information System (INIS)

    To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant

  13. Metabolic fingerprint of ischaemic cardioprotection: importance of the malate-aspartate shuttle

    DEFF Research Database (Denmark)

    Nielsen, Torsten Toftegård; StØttrup, Nicolaj Brejnholt

    2011-01-01

    The convergence of cardioprotective intracellular signalling pathways to modulate mitochondrial function as an end-target of cytoprotective stimuli is well described. However, our understanding of whether the complementary changes in mitochondrial energy metabolism are secondary responses or inherent mechanisms of ischaemic cardioprotection remains incomplete. In the heart, the malate-aspartate shuttle (MAS) constitutes the primary metabolic pathway for transfer of reducing equivalents from the cytosol into the mitochondria for oxidation. The flux of MAS is tightly linked to the flux of the tricarboxylic acid cycle and the electron transport chain, partly by the amino acid l-glutamate. In addition, emerging evidence suggests the MAS is an important regulator of cytosolic and mitochondrial calcium homeostasis. In the isolated rat heart, inhibition of MAS during ischaemia and early reperfusion by the aminotransferase inhibitor aminooxyacetate induces infarct limitation, improves haemodynamic responses, and modulates glucose metabolism, analogous to effects observed in classical ischaemic preconditioning. On the basis of these findings, the mechanisms through which MAS preserves mitochondrial function and cell survival are reviewed. We conclude that the available evidence is supportive of a down-regulation of mitochondrial respiration during lethal ischaemia with a gradual 'wake-up' during reperfusion as a pivotal feature of ischaemic cardioprotection. Finally, comments on modulating myocardial energy metabolism by the cardioprotective amino acids glutamate and glutamine are given.

  14. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  15. Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase

    International Nuclear Information System (INIS)

    The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in ?gt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also ia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases

  16. Essential Protein-Protein Interactions between Plasmodium falciparum Thymidylate Synthase and Dihydrofolate Reductase Domains*

    OpenAIRE

    Shallom, Shamira; Zhang, Kai; JIANG, Lei; Rathod, Pradipsinh K

    1999-01-01

    In Plasmodium falciparum, dihydrofolate reductase and thymidylate synthase activities are conferred by a single 70-kDa bifunctional polypeptide (DHFR-TS, dihydrofolate reductase-thymidylate synthase) which assembles into a functional 140-kDa homodimer. In mammals, the two enzymes are smaller distinct molecules encoded on different genes. A 27-kDa amino domain of malarial DHFR-TS is sufficient to provide DHFR activity, but the structural requirements for TS function have not been established. ...

  17. 1L-myo-inositol-1-phosphate synthase.

    Science.gov (United States)

    Majumder, A L; Johnson, M D; Henry, S A

    1997-09-01

    1L-myo-Inositol-1-phosphate synthase catalyzes the conversion of D-glucose 6-phosphate to 1L-myo-inositol-1-phosphate, the first committed step in the production of all inositol-containing compounds, including phospholipids, either directly or by salvage. The enzyme exists in a cytoplasmic form in a wide range of plants, animals, and fungi. It has also been detected in several bacteria and a chloroplast form is observed in alga and higher plants. The enzyme has been purified from a wide range of organisms and its active form is a multimer of identical subunits ranging in molecular weight from 58,000 to 67,000. The activity of the synthase is stimulated by NH4Cl and inhibited by glucitol 6-phosphate and 2-deoxyglucose 6-phosphate. Structural genes (INO1) encoding the 1L-myo-inositol-1-phosphate synthase subunit have been isolated from several eukaryotic microorganisms and higher plants. In baker's yeast, Saccharomyces cerevisiae, the transcriptional regulation of the INO1 gene has been studied in detail and its expression is sensitive to the availability of phospholipid precursors as well as growth phase. The regulation of the structural gene encoding 1L-myo-inositol-1-phosphate synthase has also been analyzed at the transcriptional level in the aquatic angiosperm, Spirodela polyrrhiza and the halophyte, Mesembryanthemum crystallinum. PMID:9370339

  18. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-02-26

    Covering: up to 2014The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions. PMID:25310997

  19. Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

    Science.gov (United States)

    Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

    2013-04-01

    Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-?-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. PMID:23550753

  20. Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger?

    OpenAIRE

    Jensen-urstad, Anne P. L.; Semenkovich, Clay F.

    2011-01-01

    Fatty acid synthase (FAS) catalyzes the de novo synthesis of fatty acids. In the liver, FAS has long been categorized as a housekeeping protein, producing fat for storage of energy when nutrients are present in excess. Most previous studies of FAS regulation have focused on the control of gene expression. However, recent findings suggest that hepatic FAS may also be involved in signaling processes that include activation of peroxisome proliferator-activated receptor ? (PPAR?). Moreover, rep...

  1. Inheritance of malate dehydrogenase in wild pepper Herança da malato desidrogenase em pimenta-silvestre

    Directory of Open Access Journals (Sweden)

    ADILSON RICKEN SCHUELTER

    1999-01-01

    Full Text Available Leaf extracts from wild pepper (Capsicum flexuosum Sendt were analysed for the presence of malate dehydrogenase (E.C. 1.1.1.37; MDH isozymes using starch gel electrophoresis. Seven phenotypes for MDH isozymes were observed in the genitors. Genetic analysis in F1 progenies revealed five loci coding for MDH. Isozyme banding patterns of hybrids indicated that MDH-3 and MDH-4 genes code for monomeric enzymes, while MDH-5 for a dimeric isoform. In MDH-2 loci, one particular F1 progeny showed a significant deviation from the expected isozyme pattern. It is possible that other genes are controlling the expression of MDH-2 in pepper. Also, there are two alleles coding for MDH-2 isozyme. On the other hand, MDH-1 was monomorphic for all genotypes used in the experiment.Extratos de folhas de pimenta silvestre (Capsicum flexuosum Sendt foram analisados para a presença do sistema isoenzimático malato desidrogenase (E.C. 1.1.1.37; MDH, usando a técnica eletroforese em gel de amido hidrolisado. Sete fenótipos de malato desidrogenase foram observados entre os genitores. As análises de segregação em progênies F1 revelaram que cinco locos gênicos estavam envolvidos na codificação de MDH. Os padrões de bandeamento dos híbridos indicaram que os genes MDH-3 e MDH-4 codificavam para enzimas monoméricas, enquanto o MDH-5, para uma isoforma dimérica. Para o loco MDH-2, detectou-se desvio significativo para proporção de segregação esperada. Outros genes podem estar controlando a expressão de MDH-2 em pimenta. Como nos outros locos MDH, detectaram-se dois alelos codificando para MDH-2. Por outro lado, o MDH-1 foi monomórfico para todos os genótipos avaliados no experimento.

  2. Inheritance of malate dehydrogenase in wild pepper / Herança da malato desidrogenase em pimenta-silvestre

    Scientific Electronic Library Online (English)

    ADILSON RICKEN, SCHUELTER; VICENTE WAGNER DIAS, CASALI; FERNANDO LUIZ, FINGER.

    Full Text Available Extratos de folhas de pimenta silvestre (Capsicum flexuosum Sendt) foram analisados para a presença do sistema isoenzimático malato desidrogenase (E.C. 1.1.1.37; MDH), usando a técnica eletroforese em gel de amido hidrolisado. Sete fenótipos de malato desidrogenase foram observados entre os genitore [...] s. As análises de segregação em progênies F1 revelaram que cinco locos gênicos estavam envolvidos na codificação de MDH. Os padrões de bandeamento dos híbridos indicaram que os genes MDH-3 e MDH-4 codificavam para enzimas monoméricas, enquanto o MDH-5, para uma isoforma dimérica. Para o loco MDH-2, detectou-se desvio significativo para proporção de segregação esperada. Outros genes podem estar controlando a expressão de MDH-2 em pimenta. Como nos outros locos MDH, detectaram-se dois alelos codificando para MDH-2. Por outro lado, o MDH-1 foi monomórfico para todos os genótipos avaliados no experimento. Abstract in english Leaf extracts from wild pepper (Capsicum flexuosum Sendt) were analysed for the presence of malate dehydrogenase (E.C. 1.1.1.37; MDH) isozymes using starch gel electrophoresis. Seven phenotypes for MDH isozymes were observed in the genitors. Genetic analysis in F1 progenies revealed five loci coding [...] for MDH. Isozyme banding patterns of hybrids indicated that MDH-3 and MDH-4 genes code for monomeric enzymes, while MDH-5 for a dimeric isoform. In MDH-2 loci, one particular F1 progeny showed a significant deviation from the expected isozyme pattern. It is possible that other genes are controlling the expression of MDH-2 in pepper. Also, there are two alleles coding for MDH-2 isozyme. On the other hand, MDH-1 was monomorphic for all genotypes used in the experiment.

  3. Integration and expression of Sorghum C(4) phosphoenolpyruvate carboxylase and chloroplastic NADP(+)-malate dehydrogenase separately or together in C(3) potato plants(1).

    Science.gov (United States)

    Beaujean, A; Issakidis-Bourguet, E; Catterou, M; Dubois, F; Sangwan, R S.; Sangwan-Norreel, B S.

    2001-05-01

    We have integrated two cDNAs expressing Sorghum photosynthetic phosphoenolpyruvate carboxylase (C(4)-PEPC) and NADP-malate dehydrogenase (cpMDH), two key enzymes involved in the primary carbon fixation pathway of NADP-malic enzyme-type C(4) plants, separately or together into a C(3) plant (potato). Analysis of the transgenic plants showed a 1.5-fold increase in PEPC and cpMDH activities compared to untransformed plants. Immunolocalization confirmed an increase at the protein level of these two enzymes in the transgenic plants and indicated that the Sorghum cpMDH was specifically addressed to the chloroplasts of potato mesophyll cells. However, integration of either or both of the cDNAs into the potato genome did not appear to significantly modify either tuber starch grain content or the rate of photosynthetic O(2) production compared to control untransformed plants. The low level of transgene expression probably explains the lack of influence on carbon metabolism and photosynthetic rates. This general observation suggests that some complex mechanism may regulate the level of production of foreign C(4) metabolism enzymes in C(3) plants. PMID:11337077

  4. Synthesis of N-(Methoxycarbonylthienylmethyl)thioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    OpenAIRE

    Threadgill, Michael D.; Goodyer, Claire L. M.; Suaifan, Ghadeer A. R. Y.

    2010-01-01

    Two isomeric N-(methoxycarbonylthienylmethyl)thioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS).

  5. SLOW DARK DEACTIVATION OF ARABIDOPSIS CHLOROPLAST ATP SYNTHASE CAUSED BY A MUTATION IN A NONPLASTIC SAC DOMAIN PROTEIN

    Science.gov (United States)

    A recessive mutant of Arabidopsis showed anomalous ATP synthase activation/deactivation characteristics as well as a distinct growth phenotype compared to the wild type. The most significant feature of this mutant is that the deactivation of ATP synthase induced by dark adaptation is very slow, indi...

  6. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    Science.gov (United States)

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. PMID:23911730

  7. Biosynthesis of P(3HB-co-3HV-co-3HHp terpolymer by Cupriavidus necator PHB-4 transformant harboring the highly active PHA synthase gene of Chromobacterium sp. USM2

    Directory of Open Access Journals (Sweden)

    Rathi, D-N.

    2013-01-01

    Full Text Available Aims: This study evaluates potentials of Cupriavidus necator PHB?4 transformant harboring the highly activepolyhydroxyalkanoate synthase gene (phaC of a locally isolated Chromobacterium sp. USM2 for its ability toincorporate 3-hydroxyheptanoate (3HHp monomer.Methodology and results: A mixture of fructose and sodium heptanoate fed to the culture gave rise to poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyheptanoate, [P(3HB-co-3HV-co-3HHp] terpolymer synthesis, withtraces of 3HHp monomers confirmed through gas chromatography (GC, proton (1H and carbon (13C NMR spectra.Conclusion, significance and impact of study: This study has revealed that the PHA synthase of Chromobacteriumsp. USM2 has a broad range of substrate specificity. The synthase is able to polymerize 3-hydroxyalkanoate monomershaving 4–7 carbon atoms.

  8. Bacterial selenocysteine synthase--structural and functional properties.

    Science.gov (United States)

    Tormay, P; Wilting, R; Lottspeich, F; Mehta, P K; Christen, P; Böck, A

    1998-06-15

    Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur. PMID:9688279

  9. QUANTITATIVE ASSAY OF ALMOTRIPTAN MALATE IN PURE DRUG AND PHARMACEUTICAL PREPARATIONS USING SIMPLE AND CONVENIENT VISIBLE SPECTROPHOTOMETRIC METHODS

    OpenAIRE

    U. VIPLOVA PRASAD; M. SYAM BAB; B. KALYANA RAMU

    2012-01-01

    Two direct, simple and sensitive visible spectrophotometric methods (M1&M2) are described for the assay of almotripan malate in pure and solid dosage forms. The method M1 involves oxidative coupling of drug with brucine in presence of sodium meta periodate and purple red colored species is formed and exhibits absorption maxima at 520nm. The method M2 is based on the formation of yellowish brown colored species by the drug with Folin reagent and exhibits absorption maxima at 450nm. Regression ...

  10. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  11. Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

    Science.gov (United States)

    Raharjo, Tri J; Chang, Wen-Te; Verberne, Marianne C; Peltenburg-Looman, Anja M G; Linthorst, Huub J M; Verpoorte, Robert

    2004-04-01

    A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product. PMID:15120113

  12. CHARACTERIZATION OF BARLEY SUCROSE PHOSPHATE SYNTHASE

    Directory of Open Access Journals (Sweden)

    Amani Abdel-Latif

    2014-08-01

    Full Text Available Sucrose phosphate synthase (SPS is one of a number of sucrose-metabolizing enzymes that regulates the sucrose synthesis pathway. SPS was assayed from green barley(HordeurnvulgareL. seedlings (GBS,from etiolated barley seedlings (DBS that were continuously grown in darkness, and barley seedlings that were grown in darkness and illuminated only for 30 minutes before returning to the dark conditions again (EBS.Except for DBS, both GBS and EBSSPS activities wereallosterically regulated by G-6-P(activator or Pi (inhibitor.Thiol reagents became sensitized to the enzyme activity, but could be restored with DTT or ?-ME. Glucose, maltose and lactose activated the enzymewhile ?-gluconolactone and mannose inhibited it. When compared to those plants which were maintained in total darkness, extractable sucrose-Psynthase activity of 30-min.illuminated seedlings increased about 4 folds by 1h .The activity remained constant for an additional two hours and then decreased to about 50% of maximal 5 h post illumination.

  13. Nitric oxide synthases and diabetic cardiomyopathy.

    Science.gov (United States)

    Khanna, Sanskriti; Singh, Gurinder Bir; Khullar, Madhu

    2014-12-01

    Cardiovascular complications associated with diabetes significantly contribute to high mortality and morbidity worldwide. The pathophysiology of diabetic cardiomyopathy (DCM), although extensively researched upon, is partially understood. Impairment in various signaling pathways including nitric oxide (NO) signaling has been implicated in the pathogenesis of diabetes induced myocardial damage. Nitric oxide synthases (NOS), the enzymes responsible for NO generation, play an important role in various physiological processes. Altered expression and activity of NOS have been implicated in cardiovascular diseases, however, the role of NOS and their regulation in the pathogenesis of DCM remain poorly understood. In the present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS. PMID:25153033

  14. Etude de la saccharose synthase dans la graine d'arabidopsis thaliana : localisation, regulations et fonctions

    OpenAIRE

    Angeles NÚÑez, Juan Gabriel

    2009-01-01

    Sucrose synthase (SUS) is a key enzyme in sucrose metabolism. This enzyme catalysis the reversible conversion of sucrose and UDP to UDP-glucose and fructose, which are both used in multiple biochemical pathways. The Arabidopsis genome contains six sucrose synthase genes. A member of this family (SUS2) is strongly and specifically expressed in Arabidopsis seeds during the maturation phase, corresponding to reserve accumulation period. Total SUS activity reached a peak during seed maturation, a...

  15. ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl.

    OpenAIRE

    Larossa, R. A.; Smulski, D. R.

    1984-01-01

    The herbicide sulfometuron methyl is a potent inhibitor of the branched-chain amino acid biosynthetic enzyme acetolactate synthase (ALS) isolated from bacteria, fungi, and plants. However, it did not prevent growth of wild-type Salmonella typhimurium LT2 or Escherichia coli K-12. These species each contain two acetolactate synthase isozymes. Growth of S. typhimurium and E. coli mutants lacking ALS I was prevented by the herbicide, suggesting that activity of the remaining ALS isoenzyme (II or...

  16. Does egg deposition by herbivorous pine sawflies affect transcription of sesquiterpene synthases in pine?

    OpenAIRE

    Köpke, Diana; Schröder, Roland; Fischer, Hanna M.; Gershenzon, Jonathan; Hilker, Monika; Schmidt, Axel

    2008-01-01

    Scots pine (Pinus sylvestris; Pinaceae, Pinales) is known to defend against egg deposition by herbivorous sawflies by changing its terpenoid volatile blend. The oviposition-induced pine odor attracts egg parasitoids that kill the sawfly eggs. Here, we investigated whether sawfly egg deposition activates genes encoding pine terpene synthases by extracting mRNA from oviposition-induced P. sylvestris. Three new sesquiterpene synthases, PsTPS 1, PsTPS 2, and PsTPS 3, were isolated that were shown...

  17. Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

    OpenAIRE

    Dunn-coleman, N. S.; Robey, E. A.; Tomsett, A. B.; Garrett, R. H.

    1981-01-01

    Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glu...

  18. Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis

    International Nuclear Information System (INIS)

    Acetohyroxyacid synthease I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. The authors confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site

  19. Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, P.M.; Eoyang, L.

    1987-06-01

    Acetohyroxyacid synthease I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo(2-/sup 14/C)pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of /sup 14/C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. The authors confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.

  20. Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

    Scientific Electronic Library Online (English)

    S.R., Martins; R.B., de Oliveira; G., Ballejo.

    2006-02-01

    Full Text Available Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the [...] rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

  1. Effects of essential oils, yeast culture and malate on rumen fermentation, blood metabolites, growth performance and nutrient digestibility of Baluchi lambs fed high-concentrate diets.

    Science.gov (United States)

    Malekkhahi, M; Tahmasbi, A M; Naserian, A A; Danesh Mesgaran, M; Kleen, J L; Parand, A A

    2015-04-01

    The experiment was conducted to evaluate the effects of dietary supplementation with a mixture of essential oils (MEO), yeast culture (YC) and malate on performance, nutrient digestion, rumen fermentation and blood metabolites of lambs fed high-concentrate growing diets. For this purpose, twenty Baluchi lambs (17.3 ± 0.5 kg body weight and 3 months old) were randomly assigned to four dietary treatments in a completely randomized design with five lambs per treatment. The treatment groups were as follows: (i) control: basal diet without any additive, (ii) basal diet plus 400 mg/day MEO (thymol, carvacrol, eugenol, limonene and cinnamaldehyde), (iii) basal diet with 4 g/day YC and (iv) basal diet plus 4 g/day malate. No differences between the dietary treatments were observed in dry matter intake, average daily gain or feed conversion ratio (p > 0.05). Compared with control and malate treatment, lambs fed MEO and YC had an improved crude protein digestibility (p  0.05) cell wall digestibility compared to the other treatments. No differences were observed between treatments with respect to nitrogen balance or ruminal pH and ammonia concentrations (p > 0.05). No differences were observed between treatments with respect to ruminal total volatile fatty acid concentration and molar proportions of acetate, butyrate and valerate. Molar proportion of propionate was higher (p < 0.05) for YC and malate compared to control and MEO. Plasma glucose concentration was higher (p < 0.05) in lambs fed YC and malate than in lambs fed the control or the MEO diet. Blood concentration of triglycerides significantly decreased when feeding the MEO and YC diets (p < 0.05). It was concluded that YC may be more useful as a feed additive for manipulation of rumen fermentation in lambs fed with high-concentrate diets than MEO and malate, because YC enhanced crude protein and cell wall digestibility, ruminal molar proportion of propionate and plasma glucose concentration. PMID:25060172

  2. Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features.

    Science.gov (United States)

    Liu, Ya-Jun; Li, Pan-Pan; Zhao, Ke-Xin; Wang, Bao-Jun; Jiang, Cheng-Ying; Drake, Harold L; Liu, Shuang-Jiang

    2008-09-01

    3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity. PMID:18621870

  3. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

    Directory of Open Access Journals (Sweden)

    Delnia Arshadi

    2007-06-01

    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  4. QUANTITATIVE ASSAY OF ALMOTRIPTAN MALATE IN PURE DRUG AND PHARMACEUTICAL PREPARATIONS USING SIMPLE AND CONVENIENT VISIBLE SPECTROPHOTOMETRIC METHODS

    Directory of Open Access Journals (Sweden)

    U. VIPLOVA PRASAD

    2012-05-01

    Full Text Available Two direct, simple and sensitive visible spectrophotometric methods (M1&M2 are described for the assay of almotripan malate in pure and solid dosage forms. The method M1 involves oxidative coupling of drug with brucine in presence of sodium meta periodate and purple red colored species is formed and exhibits absorption maxima at 520nm. The method M2 is based on the formation of yellowish brown colored species by the drug with Folin reagent and exhibits absorption maxima at 450nm. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (8.0-24 ?g/ml for method M1, (16-48 ?g/ml for method M2 respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the reported UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the almotriptan malate in the presence of other ingredients that are usually present in dosageforms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

  5. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    Science.gov (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-?-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion. PMID:25605536

  6. Salt Inactivates Endothelial Nitric Oxide Synthase in Endothelial Cells12

    OpenAIRE

    Li, Juan; White, James; GUO Ling; ZHAO, XIAOMIN; Wang, Jiafu; Smart, Eric J.; Li, Xiang-An

    2009-01-01

    There is a 1–4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (fro...

  7. Metabolism of aromatic amines by prostaglandin H synthase.

    OpenAIRE

    Boyd, J A; Eling, T E

    1985-01-01

    The metabolism of aromatic amines by the peroxidase activity of prostaglandin H synthase (PHS) has been studied in this laboratory by use of two model compounds, the carcinogenic primary amine 2-aminofluorene (2-AF) and the substituted amine aminopyrine (AP). 2-AF is oxidized by PHS to 2, 2-azobisfluorene, 2-aminodifluorenylamine, 2-nitrofluorene, polymeric material, and products covalently bound to macromolecules. In the presence of phenolic compounds, 2-AF oxidation results in the formation...

  8. Differential Modulation of Nitric Oxide Synthases in Aging: Therapeutic Opportunities

    OpenAIRE

    RitaTostes

    2012-01-01

    Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO) bioavailability and altered vascular expression and activity of NO synthase (NOS) enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS)-derived NO, while increased inducible ...

  9. The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function.

    Science.gov (United States)

    Cao, Sheng; Yao, Janet; Shah, Vijay

    2003-02-21

    The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed (35)S-eNOS reductase domain but not the (35)S-eNOS oxygenase domain. Furthermore GST-dyn-2 PRD binds a (35)S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.) PMID:12488320

  10. Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme’s active site closure

    Directory of Open Access Journals (Sweden)

    Park Jaeok

    2012-12-01

    Full Text Available Abstract Background Human farnesyl pyrophosphate synthase (FPPS controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme’s C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP. This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme’s active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation. Results We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi, inorganic pyrophosphate (PPi, and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade. Conclusions In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the “off” position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

  11. Acetolactate synthase activity in Euphorbia heterophylla resistant to ALS- and protox- inhibiting herbicides / Atividade da enzima acetolactato sintase em Euphorbia heterophylla com resistência múltipla aos herbicidas inibidores da ALS e da protox

    Scientific Electronic Library Online (English)

    E., Xavier; M.C., Oliveira; M.M., Trezzi; R.A., Vidal; F., Diesel; F.D., Pagnoncelli; E., Scalcon.

    2013-12-01

    Full Text Available O objetivo deste trabalho foi determinar a atividade da enzima ALS em biótipos de leiteiro (Euphorbia heterophylla) com resistência múltipla aos inibidores da ALS e da Protox na presença e ausência dos herbicidas imazapyr, imazethapyr e nicosulfuron. Efetuou-se ensaio in vitro da enzima acetolactato [...] sintase (ALS) extraída de plantas dos biótipos Vitorino, Bom Sucesso do Sul e Medianeira (com resistência múltipla aos inibidores da ALS e da Protox) e de um biótipo suscetível, na ausência e presença dos herbicidas imazapyr, imazethapyr e nicosulfuron. Na ausência dos herbicidas, os biótipos com resistência múltipla demonstraram maior afinidade da enzima pelo substrato piruvato em comparação ao biótipo suscetível. Os herbicidas imazapyr, imazethapyr e nicosulfuron produziram reduzido efeito sobre a atividade da enzima ALS dos biótipos resistentes e, ao contrário, elevado efeito inibitório sobre a ALS do biótipo suscetível. Os fatores de resistência foram elevados, superiores a 438, 963 e 474 para os biótipos Vitorino, Bom Sucesso do Sul e Medianeira, respectivamente. A resistência observada deve-se à insensibilidade da enzima ALS aos herbicidas tanto do grupo das imidazolinonas quanto das sulfonilureias, caracterizando resistência cruzada. Abstract in english The objective of this study was to determine the activity of the enzyme acetolactate synthase in biotypes of wild poinsettia (Euphorbia heterophylla) with multiple resistance to ALS- and Protox- inhibitors in the presence and absence of imazapyr, imazethapyr and nicosulfuron. We conducted in vitro a [...] ssay of ALS enzyme extracted from plants of Vitorino, Bom Sucesso do Sul and Medianeira biotypes (with multiple resistance) and a susceptible population in the absence and presence of imazapyr, imazethapyr and nicosulfuron. In the absence of herbicides, biotypes with multiple resistance showed higher affinity for the substrate of the enzyme compared with the susceptible population. The herbicides imazapyr, imazethapyr and nicosulfuron had little effect on the enzyme activity of ALS-resistant biotypes and, conversely, high inhibitory effect on ALS of the susceptible population. Resistance factors were very high, greater than 438, 963 and 474 for Vitorino, Bom Sucesso do Sul and Medianeira biotypes, respectively. The resistance to ALS inhibitors is due to the insensitivity of ALS to herbicides of both imidazolinone and sulfonylurea groups, characterizing a cross-resistance.

  12. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  13. Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

    Directory of Open Access Journals (Sweden)

    Ro Dae-Kyun

    2009-07-01

    Full Text Available Abstract Background Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development. Results Glandular trichomes of sunflower (Helianthus annuus L. were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon ?-cadinene as one of the two main products with ?-muuorlene, ?-caryophyllene, ?-humulene and ?-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well. Conclusion This study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.

  14. Radiolabeling of a wound-inducible pyridoxal phosphate utilizing protein from tomato: evidence for its identification as ACC synthase

    International Nuclear Information System (INIS)

    Aminocyclopropane 1-carboxylic acid (ACC) synthase, a pyridoxal phosphate utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate limiting step in the biosynthesis of the plant hormone, ethylene. Ethylene, besides being involved in normal plant growth processes, is also produced in response to stress, e.g. wounding, pathogen infection, etc. The authors report the partial purification (400 fold) of ACC synthase from wounded pink tomato pericarp by classical techniques including ammonium sulfate precipitation, ion exchange and phenyl sepharose chromatography. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme and the low levels present in plant tissue. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase enriched fraction was achieved. Evidence that this radiolabeled protein is ACC synthase will be presented. Amino acid sequence determination of putative ACC synthase-derived peptides is underway

  15. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  16. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC50 value of 10.3 ?mol L-1. (author)

  17. Changes in the Activity of Antioxidant and Glyoxylate Cycle Enzymes of Hydro-Primed Calendula officinalis (L. Seeds after Re-Drying Temperature Stress

    Directory of Open Access Journals (Sweden)

    Sedghi, Mohammad

    2013-04-01

    Full Text Available In order to study the effect of re-drying temperatures on the enzymatic activation of hydro-primed Calendula officinalis (L. seeds, a completely randomized design with three replications was conducted. Treatments were six different re-drying temperatures including control (without drying, 20, 30, 40, 50, and 60°C applied after hydro-priming. Results showed that rapid drying at high temperatures (40-60°C caused a significant difference comparing control. The best re-drying temperature considering many studied traits was 20-30°C. At 60°C, activity of antioxidant enzymes such as superoxide dismutase, catalase and peroxidase decreased 65.5, 75, and 62%, respectively over control. Re-drying had not significant effect on the activity of malate synthase and isocitrate lyase in comparison with control and 20-30°C that demonstrates no negative and reductive effect of re-drying with these temperatures on seed germination. It seems that seeds are more desiccation tolerant at these temperatures. In conclusion, we can dry hydro-primed seeds slowly at 20 and 30 °C and store them until utilization.

  18. Effects of Malate and Yeast Supplementation in Concentrate Containing High Cassava Chip on Rumen Ecology and Digestibility of Nutrients in Beef Cattle

    Directory of Open Access Journals (Sweden)

    Sittisak KHAMPA

    2009-01-01

    Full Text Available Four, 1-year old beef cattle were randomly assigned according to a 2 ´ 2 Factorial arrangement in a 4 ´ 4 Latin square design to study supplementation of malate level at 500 and 1,000 g with yeast (Saccharomyces cerevisiae at 1,000 and 2,000 g in concentrate containing high levels of cassava chip. The treatments were as follows: T1 is supplementation of malate at 500 g with yeast at 1,000 g; T2 is supplementation of malate at 500 g with yeast at 2,000 g; T3 is supplementation of malate at 1,000 g with yeast at 1,000 g; T4 is supplementation of malate at 1,000 g with yeast at 2,000 g in concentrate, respectively. The animals were offered the treatment concentrate at 1 % BW of dry matter basis and urea-treated rice straw. The results revealed that rumen fermentation and blood metabolites were similar for all treatments. However, the digestibility of nutrients were significantly different for the diets, especially digestible nutrient intake of crude protein (CP which was higher for cows fed cassava-based diets with T4 rather than T3, T2 and T1 (74.3, 72.5, 71.1 and 68.9 %, respectively. In addition, the concentration of volatile fatty acid was significantly different especially the concentration of propionic acid which was slightly higher in cattle receiving T4 than T3, T2 and T1 (23.3, 21.9, 20.9 and 18.0 %, respectively. The populations of protozoa and fungal zoospores were significantly different as affected by malate and yeast levels. In conclusion, the combined use of concentrate containing high levels of cassava chip at 70 % DM with malate at 1,000 g and yeast at 2,000 g in concentrate with urea-treated rice straw as a roughage improved rumen fermentation and digestibility of nutrients in beef cattle.

  19. Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

    OpenAIRE

    Kim, Jeewon; Kim, Sook-Hee; Choi, Haymie

    2008-01-01

    Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has anti...

  20. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  1. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    International Nuclear Information System (INIS)

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthaseC compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

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

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution. A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of S. solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization.

  3. Unidirectional growth of potassium hydrogen malate single crystals and its characterizations on optical, mechanical, dielectric, laser damage threshold studies

    Science.gov (United States)

    Boopathi, K.; Rajesh, P.; Ramasamy, P.

    2013-02-01

    Single crystals of potassium hydrogen malate (PHM) were successfully grown by Sankaranarayanan-Ramasamy (SR) method and conventional slow evaporation solution technique which have the sizes of 35 mm in length, 20 mm in diameter and 15 × 10 × 3 mm3 respectively. The grown PHM crystals have been subjected to single crystal X-ray diffractometer, UV-Vis NIR studies, dielectric measurements, Vickers microhardness analysis and Laser damage threshold. The range and percentage of optical transmission is represented by recording UV-Vis-NIR analysis. The dielectric constant and loss measurement was made as function of temperature in the range of 40-150°C. Mechanical strength and laser stability of the SR method grown crystals was higher than the conventional method grown crystal.

  4. Differential induction of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes in Arabidopsis thaliana by wounding and pathogenic attack.

    OpenAIRE

    Keith, B.; Dong, X. N.; Ausubel, F. M.; Fink, G. R.

    1991-01-01

    We have isolated cDNAs from two distinct genes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) in Arabidopsis thaliana. Predicted protein sequences from both genes, DHS1 and DHS2, and a potato DAHP synthase gene are highly related, but none shows significant sequence similarity to conserved microbial DAHP synthase proteins. Despite this structural difference, the DHS1 cDNA complements mutations in a yeast strain lacking DAHP synthase activity. DHS1 RNA level...

  5. Structures of human constitutive nitric oxide synthases.

    Science.gov (United States)

    Li, Huiying; Jamal, Joumana; Plaza, Carla; Pineda, Stephanie Hai; Chreifi, Georges; Jing, Qing; Cinelli, Maris A; Silverman, Richard B; Poulos, Thomas L

    2014-10-01

    Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03?Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73?Å resolution. PMID:25286850

  6. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.

    2014-01-01

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

  7. Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis.

    Science.gov (United States)

    Furdui, Cristina; Zhou, Lily; Woodard, Ronald W; Anderson, Karen S

    2004-10-29

    Escherichia coli phenylalanine-sensitive 3-deoxy-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) catalyzes the net aldol condensation of phosphoenolpyruvate and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate. For the first time, the presteady-state kinetic analysis of the Phe-sensitive DAHP synthase from E. coli is reported. The steady-state and presteady-state kinetic parameters of the DAHP synthase reconstituted with Mn(II), Cu(II), and Zn(II) were compared. These studies showed the following: 1) product release is rate-limiting for all of the three metal ions studied under physiologically relevant conditions; 2) concentration of the active sites of the metal-containing DAHP synthase is increasing from Mn- (30%) to Zn- (52%) and to Cu-DAHP synthase (88%); 3) rate constant for product formation is higher in Mn- (130-200 s(-1)) than Cu- (55 s(-1)) and Zn-DAHP synthase (6.8 s(-1)); and 4) steady-state rate (rate constant for product release) is higher for the Mn- (70 s(-1)) than for Cu- (5.6 s(-1)) and Zn-DAHP synthase (1.8 s(-1)). In addition, an examination of the reaction kinetics at lower pH reveals that for Cu-DAHP synthase, product release is no longer rate-limiting, whereas the Mn- and Zn-DAHP synthase show a slower rate of product formation, suggesting that the intermediate formation becomes rate-limiting in product formation. Also, a deuterium-isotope effect on the burst rate constant of product formation for Mn-DAHP synthase was observed at pH 6.0. This supports the hypothesis that the role of metal ion in E. coli DAHP synthase is to position the amino acids with the appropriate geometry required to coordinate and activate the water molecule. PMID:15326172

  8. Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

    OpenAIRE

    Greif, Daniel M.; David B Sacks; Michel, Thomas

    2004-01-01

    The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [32P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 pro...

  9. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  10. Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum.

    Science.gov (United States)

    Liu, Wenting; Feng, Xinxin; Zheng, Yingying; Huang, Chun-Hsiang; Nakano, Chiaki; Hoshino, Tsutomu; Bogue, Shannon; Ko, Tzu-Ping; Chen, Chun-Chi; Cui, Yunfeng; Li, Jian; Wang, Iren; Hsu, Shang-Te Danny; Oldfield, Eric; Guo, Rey-Ting

    2014-01-01

    We report the first X-ray crystal structure of ent-kaur-16-ene synthase from Bradyrhizobium japonicum, together with the results of a site-directed mutagenesis investigation into catalytic activity. The structure is very similar to that of the ? domains of modern plant terpene cyclases, a result that is of interest since it has been proposed that many plant terpene cyclases may have arisen from bacterial diterpene cyclases. The ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues. A bisphosphonate inhibitor binds to the same site. In the kaurene synthase from the moss Physcomitrella patens, 16-?-hydroxy-ent-kaurane as well as kaurene are produced since Leu and Tyr in the P. patens kaurene synthase active site are replaced by smaller residues enabling carbocation quenching by water. Overall, the results represent the first structure determination of a bacterial diterpene cyclase, providing insights into catalytic activity, as well as structural comparisons with diverse terpene synthases and cyclases which clearly separate the terpene cyclases from other terpene synthases having highly ?-helical structures. PMID:25269599

  11. Regulation of neuronal nitric oxide synthase by histone, protamine, and myelin basic protein.

    Science.gov (United States)

    Hu, J; Fridlund, J; el-Fakahany, E E

    1995-04-01

    We examined the effects of endogenous basic proteins rich in the amino acid L-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively. PMID:7544448

  12. Morphometric Analysis of Mice Testicular Tubules after Administration of Malathion and Maca Análisis Morfométrico de los Túbulos Testiculares del Ratón Después de la Administración de Malation y Maca

    Directory of Open Access Journals (Sweden)

    Eduardo Bustos-Obregón

    2007-06-01

    Full Text Available Organophosphoric (OP agropesticidas are amply used to increase food production. However, it has been verified that they induce alterations at testicular level related to the diminution of fertility in humans as in animals. On the other hand, different studies have been made to develop chemical or natural compounds that can induce an antagonistic effect to OP. In previous studies an extract from a plant, from the Peruvian Andes (Maca has been recognized by its stimulating action on spermatogenesis. In the present study the effects of both external agents were evaluated on testicular sections of testis of adult male mice on a population of 52 mice CF1, divided at random in 4 groups (Control, Malathion, Maca, Malathion-Maca, with sacrifice intervals of 1,7, 14 and 21 days. By means of morphometric technique and using the "Image Tools 3,1" software, the histology of testicular sections was evaluated, to analyze the degree of alteration induced by these agents. The epithelial height mainly has a rise in day 1, for Malathion group and then fall to day 7 to be normal by day 14. However, Malathion-Maca groups show no changes. The tubular lumen decreases at day 7 and day 14 to be normal by day 21 in Malathion group. However, in Malathion - Maca group, the tubular lumen decreases only at 14 days. The tubular diameter, at day 7 (pLos agropesticidas organofosforados (OF son ampliamente usados para incrementar la producción alimentaría. Sin embargo, se ha demostrado que inducen alteraciones a nivel testicular, relacionadas con la disminución de la fertilidad tanto en humanos como en animales. Por otra parte, diferentes estudios han sido llevados a cabo para desarrollar compuestos químicos o naturales que puedan inducir un efecto antagónico sobre los OF. En estudios anteriores, un extracto de una planta de los Andes peruanos (Maca ha sido reconocido por su acción estimulante sobre la espermatogénesis. En el presente trabajo se estudiaron los efectos de ambos agentes externos sobre el testículo. Una población de 52 ratones machos adultos de la cepa CF1 fue dividida al azar en 4 grupos (Control, Malation, Maca, Malation-Maca, con intervalos de sacrificio de 1, 7, 14 y 21 días. Para analizar el grado de alteración inducida por estos agentes, se utilizaron técnicas de histomorfometría con ayuda del programa Image tools 3.1, en secciones testiculares. Nuestros resultados muestran que la altura del epitelio aumentó al día 1 en el grupo tratado con malation, cayendo al día 7 y llegando a valores similares al control, al día 14. Sin embargo, el grupo Malation-Maca no mostró cambios significativos. El lumen tubular, disminuyó al día 7 y 14, para normalizarse al día 21 en el grupo Malation. Sin embargo, en el grupo Malation-Maca el lumen tubular sólo bajó al día 14. El diámetro tubular disminuyó a los días 7 (p < 0.01, 14 y 21 en el grupo Malation, con respecto al control (p< 0.05. Sin embargo, el grupo Malation-Maca mostró valores normales. En conclusión, es posible establecer que el daño inducido por Malation es revertido al día 21 post administración de maca

  13. The Molecular Motor F-ATP Synthase Is Targeted by the Tumoricidal Protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits ? (non-catalytic) and ? (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5?M. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active ?3?3? complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-?3?3? complex interaction. Whereas the ?3?3? complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

  14. New monomeric and dimeric uridinyl derivatives as inhibitors of chitin synthase.

    Science.gov (United States)

    Kral, Katarzyna; Bieg, Tadeusz; Nawrot, Urszula; W?odarczyk, Katarzyna; Lalik, Anna; Hahn, Przemys?aw; Wandzik, Ilona

    2015-08-01

    This study described the synthesis and in vitro evaluation of eight new derivatives of uridine as antifungal agents and inhibitors of chitin synthase. Dimeric uridinyl derivatives synthesized by us did not exhibit significant activity. One of the studied monomeric derivative, 5'-(N-succinyl)-5'-amino-5'-deoxyuridine methyl ester (compound 7) showed activities against several fungal strains (MIC range 0.06-1.00mg/mL) and inhibited chitin synthase from Saccharomyces cerevisiae (IC50=0.8mM). Moreover compound 7 exhibited synergistic interaction with caspofungin against Candida albicans (FIC index=0.28). PMID:26051755

  15. Differential Roles of Nitric Oxide Synthases in Regulation of Ultraviolet B Light-induced Apoptosis

    OpenAIRE

    Liu, Wei; Wu, Shiyong

    2010-01-01

    Ultraviolet B light (UVB) activates nitric oxide synthase(s) (NOS) and nitric oxide (NO•) production, which plays a role in regulation of apoptosis. However the role of NO• in UVB-induced apoptosis remains controversial. In this study, we analyzed expression and activation of constitutive NOSs (cNOSs) and their roles in UV-induced apoptosis of HaCaT keratinocytes. Our data showed that the expression of neuronal NOS (nNOS) was increased while endothelial NOS (eNOS) was uncoupled in the ear...

  16. Tapentadol and nitric oxide synthase systems.

    Science.gov (United States)

    Bujalska-Zadro?ny, Magdalena; Woli?ska, Renata; G?si?ska, Emilia; Nagraba, ?ukasz

    2015-04-01

    Tapentadol, a new analgesic drug with a dual mechanism of action (?-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10?mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10?mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2?mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia. PMID:25485639

  17. Scientific Opinion on the substantiation of a health claim related to citrulline-malate and faster recovery from muscle fatigue after exercise pursuant to Article 13(5) of Regulation (EC) No 1924/2006

    OpenAIRE

    EFSA Panel on Dietetic Products, Nutrition and Allergies

    2014-01-01

    Following an application from Biocodex, submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Belgium, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to citrulline-malate and faster recovery from muscle fatigue after exercise. The Panel considers that citrulline-malate is sufficiently characterised. The ...

  18. Elevation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in the Mouse Brain after Chronic Nonylphenol Exposure

    OpenAIRE

    Fei Li; Jing Li; Ling-Tong Chen; Bao-Ping Han; Yuan-Lin Zheng; Zhen Mao; Yan-Qiu Zhang

    2008-01-01

    The present study was performed to investigate the effects of chronic administration of nonylphenol (NP) on the expression of inflammation-related genes in the brains of mice. NP was given orally by gavages at 0, 50, 100, and 200 mg/kg/d. The expression of inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was evaluated by immunohistochemistry and immunoblotting assays. The nitric oxide (NO) level and nitric oxide synthase (NOS) activity were also measu...

  19. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

    OpenAIRE

    Scho?nbrunn, Ernst; Eschenburg, Susanne; Shuttleworth, Wendy A.; Schloss, John V.; Amrhein, Nikolaus; Evans, Jeremy N. S.; Kabsch, Wolfgang

    2001-01-01

    Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain c...

  20. Synthesis of N-(Methoxycarbonylthienylmethylthioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Michael D. Threadgill

    2010-04-01

    Full Text Available Two isomeric N-(methoxycarbonylthienylmethylthioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS.

  1. Knockdown of DAPIT (Diabetes-associated Protein in Insulin-sensitive Tissue) Results in Loss of ATP Synthase in Mitochondria

    OpenAIRE

    Ohsakaya, Shigenori; Fujikawa, Makoto; Hisabori, Toru; Yoshida, Masasuke

    2011-01-01

    It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of ?...

  2. Functional Characterization of the Fission Yeast Phosphatidylserine Synthase Gene, pps1, Reveals Novel Cellular Functions for Phosphatidylserine?

    OpenAIRE

    Matsuo, Yasuhiro; Fisher, Edward; Patton-Vogt, Jana; Marcus, Stevan

    2007-01-01

    To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1? mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that...

  3. Resistencia cruzada a piretroides en Aedes aegypti de Cuba inducido por la selección con el insecticida organofosforado malation

    Scientific Electronic Library Online (English)

    María Magdalena, Rodríguez; Juan A., Bisset; Cristina, Díaz; Lázaro A., Soca.

    2003-08-01

    Full Text Available Se sometió a presión de selección una cepa de Aedes aegypti (L.), colectada en el municipio de Santiago de Cuba, con niveles bajos de resistencia a malation (1,79 x), para evaluar la evolución de la resistencia a este organofosforado y su utilidad en el control. Después de 5 generaciones de selecció [...] n con malation no se logró incrementar la resistencia. Se obtuvo luego de 5 generaciones de selección (SAN-F5), un valor de factor de resistencia (FR50 de 2,22x). Se observó poca o ninguna resistencia cruzada a los insecticidas organofosforados fention, temefos y fenitrotion, sin embargo, se encontró una elevada resistencia cruzada al insecticida piretroide deltametrina, con un valor de 287,5X en la tercera generación de selección, se observó también resistencia cruzada a otros piretroides (lambdacialotrina, cipermetrina y ciflutrina). El mecanismo de esterasas elevadas no generó resistencia a los piretroides, hecho que fue corroborado a través de estudios de inhibición en gel y a través del cálculo de la frecuencia en placas de microtitulación. No obstante, hubo un incremento en la frecuencia del mecanismo de glutation-s-transferasa desde 0,049 en SANTIAGO DE CUBA hasta 0,42 en SAN-F5, lo cual puede estar asociado con la resistencia a piretroides. La resistencia cruzada a piretroides, principalmente a deltametrina, producto de la selección con malation, pudiera limitar el uso de estos insecticidas para el control, si no se lleva a cabo una buena estrategia. Abstract in english A strain from Aedes aegypti (L) collected in the municipality of Santiago de Cuba with low levels of resistance to malathion (1.79 x) was subjected to selection pressure aimed at evaluating the evolution of the resistance to this organophosphate and its usefulness for control. After 5 generations of [...] selection (SAN-F5) with malathion, it was not possible to increase the resistance and it was obtained a value of resistance factor (FR50) of 2.22x None or little cross resistance to the following organophosphate insecticides was observed: fenthion, temephos and fenitrotion; however, it was found an elevated cross resistance to deltamethrin pyrethroid, with a value of 287.5x in the third generation of selection. It was also observed cross resistance to other pyrethroids (lambda-cyhalothrin, cypermethrin and ciflutrine). The mechansim of elevated esterases did not generate resistance to pyrethroids, which was corroborated through inhibition gel studies and through the calculation of the frequency in microtitering plaques. Nevertheless, there was an increase in the frequency of the glutathione-s-transferase mechanism from 0.049 in Santiago de Cuba to 0.42 in SAN-F5, which may be associated with the resistance to pyrethroids. The cross resistance to pyrethroids, mainly to deltamethrin, resulting from the selection with malathion may limit the use of these insecticides in the control unless a good strategy for their use is laid down.

  4. Resistencia cruzada a piretroides en Aedes aegypti de Cuba inducido por la selección con el insecticida organofosforado malation

    Directory of Open Access Journals (Sweden)

    María Magdalena Rodríguez

    2003-08-01

    Full Text Available Se sometió a presión de selección una cepa de Aedes aegypti (L., colectada en el municipio de Santiago de Cuba, con niveles bajos de resistencia a malation (1,79 x, para evaluar la evolución de la resistencia a este organofosforado y su utilidad en el control. Después de 5 generaciones de selección con malation no se logró incrementar la resistencia. Se obtuvo luego de 5 generaciones de selección (SAN-F5, un valor de factor de resistencia (FR50 de 2,22x. Se observó poca o ninguna resistencia cruzada a los insecticidas organofosforados fention, temefos y fenitrotion, sin embargo, se encontró una elevada resistencia cruzada al insecticida piretroide deltametrina, con un valor de 287,5X en la tercera generación de selección, se observó también resistencia cruzada a otros piretroides (lambdacialotrina, cipermetrina y ciflutrina. El mecanismo de esterasas elevadas no generó resistencia a los piretroides, hecho que fue corroborado a través de estudios de inhibición en gel y a través del cálculo de la frecuencia en placas de microtitulación. No obstante, hubo un incremento en la frecuencia del mecanismo de glutation-s-transferasa desde 0,049 en SANTIAGO DE CUBA hasta 0,42 en SAN-F5, lo cual puede estar asociado con la resistencia a piretroides. La resistencia cruzada a piretroides, principalmente a deltametrina, producto de la selección con malation, pudiera limitar el uso de estos insecticidas para el control, si no se lleva a cabo una buena estrategia.A strain from Aedes aegypti (L collected in the municipality of Santiago de Cuba with low levels of resistance to malathion (1.79 x was subjected to selection pressure aimed at evaluating the evolution of the resistance to this organophosphate and its usefulness for control. After 5 generations of selection (SAN-F5 with malathion, it was not possible to increase the resistance and it was obtained a value of resistance factor (FR50 of 2.22x None or little cross resistance to the following organophosphate insecticides was observed: fenthion, temephos and fenitrotion; however, it was found an elevated cross resistance to deltamethrin pyrethroid, with a value of 287.5x in the third generation of selection. It was also observed cross resistance to other pyrethroids (lambda-cyhalothrin, cypermethrin and ciflutrine. The mechansim of elevated esterases did not generate resistance to pyrethroids, which was corroborated through inhibition gel studies and through the calculation of the frequency in microtitering plaques. Nevertheless, there was an increase in the frequency of the glutathione-s-transferase mechanism from 0.049 in Santiago de Cuba to 0.42 in SAN-F5, which may be associated with the resistance to pyrethroids. The cross resistance to pyrethroids, mainly to deltamethrin, resulting from the selection with malathion may limit the use of these insecticides in the control unless a good strategy for their use is laid down.

  5. Fructose-1,6-bisphosphatase, Malate Dehydrogenase, Isocitrate Lyase, Phosphoenolpyruvate Carboxykinase, Glyceraldehyde-3-phosphate Dehydrogenase, and Cyclophilin A are secreted in Saccharomyces cerevisiae grown in low glucose

    OpenAIRE

    Bennett J. Giardina; Chiang, Hui-Ling

    2013-01-01

    Our previous studies demonstrated that the key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted when Saccharomyces cerevisiae are starved of glucose for a prolonged period of time. In this study, we showed that malate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin A are also secreted in glucose-starved cells. Thus, both gluconeogenic and non-gluconeogenic enzymes are secreted via the non-classical pathway.

  6. Terpene synthases are widely distributed in bacteria

    Science.gov (United States)

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  7. Terpene synthases are widely distributed in bacteria.

    Science.gov (United States)

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-Ya, Kazuo; Omura, Satoshi; Cane, David E; Ikeda, Haruo

    2015-01-20

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  8. Calmodulin is a subunit of nitric oxide synthase from macrophages

    OpenAIRE

    1992-01-01

    A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated ...

  9. Dihydrodipicolinate synthase in opaque and floury maize mutants

    OpenAIRE

    Varisi, V. A.; Medici, L. O.; Meer, I. M.; Lea, P. J.; Azevedo, J. L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC), S-adenosylmethionine (SAM) and calcium. The results indicated that DHDPS was strongly inhibited by lysine, and that there was little variation between the mutants, indicating that lysine accumula...

  10. CTP Limitation Increases Expression of CTP Synthase in Lactococcus lactis

    OpenAIRE

    Jørgensen, Casper Møller; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which ?-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced l...

  11. Chemokine (C-C Motif) Ligand 5 is Involved in Tumor-Associated Dendritic Cell-Mediated Colon Cancer Progression Through Non-Coding RNA MALAT-1.

    Science.gov (United States)

    Kan, Jung-Yu; Wu, Deng-Chyang; Yu, Fang-Jung; Wu, Cheng-Ying; Ho, Ya-Wen; Chiu, Yen-Jung; Jian, Shu-Fang; Hung, Jen-Yu; Wang, Jaw-Yuan; Kuo, Po-Lin

    2015-08-01

    Tumor micro-environment is a critical factor in the development of cancer. The aim of this study was to investigate the inflammatory cytokines secreted by tumor-associated dendritic cells (TADCs) that contribute to enhanced migration, invasion, and epithelial-to-mesenchymal transition (EMT) in colon cancer. The administration of recombinant human chemokine (C-C motif) ligand 5 (CCL5), which is largely expressed by colon cancer surrounding TADCs, mimicked the stimulation of TADC-conditioned medium on migration, invasion, and EMT in colon cancer cells. Blocking CCL5 by neutralizing antibodies or siRNA transfection diminished the promotion of cancer progression by TADCs. Tumor-infiltrating CD11c(+) DCs in human colon cancer specimens were shown to produce CCL5. The stimulation of colon cancer progression by TADC-derived CCL5 was associated with the up-regulation of non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), which subsequently increased the expression of Snail. Blocking MALAT-1 significantly decreased the TADC-conditioned medium and CCL5-mediated migration and invasion by decreasing the enhancement of Snail, suggesting that the MALAT-1/Snail pathway plays a critical role in TADC-mediated cancer progression. In conclusion, the inhibition of CCL5 or CCL5-related signaling may be an attractive therapeutic target in colon cancer patients. J. Cell. Physiol. 230: 1883-1894, 2015. © 2014 Wiley Periodicals, Inc. PMID:25546229

  12. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    DEFF Research Database (Denmark)

    Grubmeyer, Charles; Hansen, Michael Riis

    2012-01-01

    Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 Å resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99?109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg2+ in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.

  13. Studies on the Expression of Sesquiterpene Synthases Using Promoter-?-Glucuronidase Fusions in Transgenic Artemisia annua L

    Science.gov (United States)

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

    2013-01-01

    In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for ?-caryophyllene (CPS), epi-cedrol (ECS) and ?-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

  14. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase : a bent dimer defining the adenine specificity of the substrate ATP

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila

    2015-01-01

    The enzyme 5-phosphoribosyl-1-?-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unitconsists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  15. Molecular cloning of prostacyclin (PGI2) synthase

    International Nuclear Information System (INIS)

    PGI2 synthase is a hemoprotein which may be a cytochrome P450. To test this possibility, they have begun molecular cloning of PGI2 synthase. A cDNA library has been constructed in bacteriophage lambda-gt 10 using poly(A+) RNA prepared from cultured bovine endothelial cells. They are currently screening this library with synthetic 32P-labeled oligonucleotide probes. Synthesis of these probes is based on amino acid sequence data obtained with the holoenzyme purified by immunoaffinity chromatography and with tryptic peptides isolated by HPLC. The N-terminal sequence of bovine aortic PGI2 synthase is MSWAVVFGLLAALLLLLLLTRRRRRMPGERL. This N-terminal sequence shows significant (29% and 26%) homology with rabbit and rat phenobarbital(PB)-inducible P450s, respectively, but no significant sequence homologies (2 synthase and PB-inducible P450s differ in their amino acid compositions, particularly in their contents of tryptophan, cysteine and isoleucine. The sequences of three tryptic peptides have been determined. One pentapeptide contains one of the three cysteine residues present in PGI2 synthase; this peptide shows no homology with highly conserved cysteine peptides from cytochrome P-450s. Two other peptides (a penta- and a decapeptide) also show no homology with other P450s

  16. The Fused Anthranilate Synthase from Streptomyces venezuelae Functions as a Monomer

    Science.gov (United States)

    Ashenafi, Meseret; Reddy, Prasad T.; Parsons, James F.; Byrnes, W. Malcolm

    2015-01-01

    Recently we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 ?M and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant kcat of 0.57 ± 0.02 s?1 at 30°C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but noncooperatively inhibited by tryptophan (Ki = 11.1 ± 0.1 ?M) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme’s minimal functional unit as a single TrpE-TrpG pair. PMID:25355158

  17. Nitric oxide synthase inhibitors for the treatment of chronic tension-type headache.

    Science.gov (United States)

    Ashina, Messoud

    2002-04-01

    Chronic tension-type headache may be caused by prolonged painful input from pericranial myofacial tissues, for example tender points, resulting in central sensitisation (increased excitability of neurons in the central nervous system). Animal studies have shown that sensitisation of pain pathways may be caused by or associated with the activation of neuronal nitric oxide synthase and the generation of nitric oxide. Furthermore, it has been shown that nitric oxide synthase inhibitors reduce central sensitisation in animal models of persistent pain. On the basis of this information, the analgesic effect of the nitric oxide synthase inhibitor L-N(G) methyl arginine hydrochloride was investigated. This drug significantly reduced headache and myofacial factors in patients with chronic tension-type headache. These studies show that nitric oxide plays a crucial role in the pathophysiology of tension-type headache. The analgesic effect of nitric oxide synthase inhibition in patients with chronic tension-type headache is probably due to a reduction in central sensitisation at the level of the spinal dorsal horn, trigeminal nucleus or both. Furthermore, inhibition of nitric oxide synthase may become a novel principle in the future treatment of chronic headache. PMID:11934342

  18. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  19. Enhancement of vascular targeting by inhibitors of nitric oxide synthase

    International Nuclear Information System (INIS)

    Purpose: This study investigates the enhancement of the vascular targeting activity of the tubulin-binding agent combretastatin A4 phosphate (CA4P) by various inhibitors of nitric oxide synthases. Methods and Materials: The syngeneic tumors CaNT and SaS growing in CBA mice were used for this study. Reduction in perfused vascular volume was measured by injection of Hoechst 33342 24 h after drug administration. Necrosis (hematoxylin and eosin stain) was assessed also at 24 h after treatment. Combretastatin A4 phosphate was synthesized by a modification of the published procedure and the nitric oxide synthase inhibitors L-NNA, L-NMMA, L-NIO, L-NIL, S-MTC, S-EIT, AMP, AMT, and L-TC, obtained from commercial sources. Results: A statistically significant augmentation of the reduction in perfused vascular volume by CA4P in the CaNT tumor was observed with L-NNA, AMP, and AMT. An increase in CA4P-induced necrosis in the same tumor achieved significance with L-NNA, L-NMMA, L-NIL, and AMT. CA4P induced little necrosis in the SaS tumor, but combination with the inhibitors L-NNA, L-NMMA, L-NIO, S-EIT, and L-TC was effective. Conclusions: Augmentation of CA4P activity by nitric oxide synthase inhibitors of different structural classes supports a nitric oxide-related mechanism for this effect. L-NNA was the most effective inhibitor studied

  20. Adult Ceramide Synthase 2 (CERS2)-deficient Mice Exhibit Myelin Sheath Defects, Cerebellar Degeneration, and Hepatocarcinomas*

    OpenAIRE

    Imgrund, Silke; Hartmann, Dieter; Farwanah, Hany; Eckhardt, Matthias; Sandhoff, Roger; Degen, Joachim; Gieselmann, Volkmar; Sandhoff, Konrad; Willecke, Klaus

    2009-01-01

    (Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22–C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulti...

  1. Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase

    OpenAIRE

    Singh, Salam Pradeep; Konwar, Bolin Kumar

    2012-01-01

    Nitric oxide synthases (NOS) catalyze to produce nitric oxide (NO) from L-arginine. The isoform of NOS i.e. inducible nitric oxide synthases (iNOS) expression is observed in various human malignant tumors such as breast, lung, prostate and bladder, colorectal cancer, and malignant melanoma. Also an increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, stroma of breast cancer and tumor cells of head and neck cancer. Because of its impo...

  2. Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.).

    Science.gov (United States)

    Kirby, R R

    2000-03-01

    The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences. PMID:10713448

  3. Iron-sulfur cluster dynamics in biotin synthase: a new [2Fe-2S](1+) cluster.

    Science.gov (United States)

    Lotierzo, Manuela; Bui, Bernadette Tse Sum; Leech, Helen K; Warren, Martin J; Marquet, Andrée; Rigby, Stephen E J

    2009-04-17

    Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S](2+) cluster per monomer. However, active BioB contains in addition a [4Fe-4S](2+) cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S](1+) cluster on reduction with dithionite and report the observation of a new [2Fe-2S](1+) cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion. PMID:19245793

  4. Production of squalene by squalene synthases and their truncated mutants in Escherichia coli.

    Science.gov (United States)

    Katabami, Akinori; Li, Ling; Iwasaki, Miki; Furubayashi, Maiko; Saito, Kyoichi; Umeno, Daisuke

    2015-02-01

    Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E. coli accumulated squalene up to 230 mg/L or 55 mg/g-DCW in flask culture. We also determined that a significant truncation of squalene synthase at the C-terminus retains partial cellular activity. The squalene-producing strain described herein represents a convenient platform for gene discovery and the construction of the pathway toward natural and non-natural hopanoids/steroids. PMID:25282635

  5. Na+ transport by the A1AO-ATP synthase purified from Thermococcus onnurineus and reconstituted into liposomes.

    Science.gov (United States)

    Mayer, Florian; Lim, Jae Kyu; Langer, Julian D; Kang, Sung Gyun; Müller, Volker

    2015-03-13

    The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na(+) as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na(+) transport. This is the first proof of Na(+) transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na(+) in the bioenergetics of archaea. PMID:25593316

  6. Analysis of sucrose synthas