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Sample records for malate synthase activity

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

    Directory of Open Access Journals (Sweden)

    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. Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase

    International Nuclear Information System (INIS)

    The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and postirradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established. (author). 20 refs.; 2 figs.; 1 tab

  3. Cloning and sequencing of the malate synthase gene from Hansenula polymorpha.

    Science.gov (United States)

    Bruinenberg, P G; Blaauw, M; Kazemier, B; Ab, G

    1990-01-01

    We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hansenula polymorpha. The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme. The nucleotide sequence of a 2.4-kilobase stretch of DNA covering the MAS gene was determined. The gene contains an open reading frame of 555 amino acids, amounting to a calculated molecular mass of 63,254 for the encoded protein. Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L. and Cucumis sativus L. clearly establishes the homology of all four proteins. Compared to the soluble enzyme from E. coli, the malate synthases from H. polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension. In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus. The H. polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals. PMID:2349836

  4. Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization

    International Nuclear Information System (INIS)

    Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement. Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-21

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

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

    OpenAIRE

    Afsar Shaikh

    2012-01-01

    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 exp...

  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 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. Closing Plant Stomata Requires a Homolog of an Aluminum-Activated Malate Transporter

    OpenAIRE

    Sasaki, Takayuki; Mori, Izumi C.; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

    2010-01-01

    Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by AB...

  11. Closing plant stomata requires a homolog of an aluminum-activated malate transporter.

    Science.gov (United States)

    Sasaki, Takayuki; Mori, Izumi C; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

    2010-03-01

    Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure. PMID:20154005

  12. Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

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    Singh, R; Chénier, D; Bériault, R; Mailloux, R; Hamel, R D; Appanna, V D

    2005-09-30

    We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems. PMID:16154636

  13. Extramitochondrial citrate synthase activity in bakers' yeast.

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    Rickey, T M; Lewin, A S

    1986-01-01

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

  14. 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 (...

  15. Fluorescent Assays for Ceramide Synthase Activity.

    Science.gov (United States)

    Couttas, Timothy A; Don, Anthony S

    2016-01-01

    Ceramides are the central lipid metabolite of the sphingolipid family, and exert a potent influence over cell polarity, differentiation, and survival through their biophysical properties and their specific interactions with cell signaling proteins. Literature on the importance of ceramides in physiology and pathological conditions continues to grow, with ceramides having been identified as central effectors in major human pathologies such as diabetes and neurodegenerative conditions. In mammals, ceramide synthesis from a sphingoid base and a variable length fatty acid is catalyzed by a family of six ceramide synthases (CERS1-6), whose active sites exhibit differential specificity for different length fatty acids. CERS activity has traditionally been measured using radioactive substrates. More recently mass spectrometry has been used. In this chapter, we describe a fluorescent CERS assay, the results of which can be quantified using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Methods for quantification with either TLC or HPLC are described. PMID:26552672

  16. Small-angle X-ray scattering studies on the X-ray induced aggregation of ribonnuclease, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and serum albumin. A comparison with malate synthase

    International Nuclear Information System (INIS)

    The X-ray induced aggregation of ribonuclease, lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and serum albumin in aqueous solution was monitored in situ by means of small-angle X-ray scattering. Measurements carried out with ribonuclease, LDH and serum albumin in the absence of dithiothreitol (DTT) and with GAPDH in the presence of 0.2mM DTT established the following series for the rates of aggregation of the proteins under these conditions: ribonuclease >LDH> >GAPDH> serum albumin. Within six hours from the beginning of irradiation (i.e. about the time required for the exposure of one complete scattering curve under the conditions of our experiments) the following increases of R tilde resulted: ribonuclease 9%, LDH 7%, GAPDH 4%, serum albumin <1%. Changes of R tilde exceeding 1% are, of course, too high to be tolerated in conventional scattering experiments. Measurements carried out with LDH and GAPDH in the presence of 2mM DTT established a strong protective effect of DTT against the X-ray induced aggregation of these enzymes. The initial increase of R tilde upon irradiation of LDH and GAPDH in the presence of 2mM DTT was found to be even lower than the increase of R tilde observed when serum albumin was irradiated in the absence of DTT. However, the observed decrease of anti x of LDH and GAPDH at the early stages of irradiation suggested the occurrence of fragmentation of the enzymes as another consequence of radiation damage. This finding is discussed in context with the results from previous scattering experiments and electrophoretic studies on malate synthase. (author)

  17. Disruption of Mitochondrial Malate-Aspartate Shuttle Activity in Mouse Blastocysts Impairs Viability and Fetal Growth1

    OpenAIRE

    Mitchell, Megan; Cashman, Kara S.; Gardner, David K; Thompson, Jeremy G.; Lane, Michelle

    2009-01-01

    The nutrient requirements and metabolic pathways used by the developing embryo transition from predominantly pyruvate during early cleavage stages to glucose at the blastocyst; however, the complexities involved in the regulation of metabolism at different developmental stages are not clear. The aims of this study were to examine the role of the malate-aspartate shuttle (MAS) in nutrient metabolism pathways in the developing mouse blastocyst and the consequences of impaired metabolism on embr...

  18. Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

    OpenAIRE

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

    2014-01-01

    In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa...

  19. Development Of New Visible Spectrophotometric Methods For Quantitative Determination Of Almotriptan Malate As An Active Pharmaceutical Ingredient In Formulations

    Directory of Open Access Journals (Sweden)

    U. Viplava Prasad

    2012-06-01

    Full Text Available Purpose: The aim of the investigation was to see the simple and sensitive visible spectrophotometric methods for the determination of the almotriptan malate in bulk and tablet dosage forms. Methods: Two simple, sensitive and cost effective visible spectrophotometric methods (M1-M2 were developed for the estimation of almotriptan malate in bulk and dosage forms. The first method (M1 is based on the formation of blue reduced product by treating drug with Folin Ciocalteu (FC reagent in the presence of sodium carbonate solution with an absorption maximum of 770nm. The second method (M2 is based on the complex formation product by drug with 1, 10-phenanthroline in the presence of Fe (III as an oxidant in phosphoric acid medium with an absorption maximum of 510nm. Results: Beer’s law obeyed in the concentration range of 4-12?g/ml and 1-5 ?g/ml for methodM1 and M2 respectively. No interference was observed from the usually existing additives in pharmaceutical formulations and the applicability of the methods was examined by analyzing AXERT tablets containing AM. Conclusion: The reported methods for its assay involve sophisticated equipment, which are very costly and pose problems of maintenance. To overcome these problems, the use of visible spectrophotometric technique is justifiable. The statistical data proved the accuracy, reproducibility and the precision of the proposed methods.

  20. The effect of ketotifen on nitric oxide synthase activity

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    Samuel N. Heyman; Karmeli, Fanny; Brezis, Mayer; Rachmilewitz, Daniel

    1997-01-01

    We studied the effect of ketotifen, a second generation H1-receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo.Ketotifen (100 μg ml−1) increased human colonic NOS activity from 3.7±0.6 to 14.5±1.3 nmol g−1 min−1 (P

  1. Modulation of nitric oxide synthase activity in macrophages

    OpenAIRE

    Jorens, P.G.; K. E. Matthys; Bult, H

    1995-01-01

    L-Arginine is converted to the highly reactive and unstable nitric oxide (NO) and L-citrulline by an enzyme named nitric oxide synthase (NOS). NO decomposes into other nitrogen oxides such as nitrite (NO2-) and nitrate (NO2-), and in the presence of superoxide anion to the potent oxidizing agent peroxynitrite (ONOO?). Activated rodent macrophages are capable of expressing an inducible form of this enzyme (iNOS) in response to appropriate stimuli, i.e., lipopolysaccharide (LPS) and...

  2. Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

    Science.gov (United States)

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gavva, S.R.; Harris, B.G.; Cook, P.F. (Texas Coll. of Osteopathic Medicine, Fort Worth (United States)); Weiss, P.M. (Univ. of Wisconsin, Madison (United States))

    1991-06-11

    A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K{sub NAD} and K{sub i metal} and decreases in V{sub max} as the metal size increases from Mg{sup 2+} to Mn{sup 2+} to Cd{sup 2+}, indicative of crowding in the site. The K{sub malate} value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K{sub malate} increase with all three metal ions compared to the native enzyme concomitant with a decrease in the {sup 13}C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The {sup 13}C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary {sup 13}C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd{sup 2+}. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd{sup 2+} as the divalent metal activator. Upon inactivation, only Cd{sup 2+} ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd{sup 2+}.

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

    DEFF Research Database (Denmark)

    Volke, V; Wegener, Gregers; Vasar, E; Rosenberg, R

    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-...

  5. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

    OpenAIRE

    Kida, Y; Esposito-Del Puente, A; Bogardus, C; Mott, D M

    1990-01-01

    Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatas...

  6. 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.

  7. Upregulated MALAT-1 contributes to bladder cancer cell migration by inducing epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Ying, Liang; Chen, Qi; Wang, Yawei; Zhou, Zhihua; Huang, Yiran; Qiu, Feng

    2012-09-01

    Recent studies reveal that long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in cancer biology, and lncRNA MALAT-1 expression is upregulated in some tumors. However, the contributions of MALAT-1 to bladder cancer metastasis remain largely unknown. In the present study we evaluated MALAT-1 expression in bladder cancer tissues by real-time PCR, and defined its biological functions. We verified that MALAT-1 levels were upregulated in bladder cancer tissues compared with adjacent normal tissues, and MALAT-1 expression was remarkably increased in primary tumors that subsequently metastasized, when compared to those primary tumors that did not metastasize. SiRNA-mediated MALAT-1 silencing impaired in vitro bladder cancer cell migration. Downregulation of MALAT-1 resulted in a decrease of the epithelial-mesenchymal transition (EMT)-associated ZEB1, ZEB2 and Slug levels, and an increase of E-cadherin levels. We further demonstrated that MALAT-1 promoted EMT by activating Wnt signaling in vitro. These data suggest an important role for MALAT-1 in regulating metastasis of bladder cancer and the potential application of MALAT-1 in bladder cancer therapy. PMID:22722759

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

    International Nuclear Information System (INIS)

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

  9. Nitric oxide synthase activity in Fasciola hepatica: a radiometric study.

    Science.gov (United States)

    Terenina, N B; Onufriev, M V; Gulyaeva, N V; Moiseeva, Y V; Gustafsson, M K S

    2003-06-01

    The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(omega)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica. PMID:12866797

  10. 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.

  11. In vitro stabilization and minimum active component of polygalacturonic acid synthase involved in pectin biosynthesis.

    Science.gov (United States)

    Ohashi, Takao; Ishimizu, Takeshi; Akita, Kazumasa; Hase, Sumihiro

    2007-09-01

    Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity. PMID:17827695

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

    DEFF Research Database (Denmark)

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva; Martinussen, Jan

    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 formi...

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

    DEFF Research Database (Denmark)

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva Helena; Martinussen, Jan

    2005-01-01

    GTP is an allosteric activator of CTP synthase and acts to increase the kcat 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...

  14. Malate Synthesis and Secretion Mediated by a Manganese-Enhanced Malate Dehydrogenase Confers Superior Manganese Tolerance in Stylosanthes guianensis1

    Science.gov (United States)

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

    2015-01-01

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

  15. Malate synthesis and secretion mediated by a manganese-enhanced malate dehydrogenase confers superior manganese tolerance in Stylosanthes guianensis.

    Science.gov (United States)

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

    2015-01-01

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

  16. Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase. Two amino acids before the first aspartate-rich motif solely determine eukaryotic farnesyl diphosphate synthase activity.

    Science.gov (United States)

    Ohnuma, S i; Hirooka, K; Ohto, C; Nishino, T

    1997-02-21

    Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways. PMID:9030588

  17. Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

    Science.gov (United States)

    Taillefer, M; Rydzak, T; Levin, D B; Oresnik, I J; Sparling, R

    2015-04-01

    Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme. PMID:25616802

  18. 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.

  19. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

    OpenAIRE

    RACHMILEWITZ, D; Stamler, J S; Bachwich, D; Karmeli, F; Ackerman, Z; Podolsky, D. K.

    1995-01-01

    Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in ...

  20. The Domain Responsible for Sphingomyelin Synthase (SMS) Activity

    OpenAIRE

    Yeang, Calvin; Varsheny, Shweta; Wang, Renxiao; Zhang, Ya; Ye, Deyong; Jiang, Xian-cheng

    2008-01-01

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this s...

  1. 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.

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

    OpenAIRE

    Cruz, M T; 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...

  3. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

    Science.gov (United States)

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-11-15

    Glycosylation with O-linked ?-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit ? (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as A?-treated cells. Indeed, A? bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the A?-induced impairment in ATP production and ATPase activity. These results indicate that A?-mediated reduction of ATP synthase activity in AD pathology results from direct binding between A? and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    OpenAIRE

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20??M) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn ...

  9. 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

  10. Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

    OpenAIRE

    García-Pascual, A.; COSTA, G; Labadia, A.; Persson, K.; Triguero, D

    1996-01-01

    1. To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2. NOS enzymatic activity was...

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

    OpenAIRE

    Hafke, JB; Hafke, Y; Smith, JA; Lüttge, 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-...

  12. Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Lim, Xin Ying; Pickford, Russell; Don, Anthony S

    2016-01-01

    Sphingolipids are one the major lipid families in eukaryotes, incorporating a diverse array of structural and signaling lipids such as sphingomyelin and gangliosides. The core lipid component for all complex sphingolipids is ceramide, a diacyl lipid consisting of a variable length fatty acid linked through an amide bond to a long chain base such as sphingosine or dihydrosphingosine. This reaction is catalyzed by a family of six ceramide synthases (CERS1-6), each of which preferentially catalyzes the synthesis of ceramides with different fatty acid chain lengths. Ceramides are themselves potent cellular and physiological signaling molecules heavily implicated in diabetes and neurodegenerative diseases, making it important for researchers to have access to sensitive and accurate assays for ceramide synthase activity. This chapter describes methods for assaying ceramide synthase activity in cell or tissue lysates, or in cultured cells (in situ), using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the readout. LC-MS/MS is a very sensitive and accurate means for assaying ceramide synthase reaction products. PMID:26552671

  13. 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...

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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 propo...

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

    Directory of Open Access Journals (Sweden)

    Fonovich de Schroeder T.M.

    1998-01-01

    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.

  17. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Vedula,L.; Cane, D.; Christianson, D.

    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 three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  19. 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...

  20. Thymidylate synthase expression and activity: relation to S-phase parameters and 5-fluorouracil sensitivity.

    OpenAIRE

    Mirjolet, J. F.; Barberi-Heyob, M.; Merlin, J L; Marchal, S; Etienne, M. C.; Milano, G.; Bey, P

    1998-01-01

    Six human cancer cell lines exhibiting a large range of sensitivity to 5-fluorouracil (5-FU) were evaluated for thymidylate synthase (TS) and p53 gene expression, TS and dihydropyrimidine dehydrogenase (DPD) activity, as well as cell cycle parameters, S-phase fraction (SPF), bromodeoxyuridine labelling index (LI) and S-phase duration (SPD). All these parameters were investigated for 7 days in asynchronously growing cell populations and compared with the cell sensitivity to 5-FU. No significan...

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

    OpenAIRE

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

    2003-01-01

    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. N...

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

    OpenAIRE

    1995-01-01

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

  3. Ca(2+)-independent nitric oxide synthase activity in human lung after cardiopulmonary bypass.

    OpenAIRE

    Delgado, R; A. Rojas; Glaría, L. A.; Torres, M.; Duarte, F.; Shill, R.; Nafeh, M.; E. Santin; González, N.; Palacios, M

    1995-01-01

    BACKGROUND--Because surgery involving cardiopulmonary bypass induces a systemic inflammatory response, the effect of cardiopulmonary bypass on nitric oxide (NO) generation was investigated in human lung tissue. METHODS--Nitric oxide synthase (NOS) activity was measured by the conversion of 14C-L-arginine to 14C-L-citrulline in tissue biopsy samples obtained before and after cardiopulmonary bypass. RESULTS--The Ca(2+)-independent production of NO found before cardiopulmonary bypass was extreme...

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

    OpenAIRE

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

    1998-01-01

    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 pancre...

  5. 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...

  6. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte; Ben-David, Oshrit; Klitten, Laura L; Larsen, Jan; Sabers, Anne; Vissing, John; Nielsen, Jørgen E; Hasholt, Lis; Klein, Andres D; Tsoory, Michael M; Hjalgrim, Helle; Tommerup, Niels; Futerman, Anthony H; Møller, Rikke S; Færgeman, Nils J

    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 s...

  7. 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.

  8. 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.

  9. Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2

    OpenAIRE

    Bhubalan, Kesaven; Chuah, Jo-Ann; Shozui, Fumi; Brigham, Christopher J.; Taguchi, Seiichi; Sinskey, Anthony J; Rha, ChoKyun; Sudesh, Kumar

    2010-01-01

    The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC[subscript Cs]). PhaC[subscript Cs] showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV)...

  10. 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 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)

  11. 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).

  12. Induction of long noncoding RNA MALAT1 in hypoxic mice

    Directory of Open Access Journals (Sweden)

    Lelli A

    2015-10-01

    Full Text Available Aurelia Lelli,1,2,* Karen A Nolan,1,2,* Sara Santambrogio,1,2 Ana Filipa Gonçalves,1,2 Miriam J Schönenberger,1,2 Anna Guinot,1,2 Ian J Frew,1,2 Hugo H Marti,3 David Hoogewijs,1,2,4 Roland H Wenger1,2 1Institute of Physiology and Zurich Center for Human Physiology (ZIHP, University of Zurich, Zurich, Switzerland; 2National Center of Competence in Research "Kidney.CH", Zurich, Switzerland; 3Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany; 4Institute of Physiology, University of Duisburg-Essen, Essen, Germany *These authors contributed equally to this work Abstract: Long thought to be “junk DNA”, in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA. Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1 appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function. Keywords: hypoxia-inducible factor, kidney, oxygen, proximal tubule, testis

  13. 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 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

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

    OpenAIRE

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

    1992-01-01

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

  16. Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates

    OpenAIRE

    GHIGO, Dario Antonio; BOSIA, Amalia; COSTAMAGNA, Costanzo; GAZZANO, Elena; Riganti, Chiara

    2006-01-01

    The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantifies the conversion of L-[3H]arginine to L-[3H]citrulline. We have developed a spectrophotometric procedure which continuously recycles NADPH through the addition of glucose 6-phosphate dehydrogenase to the cell lysate. This allows nitric oxide synthase to operate linearly for hours, so that nitric oxide-derived nitrite accumulates at amounts sufficient to be detected with th...

  17. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    Science.gov (United States)

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20??M) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses. PMID:22645501

  18. Zinc affects differently growth, photosynthesis, antioxidant enzyme activities and phytochelatin synthase expression of four marine diatoms.

    Science.gov (United States)

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20 ?M) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses. PMID:22645501

  19. Structure-Based Inhibitors Exhibit Differential Activities against Helicobacter pylori and Escherichia coli Undecaprenyl Pyrophosphate Synthases

    Directory of Open Access Journals (Sweden)

    Po-Huang Liang

    2008-03-01

    Full Text Available Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS, which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

  20. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-12

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

  2. 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%.

  3. PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

    OpenAIRE

    Pfeiffer, Daniel; Jendrossek, Dieter

    2014-01-01

    Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag...

  4. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    International Nuclear Information System (INIS)

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel ?-sheet flanked by four ?-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ?), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly

  5. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Soriano, Erika V.; Zhang, Yang; Colabroy, Keri L.; Sanders, Jennie M.; Settembre, Ethan C.; Dorrestein, Pieter C.; Begley, Tadhg P.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2013-09-01

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel ?-sheet flanked by four ?-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ?), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.

  6. 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.

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

    DEFF Research Database (Denmark)

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva; Martinussen, Jan

    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...... 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...

  8. Arginase Activity in Mitochondria - an Interfering Factor in Nitric Oxide Synthase Activity Assays

    OpenAIRE

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C; Miller, R. Timothy

    2009-01-01

    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 [1]. Aliquots of samples from the NOS assays were then extracted with acetone, separa...

  9. Calreticulin Transacetylase mediated activation of human platelet nitric oxide synthase by acetyl group donor compounds.

    Science.gov (United States)

    Kumar, Ajit; Sushama, Anupam; Manral, Sushma; Sinha, Rajesh; Joshi, Rini; Singh, Usha; Rohil, Vishwajeet; Prasad, Ashok K; Parmar, Virinder S; Raj, Hanumantharao G

    2012-01-01

    Polyphenols have attracted immense interest because of their diverse biological and pharmacological activities. Surprisingly, not much is documented about the biological activities of acetoxy derivatives of polyphenol called polyphenolic acetates (PA). In our previous reports, we have conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed activation of neuronal nitric oxide synthase (nNOS) and tumor necrosis factor-α (TNF-α) induced nitric oxide synthase (iNOS) by PA. In the present work, specificity of CRTAase to various classes of PA was characterized in human platelet. The effect of PA, on platelet NOS and intracellular cyclic guanosine monophosphate (cGMP), and adenosine diphosphate (ADP)-induced platelet aggregation were studied in an elaborated manner. Platelet CRTAase exhibited differential specificities to polyphenolic acetates upon incubation with l-arginine leading to activation of NOS. The intraplatelet generation of NO was studied by flowcytometry using DCFH-DA. The differential specificities of CRTAase to PA were found to positively correlate with increased production of NO upon incubation of PRP with PA and l-arginine. Further, the inhibitory effect of l-NAME on PA induced NO formation in platelets substantiated the CRTAase catalyzed activation of NOS. The real-time RT-PCR profile of NOS isoforms confirmed the preponderance of eNOS over iNOS in human platelets on treatment with PA. Western blot analysis also reiterated the differential pattern of acetylation of eNOS by PA. PA were also found effective in increasing the intraplatelet cGMP levels and inhibiting ADP-induced platelet aggregation. It is worth mentioning that the effects of PA were found to be in tune with the specificities of platelet CRTAase to PA as the substrates. PMID:22100620

  10. NK cell function triggered by multiple activating receptors is negatively regulated by glycogen synthase kinase-3?.

    Science.gov (United States)

    Kwon, Hyung-Joon; Kwon, Soon Jae; Lee, Heejae; Park, Hye-Ran; Choi, Go-Eun; Kang, Sang-Wook; Kwon, Seog Woon; Kim, Nacksung; Lee, Soo Young; Ryu, Sangryeol; Kim, Sun Chang; Kim, Hun Sik

    2015-09-01

    Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3? as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3? or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3? isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3?. Importantly, the regulation of NK cell function by GSK-3? was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3? negatively regulates NK cell activation and that modulation of GSK-3? function could be used to enhance NK cell activation. PMID:26022178

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

    DEFF Research Database (Denmark)

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

    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...

  12. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    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 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption. PMID:25900579

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

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

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

  14. 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 with...

  15. Induction of nitric oxide synthase activity by toxic shock syndrome toxin 1 in a macrophage-monocyte cell line.

    OpenAIRE

    Zembowicz, A; Vane, J R

    1992-01-01

    Toxic shock syndrome toxin 1 (TSST-1) is a Mr 22,000 protein produced by Staphylococcus aureus. It is thought to be the cause of toxic shock syndrome. We investigated the hypothesis that TSST-1 induces nitric oxide (NO) synthase and that the NO formed may be involved in the pathogenesis of toxic shock syndrome. We used the murine monocyte-macrophage cell line J744.2 that responds to TSST-1 and also expresses NO synthase activity upon immunological stimulation. J774.2 macrophages stimulated wi...

  16. Role of endothelial nitric oxide synthase in the regulation of SREBP activation by oxidized phospholipids.

    Science.gov (United States)

    Gharavi, Nima M; Baker, Nancy A; Mouillesseaux, Kevin P; Yeung, Winnie; Honda, Henry M; Hsieh, Xavier; Yeh, Michael; Smart, Eric J; Berliner, Judith A

    2006-03-31

    Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, such as interleukin (IL)-8. Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC was mediated through the activation of sterol regulatory element-binding protein (SREBP). We now present evidence for the role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment of EC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine 1177, dephosphorylation of threonine 495, and the conversion of L-arginine to L-citrulline. Activation of eNOS by Ox-PAPC was regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated that pretreatment of EC with NOS inhibitor, Nomega-nitro-L-arginine-methyl ester (L-NAME), significantly inhibited Ox-PAPC-induced IL-8 synthesis. Because SREBP activation had been previously shown to regulate IL-8 transcription by Ox-PAPC, we examined the effects of L-NAME on Ox-PAPC-induced SREBP activation. Our data demonstrated that Ox-PAPC-induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatment with L-NAME. Interestingly, treatment of EC with NO donor, S-nitroso-N-acetylpenicillamine, did not activate SREBP, suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2*-), in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2*- through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBP activation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP and SREBP-mediated inflammatory processes. PMID:16497987

  17. Substrate-bound structures of benzylsuccinate synthase reveal how toluene is activated in anaerobic hydrocarbon degradation.

    Science.gov (United States)

    Funk, Michael A; Marsh, E Neil G; Drennan, Catherine L

    2015-09-11

    Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C-C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSS?, a small protein subunit that forms a tight complex with BSS?, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these "X-succinate synthases" and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes. PMID:26224635

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

    Directory of Open Access Journals (Sweden)

    Vijay Venugopal

    2010-12-01

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

  19. 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.

  20. A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple.

    Science.gov (United States)

    Bai, Yang; Dougherty, Laura; Li, Mingjun; Fazio, Gennaro; Cheng, Lailiang; Xu, Kenong

    2012-08-01

    Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R (2) = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS(1455) that targets the SNP(1455) in Ma1 showed that the CAPS(1455A) allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for low pH or high acidity in apple. PMID:22806345

  1. Modulation of baroreceptor activity by gene transfer of nitric oxide synthase to carotid sinus adventitia.

    Science.gov (United States)

    Meyrelles, Silvana S; Sharma, Ram V; Mao, Hui Z; Abboud, Francois M; Chapleau, Mark W

    2003-05-01

    Administration of nitric oxide (NO) or NO donors to isolated carotid sinus and carotid bodies inhibits the activity of baroreceptor and chemoreceptor afferent nerves. Furthermore, NO synthase (NOS) is present in endothelial cells and in sensory nerves innervating the carotid sinus region. The major goal of this study was to determine whether overexpression of NOS in carotid sinus modulates baroreceptor activity. Rabbits were anesthetized, and adenoviral vectors (5 x 10(8) plaque-forming units) encoding genes for either beta-galactosidase (beta-Gal) or endothelial type III NOS (eNOS) were applied topically to the adventitial surface of one carotid sinus. In some experiments, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) was applied to the carotid sinus immediately after the vector. Four to five days later, baroreceptor activity and carotid sinus diameter were measured from the vascularly isolated carotid sinus of the anesthetized rabbits. Transgene expression was confirmed by X-Gal staining of beta-Gal and measurement of NOS activity by citrulline assay. The expression was restricted to the carotid sinus adventitia. Baroreceptor activity was decreased significantly, and the pressure-activity curve was shifted to higher pressures in eNOS-transduced (n = 5) compared with beta-Gal-transduced (n = 5) carotid sinuses. The pressure corresponding to 50% of maximum activity averaged 55 +/- 6 and 76 +/- 7 mmHg in beta-Gal- and eNOS-transduced carotid sinuses, respectively (P < 0.05). Decreased baroreceptor activity was accompanied by a significant increase in carotid diameter in the eNOS-transduced carotid sinuses (n = 5). l-NAME prevented the inhibition of baroreceptor activity and the increase in carotid diameter in eNOS-transduced carotid sinuses (n = 5). We conclude that adenoviral-mediated gene transfer of eNOS to carotid sinus adventitia causes sustained, NO-dependent inhibition of baroreceptor activity and resetting of the baroreceptor function curve to higher pressures. PMID:12676743

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

    Directory of Open Access Journals (Sweden)

    Rossi Adriano G

    2006-09-01

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

  4. Evolution of malate dehydrogenase in birds.

    Science.gov (United States)

    Kitto, G B; Wilson, A C

    1966-09-16

    Heart extracts from over 100 species of birds were subjected to starch-gel electrophoresis at pH 7. The "supernatant" form of malate dehydrogenase, an enzyme present in every extract, was then located on the gels by a specific staining method. The mobility of this enzyme shows very little interspecific variation. Nearly all birds tested have a supernatant malate dehydrogenase that moves as fast as the chicken enzyme. Those species with an enzyme of unusual mobility are of taxonomic interest. For example, hummingbirds and swifts, which are usually considered as two suborders of Apodiformes, are unique among the birds tested in having an enzyme that moves 63 percent as fast as the chicken enzyme. This finding appears to confirm the unity of the Apodiformes, an order whose unity has long been open to question. Similarly all families tested in the shorebird order (Charadriiformes) are unique in having an enzyme that moves 55 percent as fast as the chicken enzyme. The unity of this order was also previously open to question. PMID:5917778

  5. Valencene synthase

    OpenAIRE

    Achkar, A; Sonke, Th.; Bouwmeester, H.J.; Bosch, H.J.

    2011-01-01

    The present invention relates to a novel valencene synthase, to a nucleic acid encoding such valencene synthase, to a host cell comprising said encoding nucleic acid sequence and to a method for preparing valencene, comprising converting farnesyl diphosphate to valencene in the presence of a valencene synthase according to the invention.

  6. Identification of the first Oomycete annexin as a (1-->3)-beta-D-glucan synthase activator.

    Science.gov (United States)

    Bouzenzana, Jamel; Pelosi, Ludovic; Briolay, Anne; Briolay, Jérôme; Bulone, Vincent

    2006-10-01

    (1-->3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1-->3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1-->3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1-->3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1-->3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes. PMID:16978258

  7. 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 substrate

  8. Activation of Glycogen Synthase Kinase-3 Mediates the Olfactory Deficit-Induced Hippocampal Impairments.

    Science.gov (United States)

    Hu, Juan; Huang, He-Zhou; Wang, Xiang; Xie, Ao-Ji; Wang, Xiong; Liu, Dan; Wang, Jian-Zhi; Zhu, Ling-Qiang

    2015-12-01

    The populations with olfactory dysfunction show an increased chance for hippocampus-dependent episodic memory deficit. Although it is known that the olfactory information projects to the hippocampus through entorhinal cortex layer II, the molecular mechanisms linking olfactory deficit to the hippocampus is not understood. Using bilateral olfactory bulbectomy (OBX) as a model, we found that OBX induced memory deficits with activation of several memory-related protein kinases in the hippocampal extracts, including glycogen synthase kinase-3? (GSK-3?), protein kinase A (PKA), extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB). The OBX rats also show suppression of long-term potentiation (LTP); reduction of synapsin I, synaptophysin, NR2A/B, and PSD95; thinner presynaptic active zone and postsynaptic density with enlarged synaptic space; decreased spine numbers and mushroom-type spines; and tau hyperphosphorylation. After injection of SB216763 for several weeks by vena caudalis, selective inhibition of GSK-3? ameliorated the OBX-induced memory deficits with recovery of the synaptic components and tau phosphorylation. Furthermore, genetic ablation of GSK-3? by lentivirus-packed shRNA effectively rescued the memory deficits, synaptic disorder, and tauopathy. Our data indicate that GSK-3 activation mediates the olfactory deficits to the hippocampus, and targeting GSK-3 blocks the pathological connection. PMID:25367884

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

    Scientific Electronic Library Online (English)

    A. C., Faraldo; A, Sá-Nunes; L. H., Faccioli; E. A., Del Bel; E, Lello.

    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.

  10. Renal cortical nitric oxide synthase activity during maturational growth in the rat.

    Science.gov (United States)

    Ishii, Naohito; Fujiwara, Keiji; Lane, Pascale H; Patel, Kaushik P; Carmines, Pamela K

    2002-08-01

    The present study was designed to test the hypothesis that growth from puberty to adulthood in the rat is associated with an increase in renal cortical nitric oxide synthase (NOS) activity that results in an augmented impact of nitric oxide (NO) on hemodynamic function. Two groups of male Sprague-Dawley rats were studied: juvenile rats (approximately 2 months old) and mature rats (approximately 5 months old). NOS activity, measured as -nitro-L-arginine (NNA)-sensitive (3)H-L-citrulline production from (3)H-L-arginine, was significantly higher in the renal cortex of mature rats (57+/-2 pmol/h per mg protein) than in juveniles (42+/-3 pmol/h per mg protein). Additional animals from each group were anesthetized to determine the acute impact of NOS inhibition on arterial pressure and renal cortical blood flow, measured by single-fiber Doppler flowmetry. Cortical blood flow was higher in mature rats than in juveniles, averaging 22+/-2 and 16+/-1 perfusion units, respectively. NOS inhibition (10 mg/kg NNA i.v.) decreased renal cortical blood flow in mature rats by 35+/-7%, but only by 9+/-4% in juvenile animals. These data support the hypothesis that maturational growth in the rat is associated with augmented NOS activity coupled with an increased tonic influence of NO on renal cortical blood flow. PMID:12185463

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E; Volke, V

    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 ha...

  13. 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.

  14. 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

  15. Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

    Science.gov (United States)

    Schüll, S; Günther, S D; Brodesser, S; Seeger, J M; Tosetti, B; Wiegmann, K; Pongratz, C; Diaz, F; Witt, A; Andree, M; Brinkmann, K; Krönke, M; Wiesner, R J; Kashkar, H

    2015-01-01

    Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16?:?0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction. PMID:25766330

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

    International Nuclear Information System (INIS)

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

  17. Active-site models for complexes of quinolinate synthase with substrates and intermediates.

    Science.gov (United States)

    Soriano, Erika V; Zhang, Yang; Colabroy, Keri L; Sanders, Jennie M; Settembre, Ethan C; Dorrestein, Pieter C; Begley, Tadhg P; Ealick, Steven E

    2013-09-01

    Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8?Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel ?-sheet flanked by four ?-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe-4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005), J. Biol. Chem. 280, 26645-26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe-4S] cluster, prior to cluster assembly. PMID:23999292

  18. Chronic hyperammonemia induces tonic activation of NMDA receptors in cerebellum leading to a decrease of neuronal nitric oxide synthase activity

    Directory of Open Access Journals (Sweden)

    Hanan Ahabrach

    2010-06-01

    Full Text Available Impaired function of the glutamate-nitric oxide-cGMP pathway contributes to cognitive impairment in hyperammonemia and hepatic encephalopathy. The mechanisms by which hyperammonemia impairs this pathway remain unclear. Understanding these mechanisms would allow designing clinical treatments for cognitive deficits in hepatic encephalopathy. The aims of this work were: 1- to assess whether chronic hyperammonemia in vivo alters basal activity of neuronal nitric oxide synthase (nNOS in cerebellum and/or its activation in response to NMDA receptor activation; 2- to analyse the molecular mechanisms by which hyperammonemia induces these alterations; 3- to investigate whether tonic NMDA activation is increased in cerebellum in chronic hyperammonemia in vivo, and 4- whether this tonic activation is responsible for nNOS alterations in cerebellum. The findings show that hyperammonemia reduces both basal activity of nNOS and its activation following NMDA receptor activation. Reduced basal activity is due to increased phosphorylation of Ser847 by calcium-calmodulin-dependent protein kinases (CaMKII, which in turn is due to increased phosphorylation of Thr286. Inhibiting CaMKII, with KN-62 normalizes phosphorylation of Ser847 and basal NOS activity in hyperammonemic rats, leading to values similar to controls. Reduced activation of nNOS in response to NMDA receptor activation in hyperammonemia is due to altered subcellular localization of nNOS, with reduced amount in post-synaptic membranes and increased amount in the cytosol. Blocking NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in vivo and in slices from hyperammonemic rats, reduces phosphorylation and activity of CaMKII and normalizes nNOS phosphorylation and activity. MK-801 also increases nNOS in synaptic membranes and reduces it in cytosol. This indicates that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of the glutamate – NO – cGMP pathway.

  19. Comparison of inducible nitric oxide synthase activity in pancreatic islets of young and aged rats

    Science.gov (United States)

    Farrokhfall, Khadije; Hashtroudi, Mehri Seyed; Ghasemi, Asghar; Mehrani, Hossein

    2015-01-01

    Objective(s): Some pathologic situations such as diabetes and metabolic syndrome are associated with alternation in nitric oxide level. Incidence of these condition increases with aging. On the other hand, insulin secretion is modulated by nitric oxide, and nitric oxide synthase (NOS) activity is also altered in diabetes. In this study, modification in the enzyme activity associated with aging and also optimized procedure for islet NOS assay was investigated. Materials and Methods: Male Wistar rats were randomly divided in two experimental groups: A: adult rats; were 4 month old and B: old rats; were 12 month old. In all groups, plasma glucose, insulin and NOX (nitrite + nitrate = NOX) were measured, and also insulin secretion in isolated pancreatic islet with or without L-NAME was investigated. Furthermore, the inducible NOS activity with L-citrulline measurement in islets was measured. Results: L-citrulline was quantified using one step HPLC column. Aging induced hyperglycemia (P<0.05) and excess plasma NOX (17.74 ± 1.664 and 26.25 ± 2.166 ?mol/l in A and B groups respectively, P<0.05) with unaltered plasma insulin. Islet insulin secretion was significantly reduced in aging rats. L-NAME induced islet insulin secretion especially in aging rats (P=0.003). Inducible NOS activity in islets of aging rats was significantly higher than adult rats (1.082 ± 0.084 and 6.277 ± 0.475 pmol/min per mg protein in adult and aging rats, respectively, P<0.001). Conclusion: These findings show that decreased in islet insulin secretion may be related to increase in iNOS activity in islets, which follows impaired carbohydrate metabolism in aging. PMID:25810884

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

    Directory of Open Access Journals (Sweden)

    Fairman Robert

    2004-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Herculano A.M.

    2006-01-01

    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.

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

    International Nuclear Information System (INIS)

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

  3. Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus.

    Science.gov (United States)

    Eilert, U; De Luca, V; Constabel, F; Kurz, W G

    1987-05-01

    Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products. PMID:3579315

  4. Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen1[W

    OpenAIRE

    Bermúdez, María Ángeles; Galmés, Jeroni; Moreno, Inmaculada; Mullineaux, Philip M; Gotor, Cecilia; Romero, Luis C.

    2012-01-01

    Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants u...

  5. Skeletal muscle glucose uptake, glycogen synthase activity and GLUT 4 content during hypoglycaemia in type 1 diabetic subjects

    DEFF Research Database (Denmark)

    Orskov, L; Schmitz, O; Bak, J F; Lund, Sten; Kaal, A; Nyholm, B; Møller, N

    2001-01-01

    In healthy subjects, hypoglycaemia induces a profound 80% reduction in skeletal muscle glucose uptake and a similar suppression of glycogen synthase activity. The aim of this study was to examine the efficacy of this counterregulatory mechanism in type 1 diabetic subjects, who are especially prone to hypoglycaemic incidents. Nine type 1 diabetic male subjects were examined twice; during 120 min of hyperinsulinaemic (1.5 mU x kg(-1) x min(-1)) euglycaemia followed by (i) 240 min of graded hypogly...

  6. Multitracer Stable Isotope Quantification of Arginase and Nitric Oxide Synthase Activity in a Mouse Model of Pseudomonas Lung Infection

    OpenAIRE

    Hartmut Grasemann; Thomas Jaecklin; Anne Mehl; Hailu Huang; Mahroukh Rafii; Paul Pencharz; Felix Ratjen

    2014-01-01

    Cystic fibrosis airways are deficient for L-arginine, a substrate for nitric oxide synthases (NOSs) and arginases. The rationale for this study was to quantify NOS and arginase activity in the mouse lung. Anesthetized unventilated mice received a primed constant stable isotope intravenous infusion containing labeled L-arginine, ornithine, and citrulline. The isotopic enrichment of each of the infused isotopomers and its product amino acids were measured in plasma and organ homogenates using l...

  7. 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

  8. Glucosylceramide synthase inhibitors sensitise CLL cells to cytotoxic agents without reversing P-gp functional activity.

    Science.gov (United States)

    Gerrard, Gareth; Butters, Terry D; Ganeshaguru, Kanagasabai; Mehta, Atul B

    2009-05-01

    Malignant B-cells from most chronic lymphocytic leukaemia (CLL) patients over-express MDR1 encoded P-glycoprotein (P-gp) multidrug efflux pump. Inhibition of glucosylceramide (GC) synthesis has been shown in cell lines to correlate with the expression and function of P-gp and sensitise cancer cells to cytotoxic agents. We investigated the hypothesis that reducing intracellular GC levels will reduce P-gp expression in malignant cells from CLL patients. We studied the ability of glucosylceramide synthase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1) and N-nonyl-deoxygalactonojirimycin (OGB-2) to sensitise CLL cells to conventional cytotoxic drug 2-chlorodeoxyadenosine (CdA) and the cytostatic drugs chlorambucil and fludarabine. The effect on P-gp activity was analysed using the calcein-AM accumulation assay where a multidrug activity factor (MAF) of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. The P-gp over-expressing cell line CEM-VLB showed a MAF value of 96.4 with the P-gp inhibitor Z.3HCL, which fell to 15.7 after co-incubation with OGB-1 and 45.9 with OGB-2. The IC(50) for vincristine fell from >10 microg/ml to 55.5 ng/ml in the presence of OGB-2. In P-gp(+ve) peripheral blood mononuclear cells from three normal volunteers, the mean MAF values for Z.3HCL, OGB-1 and OGB-2 were 23.86, 1.83 and 16.2 respectively. In 9/13 CLL samples the mean P-gp functional activity was 22.15 and P-gp was over-expressed in 12/13 samples. However, the MAF value with OGB-1 and OGB-2 was <10. Nevertheless, sensitisation in CLL cells was observed by a reduction in the IC(50) in the presence of OGB-1 and OGB-2 with the conventional drugs. We conclude that although GCS inhibitors sensitize CLL cells to cytotoxic and cytostatic drugs, they do not appear to have any effect on P-gp functional activity. PMID:19285492

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

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. PMID:26592143

  10. 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

  11. Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy.

    OpenAIRE

    Poolman, B.; Molenaar, D.; Smid, E.J.; Ubbink, T; Abee, T.; Renault, P P; Konings, W. N.

    1991-01-01

    The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylca...

  12. 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.

  13. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole; Mundy, John

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially...... complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in...... the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants...

  14. Inducible nitric oxide synthase (iNOS) activity promotes ischaemic skin flap survival

    OpenAIRE

    Kane, Anthony J; Barker, Jane E.; Mitchell, Geraldine M.; Theile, David R B; Romero, Rosalind; Messina, Aurora; Wagh, Milind; Fraulin, Frankie O G; Morrison, Wayne A.; Stewart, Alastair G

    2001-01-01

    We have examined the role of nitric oxide (NO) in a model of functional angiogenesis in which survival of a skin flap depends entirely on angiogenesis to provide an arterial blood supply to maintain tissue viability.The different effects of nitric oxide synthase (NOS) inhibitors on rat skin flap survival appeared to be explained on the basis of their NOS isoform selectivity. Skin flap survival was decreased by iNOS-selective (inducible NOS) inhibitors, S-methyl-isothiourea, aminoguanidine and...

  15. In Silico Screening of the Library of Pyrimidine Derivatives as Thymidylate Synthase Inhibitors for Anticancer Activity

    OpenAIRE

    A. G. Nerkar; S. A. Ghone; A. K. Thaker

    2009-01-01

    We here report the virtual screening of several series of pyrimidine derivatives for in silico Thymidylate Synthase (TS) inhibition to arrive at possible potential inhibitors of TS with acceptable pharmacokinetic or ADME (Absorption, Distribution, Metabolism and Excretion) properties. Library of the molecules was constructed based upon structural modifications of pyrimidines nucleus. Structural modifications in descending order were performed for the series of pyrimidines, viz from pyrimidine...

  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. Prostaglandin E2 induces vascular relaxation by E-prostanoid 4 receptor-mediated activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Hristovska, Ana-Marija; Rasmussen, Lasse E; Hansen, Pernille B L; Nielsen, Susan S; Nüsing, Rolf M; Narumiya, Shuh; Vanhoutte, Paul; Skøtt, Ole; Jensen, Boye L

    2007-01-01

    The present experiments were designed to test the hypothesis that prostaglandin (PG) E(2) causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE(2) were measured in conscious mice. Single doses of PGE(2) caused concentration-dependent relaxations during contractions to phenylephrine (EC(50)=5*10(-8) mol/L). Relaxat...

  18. 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...

  19. Regulation of Aldosterone Synthase by Activator Transcription Factor/cAMP Response Element-Binding Protein Family Members

    OpenAIRE

    Nogueira, Edson F.; Rainey, William E.

    2010-01-01

    Aldosterone synthesis is regulated by angiotensin II (Ang II) and K+ acting in the adrenal zona glomerulosa, in part through the regulation of aldosterone synthase (CYP11B2). Here, we analyzed the role of cAMP response element (CRE)-binding proteins (CREBs) in the regulation of CYP11B2. Expression analysis of activator transcription factor (ATF)/CREB family members, namely the ATF1 and ATF2, the CREB, and the CRE modulator, in H295R cells and normal human adrenal tissue was performed using qu...

  20. Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinyl-CoA content in liver.

    Science.gov (United States)

    Quant, P A; Tubbs, P K; Brand, M D

    1989-08-15

    1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase. PMID:2573345

  1. 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 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.

  2. 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)

  3. 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...

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-23

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

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

    Directory of Open Access Journals (Sweden)

    Fu Y

    2014-06-01

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

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

    International Nuclear Information System (INIS)

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na6[TeW6O24] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups 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)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-22

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

  9. Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis1[W][OPEN

    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

    2014-01-01

    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

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

    Science.gov (United States)

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

    2003-01-01

    BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, Pendothelial dysfunction of aging and may therefore be a therapeutic target.

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

    Science.gov (United States)

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

    2012-10-19

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

  12. 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.

  13. 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.

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

    Science.gov (United States)

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

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-l-arginine to (14)C-l-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components. PMID:26682617

  15. 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.

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

    Science.gov (United States)

    Singh, Shailendra P.; Tao, Shixin; Fields, Timothy A.; Webb, Sydney; Harris, Raymond C.; Rao, Reena

    2015-01-01

    ABSTRACT Glycogen synthase kinase-3? (GSK3?) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3? expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-?1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-?1 treatment increased GSK3? expression and GSK3 inhibition abolished TGF-?1-induced SMAD3 activation and ?-smooth muscle actin (?-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3? stimulated ?-SMA expression even in the absence of TGF-?1 treatment. These results suggest that TGF-? regulates GSK3?, which in turn is important for TGF-?–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-? signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury. PMID:26092126

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

    Science.gov (United States)

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2?Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  18. Mechanistic insights into the bifunctional non-heme iron oxygenase carbapenem synthase by active site saturation mutagenesis.

    Science.gov (United States)

    Phelan, Ryan M; Townsend, Craig A

    2013-05-22

    The carbapenem class of ?-lactam antibiotics is known for its remarkable potency, antibacterial spectrum, and resistance to ?-lactamase-mediated inactivation. While the biosynthesis of structurally "complex" carbapenems, such as thienamycin, share initial biochemical steps with carbapenem-3-carboxylate ("simple" carbapenem), the requisite inversion at C5 and formation of the characteristic ?,?-unsaturated carboxylate are different in origin between the two groups. Here, we consider carbapenem synthase, a mechanistically distinct bifunctional non-heme iron ?-ketoglutarate-dependent enzyme responsible for the terminal reactions, C5 epimerization and desaturation, in simple carbapenem production. Interestingly, this enzyme accepts two stereoisomeric substrates and transforms each to a common active antibiotic. Owing both to enzyme and product instability, resorting to saturation mutagenesis of active site and selected second-sphere residues gave clearly differing profiles of CarC tolerance to structural modification. Guided by a crystal structure and the mutational data, in silico docking was used to suggest the positioning of each disastereomeric substrate in the active site. The two orientations relative to the reactive iron-oxo center are manifest in the two distinct reactions, C5-epimerization and C2/3-desaturation. These observations favor a two-step reaction scheme involving two complete oxidative cycles as opposed to a single catalytic cycle in which an active site tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring inversion, is further hypothesized to serve as a radical carrier. PMID:23611403

  19. High levels of nitric oxide synthase activity are associated with nasal polyp tissue from aspirin-sensitive asthmatics.

    Science.gov (United States)

    Parikh, A; Scadding, G K; Gray, P; Belvisi, M G; Mitchell, J A

    2002-04-01

    The pathogenesis of aspirin intolerance remains unclear. Inducible nitric oxide synthase (iNOS) expression is upregulated in nasal polyp epithelium, implying a role for nitric oxide (NO) in its formation. We decided to compare iNOS activity in polyp tissue from patients with and without aspirin intolerance. Nasal polyp tissue was collected from 15 patients undergoing routine nasal polypectomy. These patients were classified into three groups: Group A comprised patients with nasal polyps without asthma; Group B contained patients with nasal polyps and asthma; and Group C comprised patients with nasal polyps, asthma and aspirin sensitivity. All subjects in Group C had a history of aspirin-induced reaction and a confirmatory intranasal challenge with lysine-aspirin. NOS activity was measured by the ability of tissue homogenates to convert 3,4-L-arginine to L-citrulline in an L-N(G)-nitro-L-arginine-inhibitable fashion. The iNOS activity (picomoles) in polyp tissue from the 3 groups was: A, 248.72+/-220.79; B, 23.71+/-41.06; and C, 549.71+/-132.11. Thus, nasal polyps from patients with Samter's triad had a significantly higher iNOS activity (p = 0.004; one-way ANOVA). This finding does not correlate simply with disease severity or with the occurrence of asthma and could indicate another important facet of aspirin-induced airways disease. PMID:12030579

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

    Science.gov (United States)

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

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction. PMID:25595727

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

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

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

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

    Directory of Open Access Journals (Sweden)

    L. PANEPUCCI

    2000-05-01

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

  3. Structural insight into the molecular mechanism of allosteric activation of human cystathionine ?-synthase by S-adenosylmethionine.

    Science.gov (United States)

    Ereño-Orbea, June; Majtan, Tomas; Oyenarte, Iker; Kraus, Jan P; Martínez-Cruz, Luis Alfonso

    2014-09-16

    Cystathionine ?-synthase (CBS) is a heme-dependent and pyridoxal-5'-phosphate-dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H2S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H2S biogenesis. PMID:25197074

  4. The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

    Science.gov (United States)

    Singh, Dhirendra; Sielaff, Hendrik; Sundararaman, Lavanya; Bhushan, Shashi; Grüber, Gerhard

    2016-02-01

    A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9s(-1) and 30.4s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ? of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. PMID:26682760

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

    International Nuclear Information System (INIS)

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

  6. Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis

    Science.gov (United States)

    Nardone, Glenn; Ikeda, Allison K.; Cunnington, Aubrey J.; Okebe, Joseph; Ebonyi, Augustine O.; Njie, Madi; Correa, Simon; Jayasooriya, Shamanthi; Casals-Pascual, Climent; Billker, Oliver; Conway, David J.; Walther, Michael; Ackerman, Hans

    2015-01-01

    Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis. PMID:26407009

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

    Science.gov (United States)

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

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having ?M antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. PMID:25900220

  8. Potential Role of Glycogen Synthase Kinase-3? in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Zhou, Yi-Xia; Shi, Zhan; Singh, Pavneet; Yin, Hao; Yu, Yan-Ni; Li, Long; Walsh, Michael P; Gui, Yu; Zheng, Xi-Long

    2016-02-01

    Glycogen synthase kinase (GSK)-3?, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3? results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3? regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3? inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM ?-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3? inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3? inhibitors were mimicked by transfection with GSK-3? siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3? mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM ?-actin. As expected, overexpression of constitutively active or wild-type GSK-3? in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3? reduces myocardin transcriptional activity, suggesting a role for GSK-3? in myocardin transcriptional activity and smooth muscle differentiation. PMID:26129946

  9. Sp1 cooperates with Sp3 to upregulate MALAT1 expression in human hepatocellular carcinoma.

    Science.gov (United States)

    Huang, Ziling; Huang, Lanshan; Shen, Siqiao; Li, Jia; Lu, Huiping; Mo, Weijia; Dang, Yiwu; Luo, Dianzhong; Chen, Gang; Feng, Zhenbo

    2015-11-01

    Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched transcript 2 (NEAT2), is highly conserved among mammals and highly expressed in the nucleus. It was first identified in lung cancer as a prognostic marker for metastasis but is also associated with several other solid tumors. In hepatocellular carcinoma (HCC), MALAT1 is a novel biomarker for predicting tumor recurrence after liver transplantation. The mechanism of overexpression in tumor progression remains unclear. In the present study, we investigated the role of specificity protein 1/3 (Sp1/3) in regulation of MALAT1 transcription in HCC cells. The results showed a high expression of Sp1, Sp3 and MALAT1 in HCC vs. paired non-tumor liver tissues, which was associated with the AFP level (Sp1, r=7.44, P=0.0064; MALAT1, r=12.37, P=0.0004). Co-silencing of Sp1 and Sp3 synergistically repressed MALAT1 expression. Sp1 binding inhibitor, mithramycin A (MIT), also inhibited MALAT1 expression in HCC cells. In conclusion, the upstream of MALAT1 contains five Sp1/3 binding sites, which may be responsible for MALAT1 transcription. Inhibitors, such as MIT, provide a potential therapeutic strategy for HCC patients with MALAT1 overexpression. PMID:26352013

  10. Pharmacologic Inhibition of Sphingomyelin Synthase (SMS) Activity Reduces Apolipoprotein-B Secretion from Hepatocytes and Attenuates Endotoxin-Mediated Macrophage Inflammation

    OpenAIRE

    Lou, Bin; Dong, Jibin; LI, YALI; Ding, Tingbo; Bi, Tingting; Li, Yue; Deng, Xiaodong; Ye, Deyong; Jiang, Xian-cheng

    2014-01-01

    Sphingomyelin synthase (SMS) plays an important role in plasma atherogenic lipoprotein metabolism, inflammation, and the development of atherosclerosis. To understand whether the impaired apoB secretion and inflammation response is a direct result from lack of SMS activity, in this study, we prepared a series of compounds that inhibit SMS activity. Further, we characterized Dy105, the most potent inhibitor. We found that Dy105 treatment significantly reduces SM levels in SM-rich microdomain o...

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

    OpenAIRE

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

    1998-01-01

    Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation. ?Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 wi...

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

    OpenAIRE

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

    2004-01-01

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

  13. Estrogen, but not progesterone, induces the activity of nitric oxide synthase within the medial preoptic area in female rats.

    Science.gov (United States)

    Lima, Fernanda Barbosa; Ota, Fábio Honda; Cabral, Fernanda Jankur; Del Bianco Borges, Bruno; Franci, Celso Rodrigues

    2014-08-26

    The control of gonadotropin-releasing hormone (GnRH) secretion depends on the action of ovarian steroids and several substances, including nitric oxide (NO). NO in the medial preoptic area (MPOA) stimulates the proestrus surge of luteinizing hormone (LH). We studied the effect of estrogen (Tamoxifen-TMX) and progesterone (RU-486) antagonists on mRNA and protein expression of NO synthase (NOS), the enzyme that produces NO, as well as its activity within MPOA. Female rats received s.c. injections of TMX (3mg/animal) on first and second days of the estrous cycle (9 am), RU-486 (2mg/animal) on first, second, (8 am and 5 pm) and third days of the estrous cycle (8 am) or oil (controls) and were killed on the third day (5 pm). Real time-PCR and western blotting were performed to study NOS mRNA and protein expressions. The NOS activity was indirectly assessed by measuring the conversion from [(14)C]-L-arginine into [(14)C]-L-citrulline. TMX significantly decreased neuronal NOS (nNOS) mRNA expression (90%), and the activity of NOS, but did not alter nNOS protein expression. Also, TMX significantly decreased LH, FSH, estrogen and progesterone plasma levels. RU-486 nor affected NOS mRNA and protein expressions neither the NOS activity in the MPOA, but reduced FSH levels. The nitrergic system in the MPOA can be stimulated by estrogen whereas TMX decreased NOS activity and mRNA expression. In conclusion, the involvement of the nitrergic system in the MPOA to induce the surge of LH on proestrus depends on the estrogen action to stimulate the mRNA-nNOS expression and the activity of nNOS but it does not seem to depend on progesterone action. PMID:25044408

  14. Hypoxia-reduced nitric oxide synthase activity is partially explained by higher arginase-2 activity and cellular redistribution in human umbilical vein endothelium.

    Science.gov (United States)

    Prieto, C P; Krause, B J; Quezada, C; San Martin, R; Sobrevia, L; Casanello, P

    2011-12-01

    Hypoxia relates with altered placental vasodilation, and in isolated endothelial cells, it reduces activity of the endothelial nitric oxide synthase (eNOS) and l-arginine transport. It has been reported that arginase-2 expression, an alternative pathway for l-arginine metabolism, is increased in adult endothelial cells exposed to hypoxia as well as in pre-eclamptic placentae. We studied in human umbilical vein endothelial cells (HUVEC) whether hypoxia-reduced NO synthesis results from increased arginase-mediated l-arginine metabolism and changes in subcellular localization of eNOS and arginase-2. In HUVEC exposed (24 h) to 5% (normoxia) or 2% (hypoxia) oxygen, l-arginine transport kinetics, arginase activity (urea assay), and NO synthase (NOS) activity (l-citrulline assay) were determined. Arginase-1, arginase-2 and eNOS expression were determined by RT-PCR and Western blot. Subcellular localization of arginase-2 and eNOS were studied using confocal microscopy and indirect immunofluorescence. Experiments were done in absence or presence of S-(2-boronoethyl)-l-cysteine-HCl (BEC, arginase inhibitor) or N(G)-nitro-l-arginine methyl ester (l-NAME). Hypoxia-induced reduction in eNOS activity was associated with a reduction in eNOS phosphorylation at Serine-1177 and increased phosphorylation at Threonine-495. This was paralleled with an induction in arginase-2 expression and activity, and decreased l-arginine transport. In hypoxia the arginase inhibition, restored NO synthesis and l-arginine transport, without changes in the eNOS post-translational modification status. Hypoxia increased arginase-2/eNOS colocalization, and eNOS redistribution to the cell periphery. Altogether these data reinforce the thought that eNOS cell location, post-translational modification and substrate availability are important mechanisms regulating eNOS activity. If these mechanisms occur in pregnancy diseases where feto-placental oxygen levels are reduced remains to be clarified. PMID:21962305

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

    Science.gov (United States)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by "Biotin-switch" method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. PMID:25680459

  16. Inter-domain communication of human cystathionine ?-synthase: structural basis of S-adenosyl-L-methionine activation.

    Science.gov (United States)

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

    2014-12-26

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

  17. Nitric Oxide Synthase Activation as a Trigger of N-methyl-N-nitrosourea-Induced Photoreceptor Cell Death.

    Science.gov (United States)

    Hisano, Suguru; Koriyama, Yoshiki; Ogai, Kazuhiro; Sugitani, Kayo; Kato, Satoru

    2016-01-01

    Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death. PMID:26427435

  18. Synthesis of alkyl-ether glycerophospholipids in rat glomerular mesangial cells: evidence for alkyldihydroxyacetone phosphate synthase activity

    International Nuclear Information System (INIS)

    We studied the ability of rat glomerular mesangial cells and their microsomal fractions to incorporate 1-[14C]hexadecanol to glycerophospholipids via an O-alkyl ether linkage and assessed the presence and activity of the required enzyme: alkyl-dihydroxy acetone phosphate synthase. Suspensions of cultured mesangial cells incorporated 1-[14C]hexadecanol to the phosphatidyl ethanolamine and phosphatidyl choline lipid pools, via a bond resistant to acid and base hydrolysis. When cell homogenates or microsomal fractions were incubated with palmitoyl-DHAP and 1-[14C]hexadecanol, alkyl-DHAP and 1-O-alkyl glycerol were formed (alkyl:hexadecyl). The activity of the enzyme responsible for the O-alkyl product formation was calculated to be 2.5 +/- 0.3 and 544 +/- 50 pmoles/min/mg protein for mesangial cell homogenates and mesangial cell microsomes, respectively. These observations provide evidence that mesangial cells may elaborate either linked lipid precursors de novo for the biosynthesis of O-alkyl glycerophospholipids

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

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers

    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

  20. Nitric oxide synthase isoenzyme activities in a premature piglet model of necrotizing enterocolitis: effects of nitrergic manipulation.

    Science.gov (United States)

    Di Lorenzo, Maria; Krantis, Anthony

    2002-10-01

    Nitric oxide (NO) plays a major role in gut mucosal protection and motility. Having demonstrated the protective effects of intravenous L-arginine (L-arg) and the NO donor, sodium nitroprusside (SNP), in an in-vivo premature piglet intraluminal model of necrotizing enterocolitis (NEC) that incorporates both mucosal damage and intestinal dysmotility, we measured the effects on NO synthase (NOS) isoenzyme activities during i.v. manipulation of the nitrergic system in the NEC-injured gut. In newborn premature Yorkshire piglets, NEC was induced in four groups by intraluminal injection of acidified casein solution in closed test loops of bowel separated by normal saline-injected control loops. Group 1 (n = 4) underwent no further treatment. Group 2 (n = 4) received concomitant continuous i.v. L-arg, a NO substrate. Group 3 (n = 6) received concomitant continuous i.v. SNP, a NO donor. Group 4 (n = 5) received concomitant continuous i.v. N-omega-nitro-L-arginine-methyl-ester (L-NAME), a non-selective NO inhibitor. Control and test gut specimens were harvested after 3 h. NO synthase activity in frozen gut segments was assessed using the (14)C-L-arg to (14)C-L-citrulline conversion assay. Total NOS (TNOS), constitutive NOS (cNOS), and inducible NOS (iNOS) activities were compared. The mean and standard error were calculated for each specimen. Group means were used to compare test and control gut enzyme activities in the different treatment groups. One-way analysis of variance and the Bonferroni post test were used to compare differences among groups. A P value of less than 0.05 was considered significant. In the L-NAME group, cNOS activity was lower than in the untreated NEC group. The SNP group had higher iNOS and TNOS activities than the L-arg group; cNOS was also higher in test and control loops in the SNP versus both L-arg and L-NAME groups. However, in L-arg control loops, cNOS activity was greater than in the L-NAME group. SNP and L-arg treatment of NEC did not significantly modify NOS isoenzyme activities. Thus, in this premature piglet 3-h model of NEC, i.v. L-NAME significantly decreases cNOS activity and correlates with our previously published histopathologic findings confirming the protective role of cNOS-derived NO in NEC-injured gut mucosa. In order to further elucidate the mechanisms involved in the mucosal protection afforded by i.v. L-arg and SNP in this NEC model, studies of a longer duration have been undertaken. PMID:12471479

  1. 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)

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

    OpenAIRE

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

  4. Macromolecular crowding and the steady-state kinetics of malate dehydrogenase.

    Science.gov (United States)

    Poggi, Christopher G; Slade, Kristin M

    2015-01-20

    To understand how macromolecular crowding affects enzyme activity, we quantified the Michaelis-Menten kinetics of mitochondrial malate dehydrogenase (MDH) in the presence of hen egg white (HEW), lysozyme, bovine serum albumin (BSA), gum arabic, poly(vinylpyrrolidone) (PVP), and dextrans of various molecular weights. Although crowding tended to decrease Km and Vmax values, the magnitude depended on the crowding agent, reaction direction, and isozyme (mitochondrial porcine heart or thermophlic TaqMDH from Thermus flavus). Crowding slowed oxaloacetate reduction more significantly than malate oxidation, which may suggest that mitochondrial enzymes have evolved to function optimally under the crowded constraints in which they are immersed. Since direct comparisons of neutral to charged crowders are underrepresented in the literature, we performed these studies and found that neutral crowding agents lowered Vmax values more than charged crowders of similar size. The exception was hen egg white, a mixture of charged proteins that caused the largest observed decreases in both Km and Vmax. Finally, the data provide insight about the mechanism by corroborating MDH subunit dependence. PMID:25478785

  5. L-Malate content in irradiated onions (Allium Cepa L.) cv. Valenciana sintetica 14

    International Nuclear Information System (INIS)

    Results of L-malate evaluation in control and irradiated onions, (v. 'Valenciana sintetica 14') and its correlation with sprouting cumulative values are reported. It was concluded that if on the 150th day of storage, the malate content reaches a maximum value and the sprouting is 1 per cent or less, then it would indicate that the samples have been irradiated. L-malate values are positively correlated to sprouting in control samples, while for irradiated ones correlation was negative. (author)

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-17

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

  8. Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis.

    Science.gov (United States)

    Wang, Jing; Shen, You-Ming; Li, Bing; Zhou, Xu-zheng; Liu, Cui-cui; Zhang, Ji-yu

    2014-07-01

    The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme has been characterized in other species, but not in the genus Babesia, which causes major losses in the livestock industries worldwide. Therefore, we isolated, cloned and expressed the wild-type B. bovis dxs cDNA in Escherichia coli and evaluated its enzymatic activity in vitro. DNA sequence analysis revealed an open reading frame of 2061 bp capable of encoding a polypeptide of 686 amino acid residues with a calculated isoelectric point of pH 6.93 and a molecular mass of 75 kDa. The expressed soluble recombinant fusion DXS protein was approximately 78 kDa, which is similar to the native enzyme identified from the parasite merozoite using anti-rDXS serum. The recombinant fusion DXS enzyme exhibited Km values of 380 ± 46 µM and 790 ± 52 µM for D,L-glyceraldehyde 3-phosphate and pyruvate, respectively. In this work, we present the first cloning, expression and characterization of DXS enzyme from B. bovis. PMID:24739240

  9. 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

  10. 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; Ploug, Kenneth Beri; Hay-Schmidt, Anders; Jansen-Olesen, Inger; Gupta, Saurabh; Olesen, Jes

    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, intrave...

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

    OpenAIRE

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

    1995-01-01

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

  12. Increased nitric oxide synthase activity despite lack of response to endothelium-dependent vasodilators in postischemic acute renal failure in rats.

    OpenAIRE

    Conger, J; Robinette, J; A Villar; Raij, L; Shultz, P

    1995-01-01

    Lack of response to endothelium-dependent vasodilators generally has been considered to be evidence for decreased nitric oxide synthase (NOS) activity and NO generation after ischemic or hypoxic injury to vital organs including the kidney. In this study, renal blood flow (RBF) responses to endothelium-dependent vasodilators acetylcholine and bradykinin and the endothelium-independent vasodilator prostacyclin, the nonselective NOS inhibitor L-NAME (without and with L-arginine), the inducible N...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-08-25

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

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

    International Nuclear Information System (INIS)

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

  15. Tl+ induces the permeability transition pore in Ca2+-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 Ca2+ 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 Ca2+ 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 Ca2+, 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 Ca2+-loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl+ to Tl3+ by mitochondrial oxygen reactive species is proposed for the mechanism. PMID:25910914

  16. Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice

    Science.gov (United States)

    Peters, Tim; Hermans-Beijnsberger, Steffie; Beqqali, Abdelaziz; Bitsch, Nicole; Nakagawa, Shinichi; Prasanth, Kannanganattu V.; de Windt, Leon J.; van Oort, Ralph J.; Heymans, Stephane; Schroen, Blanche

    2016-01-01

    Background Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice. Results Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97±0.26, TAC 1.57±0.40; p<0.0001) and KO mice (sham: 3.64±0.37; TAC: 2.24±0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each). Conclusion These findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133. PMID:26919721

  17. Efficient heterocyclisation by (di)terpene synthases.

    Science.gov (United States)

    Mafu, S; Potter, K C; Hillwig, M L; Schulte, S; Criswell, J; Peters, R J

    2015-09-11

    While cyclic ether forming terpene synthases are known, the basis for such heterocyclisation is unclear. Here it is reported that numerous (di)terpene synthases, particularly including the ancestral ent-kaurene synthase, efficiently produce isomers of manoyl oxide from the stereochemically appropriate substrate. Accordingly, such heterocyclisation is easily accomplished by terpene synthases. Indeed, the use of single residue changes to induce production of the appropriate substrate in the upstream active site leads to efficient bifunctional enzymes producing isomers of manoyl oxide, representing novel enzymatic activity. PMID:26214384

  18. Reduced spinal microglial activation and neuropathic pain after nerve injury in mice lacking all three nitric oxide synthases

    Directory of Open Access Journals (Sweden)

    Shimokawa Hiroaki

    2011-07-01

    Full Text Available Abstract Background Several studies have investigated the involvement of nitric oxide (NO in acute and chronic pain using mice lacking a single NO synthase (NOS gene among the three isoforms: neuronal (nNOS, inducible (iNOS and endothelial (eNOS. However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (n/i/eNOS-/-mice and investigated the behavioral phenotypes in a series of acute and chronic pain assays. Results In a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both iNOS-/-and n/i/eNOS-/-mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation were reduced in n/i/eNOS-/-but not iNOS-/-mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line, interferon-? enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor. Conclusions Our results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.

  19. Lipid A-activated inducible nitric oxide synthase expression via nuclear factor-?B in mouse choroid plexus cells.

    Science.gov (United States)

    Takano, Masaoki; Ohkusa, Mami; Otani, Mieko; Min, Kyong-Son; Kadoyama, Keiichi; Minami, Katori; Sano, Keiji; Matsuyama, Shogo

    2015-10-01

    Choroid plexus (CP) which is responsible for the inflammatory mediators including nitric oxide (NO) are thought to play a crucial role in the process of bacterial meningitis. The present study investigated the mechanisms regulating inducible nitric oxide synthase (iNOS) expression in the choroid plexus epithelium (CPe) in mice. Initially, the expression of iNOS in mouse CPe was strengthened by intracerebroventriclar (i.c.v.) administration of lipid A, which is part of a Gram-negative bacterial endotoxin located at one end of the lipopolysaccharide (LPS) molecule. Next, the expression of iNOS in the CP epithelial cell line ECPC-4 cells was increased from 24 to 48h after lipid A treatment, although mRNA and proteins of toll-like receptor (TLR)-2 and -4 expressed in ECPC-4 cells were not changed by lipid A. The expression of total nuclear factor ?B (NF?B), an inflammatory transcriptional factor, in ECPC-4 cells was not changed for 72h after lipid A treatment, while cytoplasmic NF?B was decreased and nuclear NF?B was increased from 1 to 2h. In addition, the phosphorylation of inhibitor ?B (I?B) was peaked at 10min, and the level of I?B was attenuated from 10 to 45min after lipid A treatment. Moreover, the RNA interference (RNAi) of NF?B suppressed the expression of iNOS induced by lipid A. We demonstrated that lipid A-induced iNOS expression in ECPC-4 cells was mainly regulated by the activation of NF?B-I?B intracellular signaling pathway. Thus, we propose that the CPe plays a pivotal role in innate immunity responses of the brain, that is, the signal pathway TLRs on the CPe following inflammatory stimulation such as meningitis is activated, leading to iNOS expression through NF?B. PMID:26235132

  20. 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; Søndergaard, Teis; Stærk, Dan; Lee, T Verne; Wimmer, Reinhard; Klitgaard, Louise Graabæk; Purup, Stig; Giese, Henriette; Frandsen, Rasmus John Normand

    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 pigme...

  1. 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.

  2. Safety Assessment of Dialkyl Malates as Used in Cosmetics.

    Science.gov (United States)

    Becker, Lillian C; Bergfeld, Wilma F; Belsito, Donald V; Hill, Ronald A; Klaassen, Curtis D; Liebler, Daniel C; Marks, James G; Shank, Ronald C; Slaga, Thomas J; Snyder, Paul W; Andersen, F Alan

    2015-01-01

    The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of 6 dialkyl malate compounds used in cosmetics. These ingredients function mostly as skin-conditioning agents-emollients. The Panel reviewed relevant animal and human data related to the ingredients along with a previous safety assessment of malic acid. The similar structure, properties, functions, and uses of these ingredients enabled grouping them and using the available toxicological data to assess the safety of the entire group. The Panel concluded that these dialkyl maleate compounds are safe in the present practices of use and concentration as given in this safety assessment. PMID:26227891

  3. Spectroscopic, thermal and structural studies on manganous malate crystals

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-15

    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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  5. 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.

  6. A stable blue-light-derived signal modulates ultraviolet-light-induced activation of the chalcone-synthase gene in cultured parsley cells

    International Nuclear Information System (INIS)

    Run-off transcription assays were used to demonstrate that both the ultraviolet (UV)-B and blue-light receptors control transcription rates for chalcone-synthase mRNA in the course of light-induced flavonoid synthesis in parsley (Petroselinum crispum Miller (A.W. Hill)) cell-suspension cultures. Blue and red light alone, presumably acting via a blue-light receptor and active phytochrome (far-red absorbing form) respectively, can induce accumulation of chalcone-synthase mRNA. The extent of the response is however considerably smaller than that obtained when these wavebands are applied in combination with UV light. A preirradiation with blue light strongly increases the response to a subsequent UV pulse and this modulating effect of blue light is stable for at least 20 h. The modulating effect is abolished by a UV induction but can be reestablished by a second irradiation with blue light. (author)

  7. 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.

  8. Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants.

    Science.gov (United States)

    Rathnam, C K

    1978-01-01

    The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C4 plant, exhibited a temperature optimum of 35-37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17-20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C4 plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C4 acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C4 acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C4 enzyme while its activators showed no significant effect. PEP carboxylase from C3 species was not protected from thermal inactivation by the C4 acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C4 plants. PMID:24414875

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

    OpenAIRE

    McCool, Gabriel J.; Maura C. Cannon

    2001-01-01

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

  10. Nitric oxide enhances MPP(+)-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum.

    Science.gov (United States)

    Obata, T; Yamanaka, Y

    2001-06-01

    We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis. PMID:11384616

  11. Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana.

    Science.gov (United States)

    Mourad, G; King, J

    1992-11-01

    We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5' end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3' end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K. PMID:24178380

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

    OpenAIRE

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

    1984-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kankaanranta Hannu

    2006-02-01

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

  14. 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

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Seyffarth Gunter

    2004-06-01

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

  17. Independent fluctuations of malate and citrate in the CAM species Clusia hilariana Schltdl. under low light and high light in relation to photoprotection.

    Science.gov (United States)

    Miszalski, Zbigniew; Kornas, Andrzej; Rozpądek, Piotr; Fischer-Schliebs, Elke; Lüttge, Ulrich

    2013-03-15

    Clusia hilariana Schltdl. is described in literature as an obligate Crassulacean acid metabolism (CAM) species. In the present study we assessed the effect of irradiance with low light (LL, 200μmolm(-2)s(-1)) and high light (HL, 650-740μmolm(-2)s(-1)), on the interdependency of citrate and malate diurnal fluctuations. In plants grown at HL CAM-type oscillations of concentration of citrate and malate were obvious. However, at LL daily courses of both acids do not seem to indicate efficient utilization of these compounds as CO2 and NADPH sources. One week after transferring plants from LL to HL decarboxylation of malate was accelerated. Thus, in the CAM plant C. hilariana two independent rhythms of accumulation and decarboxylation of malate and citrate take place, which appear to be related to photosynthesis and respiration, respectively. Non photochemical quenching (NPQ) of photosystem II, especially well expressed during the evening hours was enhanced. Exposure to HL for 7 d activated oxidative stress protection mechanisms such as the interconversion of violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) (epoxydation/de-epoxydation) measured as epoxydation state (EPS). This was accompanied by a slight increase in the total amount of these pigments. However, all these changes were not observed in plants exposed to HL for only 2 d. Besides violaxanthin cycle components also lutein, which shows a small, but not significant increase, may be involved in dissipating excess light energy in C. hilariana. PMID:23253483

  18. 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.

  19. 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.

  20. 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.

  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. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K; Prats, C; Hirshman, M F; Dufresne, S D; Sakamoto, K; Vilardo, P G; Steele, M; Kim, J H; Jing, S L; Goodyear, L J; DePaoli-Roach, A A

    2001-01-01

    In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., St...

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

    OpenAIRE

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

    1994-01-01

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

  4. Metabolic activation of the antitumor drug 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by NO synthases.

    Science.gov (United States)

    Chandor, Alexia; Dijols, Sylvie; Ramassamy, Booma; Frapart, Yves; Mansuy, Daniel; Stuehr, Dennis; Helsby, Nuala; Boucher, Jean-Luc

    2008-04-01

    Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of L-arginine to L-citrulline with formation of the signaling molecule nitric oxide (NO). In addition to their fundamental role in NO biosynthesis, NOSs are also involved in the formation of reactive oxygen and nitrogen species (RONS) and in the interactions with some drugs. 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) is a dinitroaromatic compound tested as an antitumor prodrug that requires reduction to the 2- and 4-hydroxylamines to be cytotoxic. Here, we studied the interaction of neuronal, inducible, and endothelial NOSs with CB1954. Our results showed that the three purified recombinant NOSs selectively reduced the 4-nitro group of CB1954 to the corresponding 4-hydroxylamine with minimal 2-nitroreduction. Little further two-electron reduction of the hydroxylamines to the corresponding 2- and 4-amines was observed. The reduction of CB1954 catalyzed by the neuronal NOS (nNOS) was inhibited by O 2 and a flavin/NADPH binding inhibitor, diphenyliodonium (DPI), but insensitive to the addition of the heme ligands imidazole and carbon monoxide and of l-arginine analogues. This reduction proceeded with intermediate formation of a nitro-anion free radical observed by EPR. Involvement of the reductase domain of nNOS in the reduction of CB1954 was confirmed by the ability of the isolated reductase domain of nNOS to catalyze the reaction and by the stimulating effect of Ca (2+)/calmodulin on the accumulation of 4- and 2-hydroxylamines. The recombinant inducible and endothelial NOS isoforms reduced CB1954 with lower activity but higher selectivity for the cytotoxic 4-hydroxylamine compared with nNOS. Finally, CB1954 did not modify the formation of l-citrulline and RONS catalyzed by nNOS. Our results show that all three NOS isoforms are involved in the nitroreduction of CB1954, with predominant formation of the cytotoxic 4-hydroxylamine derivative. This nitroreduction could be of interest for the selective activation of prodrugs by NOSs overexpressed in tumor cells. PMID:18370414

  5. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole; Mundy, John

    2002-01-01

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

  6. Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply.

    Science.gov (United States)

    Hedrich, R; Neimanis, S; Savchenko, G; Felle, H H; Kaiser, W M; Heber, U

    2001-08-01

    Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells. PMID:11556792

  7. The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Manipulates the Activity of Glycogen Synthase Kinase-3?

    OpenAIRE

    Fujimuro, Masahiro; Hayward, S Diane

    2003-01-01

    The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in all KSHV-associated malignancies. LANA is essential for replication and maintenance of the viral episomes during latent infection. However, LANA also has a transcriptional regulatory role and can affect gene expression both positively and negatively. A previously performed yeast two-hybrid screen identified glycogen synthase kinase 3 (GSK-3) as a LANA-interacting protein. Interactio...

  8. Kaposi's Sarcoma-Associated Herpesvirus LANA Protein Downregulates Nuclear Glycogen Synthase Kinase 3 Activity and Consequently Blocks Differentiation?

    OpenAIRE

    Liu, Jianyong; Martin, Heather; Shamay, Meir; Woodard, Crystal; Tang, Qi-Qun; Hayward, S Diane

    2007-01-01

    The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) protein interacts with glycogen synthase kinase 3 (GSK-3) and relocalizes GSK-3 in a manner that leads to stabilization of ?-catenin and upregulation of ?-catenin-responsive cell genes. The LANA-GSK-3 interaction was further examined to determine whether there were additional downstream consequences. In the present study, the nuclear GSK-3 bound to LANA in transfected cells and in BCBL1 primary effusion lymp...

  9. Citrulline/malate promotes aerobic energy production in human exercising muscle

    Science.gov (United States)

    Bendahan, D; Mattei, J; Ghattas, B; Confort-Gouny, S; Le Guern, M E; Cozzone, P

    2002-01-01

    Background: Previous studies have shown an antiasthenic effect of citrulline/malate (CM) but the mechanism of action at the muscular level remains unknown. Objective: To investigate the effects of CM supplementation on muscle energetics. Methods: Eighteen men complaining of fatigue but with no documented disease were included in the study. A rest-exercise (finger flexions)-recovery protocol was performed twice before (D-7 and D0), three times during (D3, D8, D15), and once after (D22) 15 days of oral supplementation with 6 g/day CM. Metabolism of the flexor digitorum superficialis was analysed by 31P magnetic resonance spectroscopy at 4.7 T. Results: Metabolic variables measured twice before CM ingestion showed no differences, indicating good reproducibility of measurements and no learning effect from repeating the exercise protocol. CM ingestion resulted in a significant reduction in the sensation of fatigue, a 34% increase in the rate of oxidative ATP production during exercise, and a 20% increase in the rate of phosphocreatine recovery after exercise, indicating a larger contribution of oxidative ATP synthesis to energy production. Considering subjects individually and variables characterising aerobic function, extrema were measured after either eight or 15 days of treatment, indicating chronological heterogeneity of treatment induced changes. One way analysis of variance confirmed improved aerobic function, which may be the result of an enhanced malate supply activating ATP production from the tricarboxylic acid cycle through anaplerotic reactions. Conclusion: The changes in muscle metabolism produced by CM treatment indicate that CM may promote aerobic energy production. PMID:12145119

  10. 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.

  11. The Malat1 long non-coding RNA is upregulated by signalling through the PERK axis of unfolded protein response during flavivirus infection.

    Science.gov (United States)

    Bhattacharyya, Sankar; Vrati, Sudhanshu

    2015-01-01

    Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). UPR is initiated following activation of three ER-membrane resident sensors, PERK, IRE1? and ATF6, which are otherwise kept inactive through association with the ER-chaperone GRP78. Activation precedes cellular and molecular changes that act to restore homeostasis but might eventually initiate apoptosis. These changes involve influencing function of multiple genes by either transcriptional or post-transcriptional or post-translational mechanisms. Transcriptional control includes expression of transcription factor cascades, which influence cognate gene expression. Malat1 is a long non-coding RNA which is over-expressed in many human oncogenic tissues and regulates cell cycle and survival. In this report, for the first time we show activation of Malat1 following infection by two flaviviruses, both of which activate the UPR in host cells. The temporal kinetics of expression was restricted to later time points. Further, Malat1 was also activated by pharmacological inducer of UPR, to a similar degree. Using drugs that specifically inhibit or activate the PERK or IRE1? sensors, we demonstrate that signalling through the PERK axis activates this expression, through a transcriptional mechanism. To our knowledge, this is the first report of an UPR pathway regulating the expression of an lncRNA. PMID:26634309

  12. Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury

    Directory of Open Access Journals (Sweden)

    Okubo Masamichi

    2012-02-01

    Full Text Available Abstract Background Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown. Methods Using the rat spared nerve injury (SNI model, we investigated the expression of PAF synthases (LPCAT1 and 2 and PAF receptor (PAFr mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR and double-labeling analysis of in situ hybridization histochemistry (ISHH with immunohistochemistry (IHC were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086. Results RT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia. Conclusions Our data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development of neuropathic pain.

  13. In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

    Science.gov (United States)

    Urbina, Julio A.; Concepcion, Juan Luis; Caldera, Aura; Payares, Gilberto; Sanoja, Cristina; Otomo, Takeshi; Hiyoshi, Hironobu

    2004-01-01

    Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with Ki values in the low nanomolar to subnanomolar range in the absence or presence of 20 ?M inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease. PMID:15215084

  14. Sucrose Synthase: Expanding Protein Function

    Science.gov (United States)

    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. Th...

  15. Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer

    OpenAIRE

    Kawabata, Shigeru; Chiang, Chun-Te; Tsurutani, Junji; Shiga, Hideaki; Arwood, Matthew L.; Komiya, Takefumi; Gills, Joell J.; Memmott, Regan M.; Dennis, Phillip A

    2014-01-01

    Non-small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that...

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

    DEFF Research Database (Denmark)

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

    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-k...

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

    Science.gov (United States)

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

    2016-01-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine ?-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration. PMID:26803480

  18. [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

  19. Processes of malate catabolism during the anaerobic metabolism of grape berries

    International Nuclear Information System (INIS)

    In order to precise malate fate during the anaerobic metabolism of grape, malate-3-14C was injected into Carignan berries kept in darkness at 350C under carbon dioxide atmosphere. The injection of labelled malate was effected in presence or not of non-labelled oxalate which inhibits malic enzyme (EC I.I.I.40). The analyses of the samples fixed after 3 and 7 days anaerobiosis concerned the titration of various substrates, organic acids, amino-acids and glycolysis products, and the measuring of the NADP+-malic enzyme (EC I.I.I.40) and malate dehydrogenase (EC I.I.I.40). Radioactivity is mainly observed in ethanol, amino-butyrate the non-separated group glycerate-shikimate and succinate. Malic enzyme acts in the first sequence of a process leading from malate to ethanol. Alanin synthesis seems to be stimulated in presence of oxalate. The results obtained and some hypotheses presented in the literature induce to suggest a utilization scheme for malate in the anaerobic metabolism of grape

  20. Long Noncoding RNA MALAT-1 Can Predict Poor Prognosis: A Meta-Analysis

    Science.gov (United States)

    Tang, Ruixue; Jiang, Mengtong; Liang, Lu; Xiong, Dandan; Dang, Yiwu; Chen, Gang

    2016-01-01

    Background MALAT-1 is a highly conserved nuclear long non-coding RNA (lncRNA). The overexpression of MALAT-1 has been reported in several types of cancers. This meta-analysis was conducted to further investigate its potential role as a prognostic indicator in various cancers. Material/Methods The meta-analysis was performed by use of systematic search terms in 13 databases for qualified papers on prognosis in cancer from inception to June 30, 2015. The combined hazard ratios (HRs) with 95% confidence interval (95% CI) were computed to demonstrate the effect of MALAT-1 on prognosis of cancers. Results A total of 590 papers were initially identified, and 17 studies were finally included in this paper. Meta-analysis was accomplished with a total of 1626 patients. Combined HRs and 95% CI were calculated by fixed-effects or random-effects models. The quality assessment of included studies was performed by the Newcastle-Ottawa scale (NOS). High expression of MALAT-1 was found to be an indicator of poor prognosis in overall survival (OS) (HR=1.84, 95% CI: 1.27–2.67) and disease-free survival (DFS) (HR=2.37, 95% CI: 1.55–3.62). In subgroups, the associations between MALAT-1 and survival were also apparent, for instance, in country subgroup: China (HR=1.85, 95% CI: 1.14–2.99). Conclusions The overexpression of MALAT-1 may be a potential prognostic indicator for various human cancers. PMID:26821178

  1. 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

  2. Vesicocutaneous fistula formation during treatment with sunitinib malate: Case report

    Directory of Open Access Journals (Sweden)

    Sakashita Hiroyuki

    2010-11-01

    Full Text Available Abstract Background The oral multi-kinase inhibitor sunitinib malate improves the survival of patients with gastrointestinal stromal tumors (GIST after the disease progresses or intolerance to imatinib mesylate develops. Urinary fistulae arising during treatment with sunitinib for GIST have not been described. Case presentation We describe a 62-year-old female patient diagnosed with unresectable GIST that involved the abdominal wall, urinary bladder wall, bowel, mesentery and peritoneum in the pelvic cavity. Intestinocutaneous fistulae developed on a surgical lesion after orally administered imatinib was supplemented by an arterial infusion of 5-flurouracil. Sunitinib was started after the patient developed resistance to imatinib. On day 4 of the fourth course of sunitinib, a widely dilated cutaneous fistula discharged large amounts of fluid accompanied by severe abdominal pain. Urinary communication was indicated based on the results of an intravenous injection of indigo carmine. Computed tomography findings suggested a small opening on the anterior urinary bladder wall and fistulous communication between the bladder and abdominal walls bridged by a subcutaneous cavity. The fistula closed and the amount of discharge decreased when sunitinib was discontinued. Therefore, sunitinib might have been associated with the development of the vesicocutaneous fistula in our patient. Conclusion This is the first description of a vesicocutaneous fistula forming while under sunitinib treatment. Clinicians should be aware of the possible complication of vesicocutaneous fistula formation during treatment with molecular targeting agents in patients with extravesical invasion and peritoneal dissemination of GIST.

  3. 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.

  4. cAMP regulates the functional activity, coupling efficiency and structural organization of mammalian FOF1 ATP synthase.

    Science.gov (United States)

    De Rasmo, Domenico; Micelli, Loris; Santeramo, Arcangela; Signorile, Anna; Lattanzio, Paolo; Papa, Sergio

    2016-04-01

    The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death. PMID:26775111

  5. 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.

  6. BAK activation is necessary and sufficient to drive ceramide synthase-dependent ceramide accumulation following inhibition of BCL2-like proteins.

    Science.gov (United States)

    Beverly, Levi J; Howell, Lauren A; Hernandez-Corbacho, Maria; Casson, Lavona; Chipuk, Jerry E; Siskind, Leah J

    2013-05-15

    Determining mechanistic details about how drugs kill cancer cells is critical for predicting which cancers will respond to given therapeutic regimens and for identifying effective combinations of drugs that more potently kill cancer cells while sparing normal cells. The BCL2 family of proteins and bioactive sphingolipids are intricately linked during apoptotic cell death. In fact, many chemotherapeutic drugs are known to cause accumulation of the pro-apoptotic sphingolipid ceramide; however, the mechanism by which this occurs is not completely understood. In the present study we demonstrate that direct inhibition of anti-apoptotic BCL2 proteins with ABT-263 is sufficient to induce C(16)-ceramide synthesis in multiple cell lines, including human leukaemia and myeloma cells. ABT-263 activates CerS (ceramide synthase) activity only in cells expressing BAK or in cells capable of activating BAK. Importantly, recombinant BAK is sufficient to increase in vitro CerS activity in microsomes purified from Bak-KO (knockout) cells and activated BAK more potently activates CerS than inactive BAK. Likewise, ABT-263 addition to wild-type, but not Bak-deficient, microsomes increases CerS in vitro activity. Furthermore, we present a feed-forward model by which BAK activation of CerS by chemotherapeutic drugs leads to elevated ceramide levels that result in synergistic channel formation by ceramide (or one of its metabolites) and BAX/BAK. PMID:23480852

  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 <0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH?-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH?-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH?-terminal dephosphorylation may be the site for "fine-tuning" insulin-mediated GS activation in humans.

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

    Science.gov (United States)

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

    2012-01-01

    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. PMID:23203096

  9. 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.

  10. Retinoic acid activates human inducible nitric oxide synthase gene through binding of RAR?/RXR? heterodimer to a novel retinoic acid response element in the promoter

    International Nuclear Information System (INIS)

    Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RAR?/RXR? heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5 bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RAR?/RXR? directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RAR?/RXR?) in the induction of hiNOS by RA

  11. Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi.

    Science.gov (United States)

    Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

    2010-02-01

    Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism. PMID:19690851

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

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

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

  13. 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 Niklas Fabian

    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.

  14. Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids.

    Science.gov (United States)

    Khoo, Nicholas K H; Rudolph, Volker; Cole, Marsha P; Golin-Bisello, Franca; Schopfer, Francisco J; Woodcock, Steven R; Batthyany, Carlos; Freeman, Bruce A

    2010-01-15

    Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO(2)) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO(2) via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO(2)-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO(2)-FAs stimulated the phosphorylation of eNOS at Ser(1179). These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO(2)-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders. PMID:19857569

  15. The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism.

    Science.gov (United States)

    Swiderek, Katarzyna; Kohen, Amnon; Moliner, Vicent

    2015-11-18

    The hydride transfer from C6 of tetrahydrofolate to the reaction's exocyclic methylene-dUMP intermediate is the rate limiting step in thymidylate synthase (TSase) catalysis. This step has been studied by means of QM/MM molecular dynamics simulations to generate the corresponding free energy surfaces. The use of two different initial X-ray structures has allowed exploring different conformational spaces and the existence of chemical paths with not only different reactivities but also different reaction mechanisms. The results confirm that this chemical conversion takes place preferentially via a concerted mechanism where the hydride transfer is conjugated to thiol-elimination from the product. The findings also confirm the labile character of the substrate-enzyme covalent bond established between the C6 of the nucleotide substrate and a conserved cysteine residue. The calculations also reproduce and rationalize a normal H/T 2° kinetic isotope effect measured for that step. From a computational point of view, the results demonstrate that the use of an incomplete number of coordinates to describe the real reaction coordinate can render biased results. PMID:25868526

  16. 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.

  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

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-30

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

  20. 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.

  1. Constitutive activation of glycogen synthase kinase-3? correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer

    Directory of Open Access Journals (Sweden)

    Cho Yu

    2010-08-01

    Full Text Available Abstract Background Aberrant regulation of glycogen synthase kinase-3? (GSK-3? has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3? phosphorylated at Tyr216 (pGSK-3? and its relationship with other tumor-associated proteins in human gastric cancers. Methods Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3? inhibitor lithium chloride (LiCl for immunoblot analysis. Results We found that pGSK-3? was expressed in 129 (46% of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P P P P P Conclusions GSK-3? activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3? activation is a useful prognostic marker for the early-stage gastric cancer.

  2. Prion peptide PrP106-126 induces inducible nitric oxide synthase and proinflammatory cytokine gene expression through the activation of NF-?B in macrophage cells.

    Science.gov (United States)

    Lu, Yun; Liu, Ailing; Zhou, Xiangmei; Kouadir, Mohammed; Zhao, Wei; Zhang, Siming; Yin, Xiaomin; Yang, Lifeng; Zhao, Deming

    2012-05-01

    The inflammatory response in prion diseases is dominated by microglia activation. The molecular mechanisms that lie behind this inflammatory process are not very well understood. In the present study, we examined the activat2ion of nuclear factor-kappa B (NF-?B) upon exposure to PrP106-126 and its role in PrP106-126-induced upregulation of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines (interleukin [IL]-1?, tumor necrosis factor [TNF]-?, IL-6) in Ana-1 macrophages. The results showed that iNOS and proinflammatory cytokine release was significantly elevated in Ana-1 macrophages upon exposure to PrP106-126; that PrP106-126 treatment led to a significant NF-?B activation; that proinflammatory cytokines gene expression was elevated in macrophages upon exposure to PrP106-126; and that NF-?B inhibition significantly abrogated PrP106-126-induced upregulation of iNOS and inflammatory cytokine mRNA expression. These results suggest that treatment with neurotoxic prion peptides leads to the activation of transcription factor NF-?B, which in turn stimulates gene expression of iNOS and proinflammatory cytokines in Ana-1 macrophages. PMID:22149924

  3. 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.

  4. Development and validation of HPTLC method for the estimation of almotriptan malate in tablet dosage form

    Directory of Open Access Journals (Sweden)

    Suneetha A

    2010-01-01

    Full Text Available A new, simple, precise and accurate high performance thin layer chromatographic method has been proposed for the determination of almotriptan malate in a tablet dosage form. The drug was separated on aluminum plates precoated with silica gel 60 GF 254 with butanol:acetic acid:water (3:1:1 was used as mobilephase. Quantitative analysis was performed by densitometric scanning at 300 nm. The method was validated for linearity, accuracy, precision and robustness. The calibration plot was linear over the range of 100-700 ng/band for almotriptan malate. The method was successfully applied to the analysis of drug in a pharmaceutical dosage form.

  5. Malate supplementation to beef cattle: effects on growth performance and rumen fermentation products

    OpenAIRE

    Stefano Vandoni; Carlo Angelo Sgoifo Rossi

    2010-01-01

    Two trials were performed to evaluate the effects of malate supplementation on the growth performance as well as on ruminal pH and fermentation products of beef cattle. A total of 80 Charolaise bullocks were randomly allotted to one of the four experimental groups which included two experiments. The first experiment involved a control group (C) fed with a corn silage based diet and a treated group (T) fed the same diet supplemented with 20 g/head/day of malate. The second experiment involved ...

  6. Protective Effects of L-Malate against Myocardial Ischemia/Reperfusion Injury in Rats

    OpenAIRE

    Ding, Shiao; Yang, Yang; MEI, JU

    2016-01-01

    Objective. To investigate the protective effects of L-malate against myocardial ischemia/reperfusion (I/R) injury in rats. Methods. Male Sprague-Dawley rats were randomly assigned to the following groups: sham (sham), an ischemia/reperfusion (I/R) model group (model), an DMF pretreated group (DMF), and 5 L-malate pretreated groups (15, 60, 120, 240, or 480?mg/kg, gavage) before inducing myocardial ischemia. Plasma LDH, cTn-I, TNF-?, hs-CRP, SOD, and GSH-PX were measured 3?h later I/R. Areas o...

  7. Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution / Atividade de invertases e sacarose sintase em plantas de cafeeiro pulverizadas com solução de sacarose

    Scientific Electronic Library Online (English)

    José Carlos da, Silva; José Donizeti, Alves; Amauri Alves de, Alvarenga; Marcelo Murad, Magalhães; Dárlan Einstein do, Livramento; Daniela Deitos, Fries.

    Full Text Available Uma prática cuja eficiência não foi ainda comprovada cientificamente, é a pulverização dos cafeeiros com solução diluída de sacarose, como fonte de carbono para as plantas. Este trabalho visou estudar o efeito da pulverização de açúcar via folha nos teores endógenos de carboidratos e na atividade da [...] s enzimas invertases e sacarose sintase em mudas de cafeeiros (Coffea arabica L.) com baixo (baixo) e alto (normal) nível de reservas de carbono. As pulverizações ocorreram nas concentrações de 0,5 e 1% de sacarose utilizando-se água como testemunha. A aplicação de sacarose a 1% aumentou a concentração de açúcares solúveis totais (AST) em plantas depauperadas, como também aumentou as atividades das enzimas invertase ácida da parede, invertase ácida do vacúolo, invertase neutra do citosol e sacarose sintase. Em plantas com níveis normais de carboidratos não foi observada nenhuma alteração nos teores dos AST, como também nas atividades de tais enzimas. Independentemente dos tratamentos aplicados e do estado fisiológico das plantas, não foram observadas diferenças na transpiração e na condutância estomática, mostrando, desta forma, o controle estomático da transpiração. A fotossíntese foi estimulada a 0,5% e 1% em plantas depauperadas, o que não aconteceu com plantas normais. A pulverização de sacarose em mudas de cafeeiros só é eficiente tratando-se de plantas depauperadas na concentração de 1%. Abstract in english One management practice of which the efficiency has not yet been scientifically tested is spraying coffee plants with diluted sucrose solutions as a source of carbon for the plant. This paper evaluates the effect of foliar spraying with sugar on the endogenous level of carbohydrates and on the activ [...] ities of invertase and sucrose synthase in coffee (Coffea arabica L.) seedlings with reduced (low) and high (normal) levels of carbon reserve. The concentrations used were 0.5 and 1.0% sucrose, and water as a control. The use of sucrose at 1.0% caused an increase in the concentration of total soluble sugars in depauperate plants, as well as increased the activity of the following enzymes: cell wall and vacuole acid invertase, neutral cytosol invertase and sucrose synthase. In plants with high level of carbon reserve, no increments in total soluble sugar levels or in enzymatic activity were observed. Regardless of treatments or plants physiological state, no differences in transpiration or stomatal conductance were observed, demonstrating the stomatal control of transpiration. Photosynthesis was stimulated with the use of 0.5 and 1.0 % sucrose only in depauperate plants. Coffee seedling spraying with sucrose is only efficient for depauperate plants, at the concentration of 1.0%.

  8. Higher plant cellulose synthases

    OpenAIRE

    Richmond, Todd

    2000-01-01

    The sole function of cellulose synthases, which are found in plants bacteria, fungi, and animals, is to produce the biopolymer cellulose. Although no crystal structure has yet been solved, a considerable amount is known about their structure, function and evolution.

  9. Bacterial infection induces nitric oxide synthase in human neutrophils.

    OpenAIRE

    Wheeler, M A; Smith, S. D.; García-Cardeña, G; Nathan, C. F.; Weiss, R.M.; Sessa, W.C.

    1997-01-01

    The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric o...

  10. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. (Univ. of Iowa, Iowa City (United States) Western Michigan Univ., Kalamazoo (United States))

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

  11. Effect of black currant anthocyanins on the activation of endothelial nitric oxide synthase (eNOS) in vitro in human endothelial cells.

    Science.gov (United States)

    Edirisinghe, Indika; Banaszewski, Katarzyna; Cappozzo, Jack; McCarthy, Danielle; Burton-Freeman, Britt M

    2011-08-24

    Polyphenols are known to induce vasodilatory function via activation of the redox-sensitive phosphatidylinositol-3 (PI3)/protein kinase B (Akt) pathway. Black currant fruits have appreciable amounts of polyphenolic compounds including cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside, and delphinidin-3-O-rutinoside. It was hypothesized that black currant fruit extracts would cause activation of endothelial nitric oxide synthase (eNOS) through activation of redox-sensitive PI3 kinase/Akt signaling pathway. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations/times of black currant juice concentrates (Ben Gairn and Ben Hope) and the activation of Akt and eNOS was measured using immunoblotting. Vitamin C is also known to activate Akt and eNOS in in vitro models, and black currants are rich in vitamin C. Therefore, the effect of black currant extracts with and without coexisting vitamin C was investigated, using SPE columns to eliminate vitamin C content. The individual (and combined) effects of the major anthocyanins present in black currant juice samples with and without vitamin C were investigated and compared to the effects of the whole extract. Black currant juice samples (1 ?L/mL) significantly increased the phosphorylation of Akt (p-Akt) and eNOS (p-eNOS) (P Vitamin C alone significantly increased the p-Akt and p-eNOS (P vitamin C from black currant did not significantly affect p-Akt and p-eNOS compared to black currant with vitamin C. Assessment of individual anthocyanins also showed significant effects on p-Akt and p-eNOS. In summary, in the present study data suggested that black currant concentrates, Ben Gairn and Ben Hope, activated eNOS via Akt/PI3 kinase pathway in vitro in HUVECs and that the effect was not dependent on vitamin C. PMID:21761876

  12. Decreased Activity in Neuropathic Pain Form and Gene Expression of Cyclin-Dependent Kinase5 and Glycogen Synthase Kinase-3 Beta in Soleus Muscle of Wistar Male Rats

    Science.gov (United States)

    Rahmati, Masoud; Taherabadi, Seyed Jalal; Mehrabi, Mahmoud

    2015-01-01

    Background: The relationship between decreased activity/neuropathic pain and gene expression alterations in soleus muscle has remained elusive. Objectives: In this experimental study, we investigated the effects of decreased activity in neuropathic pain form on Cyclin-Dependent Kinase 5 (CDK5) and Glycogen Synthase Kinase-3 ? (GSK-3?) gene expression in soleus muscle of rats. Materials and Methods: Twelve male Wistar rats were randomly divided into three groups: (1) tight ligation of the L5 spinal nerve (SNL: n = 4); (2) sham surgery (Sham: n = 4), and (3) control (C: n = 4). The threshold to produce a withdrawal response to a mechanical and thermal stimulus was measured using von Frey filaments and radiation heat apparatus, respectively. Following 4 weeks after surgery, the left soleus muscle was removed and mRNA levels were determined by real-time Polymerase Chain Reaction (PCR). Results: Compared to control animals, L5 ligated animals developed mechanical and heat hypersensitivity during total period of study. Soleus muscle weight as well as CDK5 mRNA levels (less than ~ 0.4 fold) was decreased and GSK-3? mRNA levels (up to ~ 7 folds) increased in L5 ligated animals. Conclusions: These results showed enhanced muscle atrophy processes following peripheral nerve damage and might provide a useful approach to study underlying muscle mechanisms associated with clinical neuropathic pain syndromes. PMID:26290750

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

    Science.gov (United States)

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

    2015-11-01

    The biodiversity of plant endophytic fungi is enormous, numerous competent endophytic fungi are capable of providing different forms of fitness benefits to host plants and also could produce a wide array of bioactive natural products, which make them a largely unexplored source of novel compounds with potential bioactivity. In this study, we provided a first insights into revealing the diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff.) from China using rDNA-ITS phylogenetic analysis. Here, the potential of fungi in producing bioactive natural products was estimated based on the beta-ketosynthase detected in the polyketide synthase (PKS) gene cluster and on the bioassay of antagonistic activity against two rice phytopathogens Thanatephorus cucumeris and Xanthomonas oryzae. A total of 229 endophytic fungal strains were validated in 19 genera. Among the 24 representative strains, 13 strains displayedantagonistic activity against the phytopathogens. Furthermore, PKS genes were detected in 9 strains, indicating their potential for synthesising PKS compounds. Our study confirms the phylogenetic diversity of endophytic fungi in O. rufipogon G. and highlights that endophytic fungi are not only promising resources of biocontrol agents against phytopathogens of rice plants, but also of bioactive natural products and defensive secondary metabolites. PMID:26466878

  14. Glycogen synthase kinase 3? regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    International Nuclear Information System (INIS)

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 ? (GSK3?) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3? has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3? (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3? delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3? is required for the activation of NFAT during wound repair

  15. 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; Harholt, Jesper; Sørensen, Susanne Oxenbøll; Moller, Isabel Eva; Blaukopf, Claudia; Johansen, Bo Buchholt; Delotto, Robert; Pauly, Markus; Scheller, Henrik Vibe; Willats, William George Tycho

    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 sus...

  16. In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida.

    Science.gov (United States)

    Nagy, A H; Siddiqui, M O; Kocsis, Z G; Vida, G

    1980-07-01

    Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms. PMID:24301014

  17. Malate supplementation to beef cattle: effects on growth performance and rumen fermentation products

    Directory of Open Access Journals (Sweden)

    Stefano Vandoni

    2010-01-01

    Full Text Available Two trials were performed to evaluate the effects of malate supplementation on the growth performance as well as on ruminal pH and fermentation products of beef cattle. A total of 80 Charolaise bullocks were randomly allotted to one of the four experimental groups which included two experiments. The first experiment involved a control group (C fed with a corn silage based diet and a treated group (T fed the same diet supplemented with 20 g/head/day of malate. The second experiment involved a dry control group (DC fed with a dry diet (without corn silage and a treated group (DMS fed with the same diet supplemented with 20 g/head/day malate. Bullocks fed diets with corn silage and supplemented with malate demonstrated higher live weight in the transition, fattening (P<0.05 and finishing periods (P<0.10, and higher average daily gain in the fattening and finishing period (P<0.10 than the control group. In the second experiment no difference in growth performance were highlighted. In both trials no statistical difference was highlighted for ruminal fluid pH. In regards to fermentation products, the supplemented animals exhibited a higher propionic acid proportion, lower acetate:propionate ratio, and higher N-NH3 concentration than the controls for both trials.

  18. 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.

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

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-01

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities. Several major bottlenecks, among which the rareness of phenotypic NOS activity in meat-compatible CNS, need to be considered, which is seriously questioning the relevance of this pathway in fermented meats. PMID:25805616

  20. 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.

  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. Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

    Directory of Open Access Journals (Sweden)

    Hunter Randy

    2008-01-01

    Full Text Available Abstract Background Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-?-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS; however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1 the effect of the PPAR-? agonist pioglitazone on lipopolysaccharide (LPS-induced iNOS activity and nitric oxide (NO generation by microglia; (2 the differential role of p38 mitogen-activated protein kinase (p38 MAPK, c-Jun NH(2-terminal kinase (JNK, and phosphoinositide 3-kinase (PI3K on LPS-induced NO generation; and (3 the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. Methods Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-?, PI3K, and protein kinase B (Akt were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH-positive neurons were counted in mesencephalic neuron-microglia mixed cultures. Results Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-?, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase. Conclusion We demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-? activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

  3. 17?-Estradiol treatment inhibits breast cell proliferation, migration and invasion by decreasing MALAT-1 RNA level

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Ziyi [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China); Chen, Changjin [Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041 (China); Liu, Yu [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China); Wu, Chuanfang, E-mail: 879413966@qq.com [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China)

    2014-03-07

    Highlights: • E2 affects not only estrogen-receptor ? positive breast cells but also negative ones. • 100 nM E2 treatment affects breast cells proliferation, migration. • 100 nM E2 treatment functions in an estrogen-receptor ?-independent way. • E2 treatment decreases MALAT-1 RNA level by post-transcriptional regulation. - Abstract: Breast cancer cells, which express estrogen receptor ? (ER?), respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. But breast cancer cells without ER? show no effect on low concentration of estrogen treatment. Proliferation, migration and invasion of MCF10a, MCF7 and MB231 cells treated with low (1 nM) or high (100 nM) dose of 17?-Estradiol (E2) was performed. We identified the effects of E2 on these breast cell lines, and looked for the difference in the presence and absence of ER?. Specifically, we looked for the changes of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1), which is found extensively and highly expressed in several kinds of tumor cells, including breast carcinoma. It was observed that proliferation, migration and invasion of breast cells were greatly affected by high concentration E2 treatment and were not affected by low concentration E2 treatment in an ER? independent way. We found that the high concentration E2 treatment largely decreased MALAT-1 RNA level. Interestingly, MALAT-1 decreasing by knocking down showed similar effects on proliferation, migration and invasion. E2 treatment affects breast tumor or non-tumor cells proliferation, migration and invasion in an ER? -independent, but a dose-dependent way by decreasing the MALAT-1 RNA level.

  4. 17?-Estradiol treatment inhibits breast cell proliferation, migration and invasion by decreasing MALAT-1 RNA level

    International Nuclear Information System (INIS)

    Highlights: • E2 affects not only estrogen-receptor ? positive breast cells but also negative ones. • 100 nM E2 treatment affects breast cells proliferation, migration. • 100 nM E2 treatment functions in an estrogen-receptor ?-independent way. • E2 treatment decreases MALAT-1 RNA level by post-transcriptional regulation. - Abstract: Breast cancer cells, which express estrogen receptor ? (ER?), respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. But breast cancer cells without ER? show no effect on low concentration of estrogen treatment. Proliferation, migration and invasion of MCF10a, MCF7 and MB231 cells treated with low (1 nM) or high (100 nM) dose of 17?-Estradiol (E2) was performed. We identified the effects of E2 on these breast cell lines, and looked for the difference in the presence and absence of ER?. Specifically, we looked for the changes of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1), which is found extensively and highly expressed in several kinds of tumor cells, including breast carcinoma. It was observed that proliferation, migration and invasion of breast cells were greatly affected by high concentration E2 treatment and were not affected by low concentration E2 treatment in an ER? independent way. We found that the high concentration E2 treatment largely decreased MALAT-1 RNA level. Interestingly, MALAT-1 decreasing by knocking down showed similar effects on proliferation, migration and invasion. E2 treatment affects breast tumor or non-tumor cells proliferation, migration and invasion in an ER? -independent, but a dose-dependent way by decreasing the MALAT-1 RNA level

  5. 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)

  6. Zinc-homocysteine binding in cobalamin-dependent methionine synthase and its role in the substrate activation: DFT, ONIOM, and QM/MM molecular dynamics studies.

    Science.gov (United States)

    Abdel-Azeim, Safwat; Li, Xin; Chung, Lung Wa; Morokuma, Keiji

    2011-11-30

    Cobalamin-dependent methionine synthase (MetH) is an important metalloenzyme responsible for the biosynthesis of methionine. It catalyzes methyl transfer from N(5)-methyl-tetrahydrofolate to homocysteine (Hcy) by using a zinc ion to activate the Hcy substrate. Density functional theory (B3LYP) calculations on the active-site model in gas phase and in a polarized continuum model were performed to study the Zn coordination changes from the substrate-unbound state to the substrate-bound state. The protein effect on the Zn(2+) coordination exchange was further investigated by ONIOM (B3LYP:AMBER)-ME and EE calculations. The Zn(2+)-coordination exchange is found to be highly unfavorable in the gas phase with a high barrier and endothermicity. In the water solution, the reaction becomes exothermic and the reaction barrier is drastically decreased to about 10.0 kcal/mol. A considerable protein effect on the coordination exchange was also found; the reaction is even more exothermic and occurs without barrier. The enzyme was suggested to constrain the zinc coordination sphere in the reactant state (Hcy-unbound state) more than that in the product state (Hcy-bound state), which promotes ligation of the Hcy substrate. Molecular dynamics simulations using molecular mechanics (MM) and PM3/MM potentials suggest a correlation between the flexibility of the Zn(2+)-binding site and regulation of the enzyme function. Directed in silico mutations of selected residues in the active site were also performed. Our studies support a dissociative mechanism starting with the Zn-O(Asn234) bond breaking followed by the Zn-S((Hcy)) bond formation; the proposed associative mechanism for the Zn(2+)-coordination exchange is not supported. PMID:21837727

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Ali Ghasemzadeh

    2014-10-01

    Full Text Available In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old. The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74 was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW] and total phenolic (TP (18.21 mg/g DW were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW and gallic acid (5.96 mg/g DW were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50 values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP assay showed a higher activity in 6-month-old buds (488 ?M of Fe(II/g than in 1-year-old buds (453 ?M of Fe(II/g, in contrast to the DPPH result. Significant correlations (p < 0.05 were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity against HeLa cancer cells with IC50 value of 56.8 µg/mL. These results indicate that early harvesting of snake grass (6-month-old may yield increased concentrations of secondary metabolites, which are potent antioxidant compounds.

  9. Smoking cessation early in pregnancy and birth weight, length, head circumference, and endothelial nitric oxide synthase activity in umbilical and chorionic vessels: an observational study of healthy singleton pregnancies

    DEFF Research Database (Denmark)

    Andersen, Malene R; Simonsen, Ulf; Uldbjerg, Niels; Stender, Steen; Aalkjaer, Christian

    2009-01-01

    BACKGROUND: Reduced production of the vasodilator nitric oxide (NO) in fetal vessels in pregnant smokers may lower the blood flow to the fetus and result in lower birth weight, length, and head circumference. The present study measured endothelial NO synthase (eNOS) activity in fetal umbilical and chorionic vessels from nonsmokers, smokers, and ex-smokers and related the findings to the fetal outcome. METHODS AND RESULTS: Of 266 healthy, singleton pregnancies, 182 women were nonsmokers, 43 were ...

  10. The tomato terpene synthase gene family.

    Science.gov (United States)

    Falara, Vasiliki; Akhtar, Tariq A; Nguyen, Thuong T H; Spyropoulou, Eleni A; Bleeker, Petra M; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E; Schilmiller, Anthony L; Last, Robert L; Schuurink, Robert C; Pichersky, Eran

    2011-10-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  11. The Pb-hyperaccumulator aquatic fern Salvinia minima Baker, responds to Pb(2+) by increasing phytochelatins via changes in SmPCS expression and in phytochelatin synthase activity.

    Science.gov (United States)

    Estrella-Gómez, N; Mendoza-Cózatl, D; Moreno-Sánchez, R; González-Mendoza, D; Zapata-Pérez, O; Martínez-Hernández, A; Santamaría, J M

    2009-03-01

    The relationship between accumulation of Pb(2+) and the activation of chelation and metal sequestration mechanisms mediated by phytochelatins (PC) was analyzed in the Pb(2+) hyperaccumulator aquatic fern Salvinia minima, after exposure to 40microM Pb(NO(3))(2). The tissue accumulation pattern of lead and the phytochelatin biosynthesis responses were analyzed in both, S. minima submerged root-like modified fronds (here named "roots"), and in its aerial leaf-like fronds ("leaves"). S. minima roots accumulated a significantly higher concentrations of Pb(+2) than leaves did. Accumulation of Pb(2+) in roots was bi-phasic with a first uptake phase reached after 3h exposure and a second higher uptake phase reached after 24h exposure. In leaves, a single delayed, smaller uptake phase was attained only after 9h of exposure. In roots lead accumulation correlated with an increased phytochelatin synthase (PCS) activity and an enhanced PC production. A higher proportion of polymerized PC(4) was observed in both tissues of exposed S. minima plants relative to unexposed ones, although a higher concentration of PC(4) was found in roots than in leaves. PCS activity and Pb(2+) accumulation was also higher in roots than in leaves. The expression levels of the S. minima PCS gene (SmPCS), in response to Pb(2+) treatment, were also evaluated. In S. minima leaves, the accumulation of Pb(2+) correlated with a marked increase in expression of SmPCS, suggesting a transcriptional regulation in the PCS activation and PC accumulation in this S. minima tissue. However, in roots, the basal expression of SmPCS was down-regulated after Pb(2+) treatment. This fact did not correlate with the later but strong increase in both, PCS activity and PC production; suggesting that the PC biosynthesis activation in S. minima roots occurs only by post-translational activation of PCS. Taken together, our data suggest that the accumulation of PC in S. minima is a direct response to Pb(2+) accumulation, and phytochelatins do participate as one of the mechanism to cope with Pb(2+) of this Pb-hyperaccumulator aquatic fern. PMID:19110323

  12. Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-?B and JNK/p38 MAPK activation pathways

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chih

    2011-05-01

    Full Text Available Abstract Objectives Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl-5-hydroxy-2-(4-hydroxyphenyl-7-methoxy-4H-chromen-4-one, one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS-activated macrophages. Methods We used nitrate and prostaglandin E2 (PGE2 assays to examine inhibitory effect of aciculatin on nitric oxide (NO and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis. Results Aciculatin remarkably decreased the LPS (1 ?g/mL-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 ?M. Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-?B activation, an effect highly correlated with its inhibitory effect on LPS-induced I?B kinase (IKK activation, I?B degradation, NF-?B phosphorylation, nuclear translocation and binding of NF-?B to the ?B motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs. Conclusion Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-?B and JNK/p38 MAPK pathways.

  13. Suppression of autophagy and activation of glycogen synthase kinase 3beta facilitate the aggregate formation of tau.

    Science.gov (United States)

    Kim, Song-In; Lee, Won-Ki; Kang, Sang-Soo; Lee, Sue-Young; Jeong, Myeong-Ja; Lee, Hee Jae; Kim, Sung-Soo; Johnson, Gall V W; Chun, Wanjoo

    2011-04-01

    Neurofibrillary tangle (NFT) is a characteristic hallmark of Alzheimer's disease. GSK3? has been reported to play a major role in the NFT formation of tau. Dysfunction of autophagy might facilitate the aggregate formation of tau. The present study examined the role of GSK3?-mediated phosphorylation of tau species on their autophagic degradation. We transfected wild type tau (T4), caspase-3-cleaved tau at Asp421 (T4C3), or pseudophosphorylated tau at Ser396/Ser404 (T4-2EC) in the presence of active or enzyme-inactive GSK3?. Trehalose and 3-methyladenine (3-MA) were used to enhance or inhibit autophagic activity, respectively. All tau species showed increased accumulation with 3-MA treatment whereas reduced with trehalose, indicating that tau undergoes autophagic degradation. However, T4C3 and T4-2EC showed abundant formation of oligomers than T4. Active GSK3? in the presence of 3-MA resulted in significantly increased formation of insoluble tau aggregates. These results indicate that GSK3?-mediated phosphorylation and compromised autophagic activity significantly contribute to tau aggregation. PMID:21660151

  14. Sensitivity of the plant vacuolar malate channel to pH, Ca2+ and anion-channel blockers.

    OpenAIRE

    Pantoja, O; Smith, JA

    2002-01-01

    The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in w...

  15. Temperature Response of Isoprene Emission in Vivo Reflects a Combined Effect of Substrate Limitations and Isoprene Synthase Activity: A Kinetic Analysis1

    Science.gov (United States)

    Rasulov, Bahtijor; Hüve, Katja; Bichele, Irina; Laisk, Agu; Niinemets, Ülo

    2010-01-01

    The responses of isoprene emission rate to temperature are characterized by complex time-dependent behaviors that are currently not entirely understood. To gain insight into the temperature dependencies of isoprene emission, we studied steady-state and transient responses of isoprene emission from hybrid aspen (Populus tremula × Populus tremuloides) leaves using a fast-response gas-exchange system coupled to a proton-transfer reaction mass spectrometer. A method based on postillumination isoprene release after rapid temperature transients was developed to determine the rate constant of isoprene synthase (IspS), the pool size of its substrate dimethylallyldiphosphate (DMADP), and to separate the component processes of the temperature dependence of isoprene emission. Temperature transients indicated that over the temperature range 25°C to 45°C, IspS was thermally stable and operated in the linear range of its substrate DMADP concentration. The in vivo rate constant of IspS obeyed the Arrhenius law, with an activation energy of 42.8 kJ mol?1. In contrast, steady-state isoprene emission had a significantly lower temperature optimum than IspS and higher activation energy. The reversible temperature-dependent decrease in the rate of isoprene emission between 35°C and 44°C was caused by decreases in DMADP concentration, possibly reflecting reduced pools of energetic metabolites generated in photosynthesis, particularly of ATP. Strong control of isoprene temperature responses by the DMADP pool implies that transient temperature responses under fluctuating conditions in the field are driven by initial DMADP pool size as well as temperature-dependent modifications in DMADP pool size during temperature transients. These results have important implications for the development of process-based models of isoprene emission. PMID:20837700

  16. Temperature response of isoprene emission in vivo reflects a combined effect of substrate limitations and isoprene synthase activity: a kinetic analysis.

    Science.gov (United States)

    Rasulov, Bahtijor; Hüve, Katja; Bichele, Irina; Laisk, Agu; Niinemets, Ulo

    2010-11-01

    The responses of isoprene emission rate to temperature are characterized by complex time-dependent behaviors that are currently not entirely understood. To gain insight into the temperature dependencies of isoprene emission, we studied steady-state and transient responses of isoprene emission from hybrid aspen (Populus tremula × Populus tremuloides) leaves using a fast-response gas-exchange system coupled to a proton-transfer reaction mass spectrometer. A method based on postillumination isoprene release after rapid temperature transients was developed to determine the rate constant of isoprene synthase (IspS), the pool size of its substrate dimethylallyldiphosphate (DMADP), and to separate the component processes of the temperature dependence of isoprene emission. Temperature transients indicated that over the temperature range 25°C to 45°C, IspS was thermally stable and operated in the linear range of its substrate DMADP concentration. The in vivo rate constant of IspS obeyed the Arrhenius law, with an activation energy of 42.8 kJ mol(-1). In contrast, steady-state isoprene emission had a significantly lower temperature optimum than IspS and higher activation energy. The reversible temperature-dependent decrease in the rate of isoprene emission between 35°C and 44°C was caused by decreases in DMADP concentration, possibly reflecting reduced pools of energetic metabolites generated in photosynthesis, particularly of ATP. Strong control of isoprene temperature responses by the DMADP pool implies that transient temperature responses under fluctuating conditions in the field are driven by initial DMADP pool size as well as temperature-dependent modifications in DMADP pool size during temperature transients. These results have important implications for the development of process-based models of isoprene emission. PMID:20837700

  17. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different cellular compartments and suggest that NO may have specific actions in relation to its site of production. The localization of type I NO synthase in the vicinity of mitochondria supports a specific action of NO on mitochondrial respiration, whereas the localization of type III NO synthase in vascular endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle.

  18. Increased nitric oxide synthase activity despite lack of response to endothelium-dependent vasodilators in postischemic acute renal failure in rats.

    Science.gov (United States)

    Conger, J; Robinette, J; Villar, A; Raij, L; Shultz, P

    1995-01-01

    Lack of response to endothelium-dependent vasodilators generally has been considered to be evidence for decreased nitric oxide synthase (NOS) activity and NO generation after ischemic or hypoxic injury to vital organs including the kidney. In this study, renal blood flow (RBF) responses to endothelium-dependent vasodilators acetylcholine and bradykinin and the endothelium-independent vasodilator prostacyclin, the nonselective NOS inhibitor L-NAME (without and with L-arginine), the inducible NOS inhibitor aminoguanidine, and the NO-donor sodium nitroprusside were examined in 1-wk norepinephrine-induced (NE) and sham-induced acute renal failure (ARF) rats. Compared with sham-ARF, there was no increase in RBF to intrarenal acetylcholine and bradykinin, but a comparable RBF increase to prostacyclin in NE-ARF kidneys. However, there was a significantly greater decline in RBF to intravenous L-NAME in NE- than sham-ARF rats (-65 +/- 8 vs. -37 +/- 5%, P < 0.001) which was completely blocked by prior L-arginine infusion. There was no change in RBF to the inducible NOS specific inhibitor aminoguanidine. Unlike sham-ARF, there was no increase in RBF to intrarenal sodium nitroprusside in NE-ARF. Immunohistochemistry and immunofluorescence detection of constitutive (c) NOS using mouse monoclonal antibody were carried out to positively determine the presence of cNOS in NE-ARF. 90% of renal resistance vessels showed evidence of endothelial cNOS in both sham- and NE-ARF. Taken together, results of these experiments are consistent with the conclusion that NOS/NO activity is, in fact, maximal at baseline in 1-wk NE-ARF and cannot be increased further by exogenous stimuli of NOS activity. The increased NOS is likely of the constitutive form and of endothelial origin. It is suggested that the increased NOS activity is in response to ischemia-induced renal vasoconstrictor activity. Attenuated response to endothelium-dependent vasodilators cannot be interpreted only as evidence for decreased NOS activity. Images PMID:7542287

  19. Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

    Science.gov (United States)

    Hayashi, H; Huang, P; Inoue, K; Hiraoka, N; Ikeshiro, Y; Yazaki, K; Tanaka, S; Kushiro, T; Shibuya, M; Ebizuka, Y

    2001-12-01

    An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols. PMID:11733028

  20. Inhibition of glycogen synthase kinase 3beta activity with lithium prevents and attenuates paclitaxel-induced neuropathic pain

    OpenAIRE

    GAO, MEI; Yan, Xisheng; Weng, Han-Rong

    2013-01-01

    Paclitaxel (taxol) is a first-line chemotherapy-drug used to treat many types of cancers. Neuropathic pain and sensory dysfunction are the major toxicities, which are dose-limiting and significantly reduce the quality of life in patients. Two known critical spinal mechanisms underlying taxol-induced neuropathic pain are an increased production of pro-inflammatory cytokines including interleukin-1? (IL-1?) and suppressed glial glutamate transporter activities. In this study, we uncovered that ...

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

    OpenAIRE

    Henard, Calvin A.; Vázquez-Torres, Andrés

    2012-01-01

    In coordination with the ppGpp alarmone, the RNA polymerase regulatory protein DksA controls the stringent response of eubacteria, negatively regulating transcription of translational machinery and directly activating amino acid promoters and de novo amino acid biosynthesis. Given the effects of nitric oxide (NO) on amino acid biosynthetic pathways and the intimate relationship of DksA with amino acid synthesis and transport, we tested whether DksA contributes to the resistance of Salmonella ...

  2. 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.

  3. Geranyl diphosphate synthase from mint

    Science.gov (United States)

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

    1999-01-01

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

  4. Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

    OpenAIRE

    Ro, Y T; Eom, C Y; Song, T.; Cho, J W; Kim, Y.M.

    1997-01-01

    Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. Th...

  5. 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.

  6. Changes in phytochemical synthesis, chalcone synthase activity and pharmaceutical qualities of sabah snake grass (Clinacanthus nutans L.) in relation to plant age.

    Science.gov (United States)

    Ghasemzadeh, Ali; Nasiri, Alireza; Jaafar, Hawa Z E; Baghdadi, Ali; Ahmad, Izham

    2014-01-01

    In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old). The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74) was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF) production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW]) and total phenolic (TP) (18.21 mg/g DW) were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW) and gallic acid (5.96 mg/g DW) were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50) values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP) assay showed a higher activity in 6-month-old buds (488 ?M of Fe(II)/g) than in 1-year-old buds (453 ?M of Fe(II)/g), in contrast to the DPPH result. Significant correlations (p snake grass (6-month-old) may yield increased concentrations of secondary metabolites, which are potent antioxidant compounds. PMID:25361426

  7. 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.

  8. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

    OpenAIRE

    Tzeng, E.; Billiar, T R; Robbins, P D; Loftus, M; Stuehr, D. J.

    1995-01-01

    Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absen...

  9. 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.

  10. 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.

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

    Science.gov (United States)

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

    2011-04-01

    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 ? 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? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

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

    International Nuclear Information System (INIS)

    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. 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. 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. 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

  13. Lupus Nephritis: Glycogen Synthase Kinase 3β Promotion of Renal Damage Through Activation of the NLRP3 Inflammasome in Lupus-Prone Mice

    Science.gov (United States)

    Zhao, Jijun; Wang, Hongyue; Huang, Yuefang; Zhang, Hui; Wang, Shuang; Gaskin, Felicia; Yang, Niansheng; Fu, Shu Man

    2015-01-01

    Objective Glycogen synthase kinase 3β (GSK-3β) has been demonstrated to be involved in immune and inflammatory responses via multiple signaling pathways, leading to the production of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β in the pathogenesis of lupus nephritis in 2 mouse models. Methods Thiadiazolidinone 8 (TDZD-8), a selective inhibitor of GSK-3β, was administered intraperitoneally to 12-week-old MRL/lpr mice for 8 weeks or to 22-week-old (NZB × NZW)F1 mice for 12 weeks. The expression of GSK-3β and NLRP3 inflammasome components was analyzed. Proteinuria, biochemical parameters, proinflammatory cytokines, anti–double-stranded DNA (anti-dsDNA) antibody levels, and renal pathology were examined. In vitro, the effect of GSK-3β–directed small interfering RNA (siRNA) on NLRP3 inflammasome activation was evaluated in bone marrow–derived macrophages (BMMs) from the mice and in the J774A.1 macrophage cell line. Results The incidence of severe proteinuria and renal inflammation was significantly attenuated in both models, with a significant reduction in anti-dsDNA antibody production, immune complex deposition in the kidney, and circulating proinflammatory cytokine levels. TDZD-8 inhibited the activation of GSK-3β and caspase 1, with a concomitant decrease in interleukin-1β (IL-1β) synthesis. In vitro, GSK-3β siRNA transfection of mouse BMMs and the J774A.1 cell line with GSK-3β siRNA inhibited the expression of GSK-3β, the activation of caspase 1, and the production of IL-1β. Conclusion These results show that GSK-3β promotes lupus nephritis at least partly by activating the NLRP3/IL-1β pathway. The linking of GSK-3β to the NLRP3/IL-1β pathway is a novel observation in our study. Our results suggest that the GSK-3β/NLRP3/IL-1β pathway may be a potential therapeutic target for lupus in humans. PMID:25512114

  14. Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression.

    Science.gov (United States)

    Mann, Giovanni E; Rowlands, David J; Li, Francois Y L; de Winter, Patricia; Siow, Richard C M

    2007-07-15

    The endothelium plays a key role in the maintenance of vascular homeostasis, and increased oxidative stress in vascular disease leads to reduced nitric oxide bioavailability and impaired endothelium-dependent relaxation of resistance vessels. Although epidemiological evidence suggests that diets containing high amounts of natural antioxidants afford protection against coronary heart disease (CHD), antioxidant supplementation trials have largely reported only marginal health benefits. There is controversy concerning the cardiovascular benefits of prolonged estrogen/progestin or soy isoflavone therapy for postmenopausal women and patients with an increased risk of CHD. Research on the potential health benefits of soy isoflavones and other polyphenols contained in red wine, green and black tea and dark chocolate developed rapidly during the 1990's, and recent clinical trials and studies in animal models and cultured endothelial cells provide important and novel insights into the mechanisms by which dietary polyphenols afford protection against oxidative stress. In this review, we highlight that NO and reactive oxygen radicals may mediate dietary polyphenol induced activation of Nrf2, which in turn triggers antioxidant response element (ARE) driven transcription of phase II detoxifying and antioxidant defense enzymes in vascular cells. PMID:17498676

  15. LncRNA MALAT1 overexpression is an unfavorable prognostic factor in human cancer: evidence from a meta-analysis

    OpenAIRE

    Zhang, Jun; Zhang, Bingya; Wang, Tiejun; WANG, HONGYONG

    2015-01-01

    Long non-coding RNAs (lncRNAs) have been suggested to serve as an important role in tumor development and progression. The aim of this study was to analyse the association between lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and cancer patients’ overall survival. We systematically and carefully searched the studies from electronic databases and seriously identified according to eligibility criteria. The correlation between lncRNA MALAT1 expression and overall surviva...

  16. Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

    International Nuclear Information System (INIS)

    Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control ?9?1 signaling. Our recent studies have clearly demonstrated the role of ?9?1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of ?9?1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that ?9?1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced ?9?1 integrin and iNOS levels

  17. delta-Aminolevulinic acid synthase from Euglena gracilis.

    OpenAIRE

    Beale, S I; FOLEY, T; Dzelzkalns, V

    1981-01-01

    delta-Aminolevulinic acid (ALA) synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] activity was detected in cell extracts of the unicellular green flagellate alga Euglena gracilis. The enzyme was identified by substrate and cofactor requirements, and activity was proportional to number of cells extracted and duration of incubation. The incubation product was spectrophotometrically and chromatographically identical to ALA. ALA synthase activity is present in t...

  18. Metabolic fingerprint of ischaemic cardioprotection: importance of the malate-aspartate shuttle

    DEFF Research Database (Denmark)

    Nielsen, Torsten Toftegård; Støttrup, Nicolaj Brejnholt; Løfgren, Bo; Bøtker, Hans Erik

    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 ...

  19. STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE

    Directory of Open Access Journals (Sweden)

    T. Femlin Blessia

    2012-02-01

    Full Text Available Polyhydroxybutyrate (PHB is a polyhydroxyalkanoate (PHA, a polymer belonging to polyesters class and is composed of hydroxy fatty acids. PHB is produced by microorganisms apparently in response to conditions of physiological stress. PHB synthases are the key enzymes of PHB biosynthesis. The PHB synthases obtained from Chromobacterium violaceum, belongs to the class I PHA synthases. Due to the limited structural information of PHB synthase, its functional properties including catalysis are unknown. Therefore, this study seeks to investigate the structural and functional properties of PHB synthase (phaC by predicting its three dimensional structure using bioinformatics methods. Present 15 ns molecular dynamics study provides an overall insight about some of the parameters such as energy, RMSD (Root Mean Square Deviation, SASA (Solvent Accessible Surface Area, hydrogen bonds, etc., Protein-protein docking reveals the binding mode of the protein in the active dimer state.

  20. Characterization of the immunogenicity and pathogenicity of malate dehydrogenase in Brucella abortus.

    Science.gov (United States)

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Sun, Xiaoqing; Wang, Shaohui; Ding, Chan; Yu, Shengqing

    2014-07-01

    Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. abortus S2308 was cloned, sequenced and expressed. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein. In addition, recombinant MDH showed sero-reactivity with 30 individual bovine B. abortus-positive sera by enzyme-linked immunosorbent assay, indicates that MDH may be used as a candidate marker for sero-diagnosis of brucellosis. Furthermore, MDH exhibits fibronectin and plasminogen-binding ability in immunoblotting assay. Inhibition assays on HeLa cells demonstrated that rabbit anti-serum against MDH significantly reduced both bacterial adherence and invasion abilities (p < 0.05), suggesting that MDH play a role in B. abortus colonization. Our results indicated that MDH is not only an immunogenic protein, but is also related to bacterial pathogenesis and may act as a new virulent factor, which will benefit for further understanding the MDH's roles in B. abortus metabolism, pathogenesis and immunity. PMID:24609497

  1. 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.

  2. 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.

  3. Catalase induces the expression of inducible nitric oxide synthase through activation of NF-kappaB and PI3K signaling pathway in Raw 264.7 cells.

    Science.gov (United States)

    Jang, Byeong-Churl; Paik, Ji-Hye; Kim, Sang-Pyo; Bae, Jae-Hoon; Mun, Kyo-Chul; Song, Dae-Kyu; Cho, Chi-Heum; Shin, Dong-Hoon; Kwon, Taeg Kyu; Park, Jong-Wook; Park, Jong-Gu; Baek, Won-Ki; Suh, Min-Ho; Lee, Soo Hwan; Baek, Suk-Hwan; Lee, In-Seon; Suh, Seong-Il

    2004-12-01

    It has been reported that macrophages produce substantial amounts of nitrite and nitrate after addition of catalase, but the mechanism associated remains unclear. In present study, we investigated whether catalase modulates the expression of inducible nitric oxide synthase (iNOS), an enzyme that produces nitric oxide. Exposure of Raw 264.7 macrophages (Raw cells) to catalase induced high expression of iNOS mRNA as well as protein with enzymatic activity. Data of mechanical analyses, such as iNOS promoter-driven luciferase assay and actinomycin D chase experiments demonstrated that the induction was due to increased iNOS transcription and post-transcriptional iNOS mRNA stability. Of interest, catalase-induced iNOS protein expression was abrogated through inactivation of NF-kappaB pathway by MG132 or BAY 11-7085 and PI3K pathway by LY294002 or wortmannin, respectively. In particular, blockage of PI3K pathway by LY294002 down-regulated iNOS transcription and steady-state iNOS mRNA levels as well as iNOS mRNA stability induced by catalase, suggesting regulation of PI3K pathway in catalase-induced iNOS expression at the levels of iNOS transcription, steady-state mRNA status, and mRNA stability. Additional cell culture works in different types of cells indicated that iNOS expression by catalase might be cell type-specific, based on the facts that catalase induced iNOS expression in BV2 microglial macrophage-like cells, but not in HT-29 or A549, human colon or lung cancer epithelial-like cells. Together, these results demonstrate for the first time that catalase induces iNOS expression in Raw cells, which seems to be associated with the increase of iNOS transcription and mRNA stability as well as the activation of NF-kappaB and PI3K signaling pathways. PMID:15498507

  4. 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.

  5. Evidence for mediation of L-2-chloropropionic acid-induced delayed neuronal cell death by activation of a constitutive nitric oxide synthase.

    OpenAIRE

    Widdowson, P. S.; Farnworth, M.; Moore, R B; Dunn, D.; Wyatt, I

    1996-01-01

    1. Delayed neuronal cell death elicited by excess excitatory amino acid concentrations has been strongly implicated in many neurological disorders including head trauma, stroke, motor neurone disease and Huntington's disease. We have used the neurotoxin, L-2-chloropropionic acid (L-CPA) to model cellular events in vivo leading to delayed neuronal cell loss which is confined to the cerebellar cortex and can be prevented by inhibitors of nitric oxide synthase such as NG-nitro-L-arginine methyl ...

  6. Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors.

    Science.gov (United States)

    Shen, Yuefei; Zhang, Shiyi; Zhang, Fuwu; Loftis, Alexander; Pavía-Sanders, Adriana; Zou, Jiong; Fan, Jingwei; Taylor, John-Stephen A; Wooley, Karen L

    2013-10-18

    A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury. PMID:23999874

  7. 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.

  8. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids; Hansen, Frederik; Overgaard, Michael Toft; Giese, Henriette; Søndergaard, Teis

    2014-01-01

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been described, even fewer from fungi and none from Fusarium species. Multidomain proteins can be quite challenging to work with, which is why the project intends to solve the 3D-structures of single domains of P...

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

    OpenAIRE

    Kankaanranta Hannu; Sareila Outi; Lahti Aleksi; Moilanen Eeva

    2006-01-01

    Abstract Background Nitric oxide (NO) is an inflammatory mediator, which acts as a cytotoxic agent and modulates immune responses and inflammation. p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is activated by chemical and physical stress and regulates immune responses. Previous studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli. The aim of the present study was to investigate the mechanisms involved in the regulation of ...

  10. 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.

  11. 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.

  12. 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

  13. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase ???? heterotetramer. Plants have evolved multiple ? (TSA and ? (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase ?-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the ?-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native ?-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as ?-subunit in this complex.

  14. Sunitinib Malate plus Lomustine for Patients with Temozolomide-refractory Recurrent Anaplastic or Low-grade Glioma.

    Science.gov (United States)

    Duerinck, Johnny; Du Four, Stephanie; Sander, Wilhelm; Van Binst, Anne-Marie; Everaert, Hendrik; Michotte, Alex; Hau, Peter; Neyns, Bart

    2015-10-01

    Tyrosine kinase signaling through the vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor- ? (PDGFR-?) and KIT cell surface receptors mediates neo-angiogenesis and contributes to cancer cell survival in recurrent anaplastic and low-grade glioma. Thirteen patients with temozolomide-refractory recurrent anaplastic or low-grade glioma were treated with sunitinib malate, a small-molecule tyrosine kinase inhibitor of the VEGFR, PDGFR, and KIT receptors, in combination with lomustine. The most frequent grade 3 and 4 adverse events were fatigue, thrombocytopenia, neutropenia and lymphopenia. The best objective tumor response by Response Assessment in Neuro-Oncology (RANO) criteria was one complete response, one unconfirmed partial response and three cases of stable disease. The median progression-free survival was 1.8 months (95% confidence interval=1.0-2.7 months) with 6-month progression-free survival of 15% (95% confidence interval=0-35%). The median overall survival was 6.7 months (95% confidence interval=0.7-12 months). The investigated combination regimen of sunitinib and lomustine is well-tolerated but insufficiently active to warrant further investigation in an unselected population of patients with temozolomide-refractory recurrent anaplastic and low-grade glioma. PMID:26408725

  15. NADP-malate Dehydrogenase Isoforms of Wheat Leaves under Drought: Their Localization, and Some physicochemical and Kinetic Properties

    Directory of Open Access Journals (Sweden)

    H.G. Babayev

    2015-09-01

    Full Text Available Changes in sub-cellular localization, isoenzyme spectrum and kinetic characteristics of NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.82 in Triticum durum Desf. genotypes with contrasting drought tolerance have been studied. In chloroplast and cytosol fractions of mesophyll cells of wheat flag leaves 70-75% and 25-30% of the total NADP-MDH activity were found to be localized, respectively. Three isoforms of NADP-MDH with molecular weights of 66, 74 and 86 kDa were revealed in the chloroplast fraction, whereas in the cytosolic fraction molecular weights of the isoenzymes were found to be 42, 66 and 74 kDa. Drought caused the formation of a new 90 kDa isoform of the enzyme in the chloroplast fraction in anthesis phase of ontogenesis. In the drought-tolerant genotype the appearance of the new isoform in the chloroplast fraction was accompanied by a more rapid increase in Km and Vmax contrary to the chloroplast fraction of the drought-sensitive genotype manifesting a slight decrease in these parameters, suggesting one of the adaptive traits in forming drought tolerance in C3 plants.

  16. Long non-coding RNA MALAT-1 is downregulated in preeclampsia and regulates proliferation, apoptosis, migration and invasion of JEG-3 trophoblast cells.

    Science.gov (United States)

    Chen, Haiying; Meng, Tao; Liu, Xuemin; Sun, Manni; Tong, Chunxiao; Liu, Jing; Wang, He; Du, Juan

    2015-01-01

    Long non-coding RNA (lncRNA), as a newly identified subset of the transcriptome, has been implicated in a variety of physiological and pathological processes. Metastasis associated lung adenocarcinoma transcript-1 (MALAT-1), a lncRNA that was initially detected in the metastatic lung cancer, was reported to be overexpressed in placenta previa increta/percreta (I/P), which is caused by excessive trophoblast invasion. However, the role of MALAT-1 in the regulation of trophoblast behavior is not fully understood. In this study, we first examined the expression of MALAT-1 in the placentas from the patients with preeclampsia, the pathology of which is associated with inadequate trophoblast invasion, and found that the expression of MALAT-1 was downregulated in the preeclamptic placentas as compared to the normal placentas. We further investigated the function of MALAT-1 in JEG-3 trophoblast cell line using short interfering RNA (siRNA) against MALAT-1 transcripts. Silencing of MALAT-1 in JEG-3 cells suppressed proliferation and induced cell cycle arrest at G0/G1 phase. Reduced expression of MALAT-1 by RNA interference resulted in enhanced apoptosis in JEG-3 cells, accompanied with elevated levels of the pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). Moreover, the migration rate and the invasiveness of JEG-3 cells were suppressed when MALAT-1 was downregulated. In summary, our results suggest that MALAT-1 may play an important role in the regulation of proliferation, cell cycle, apoptosis, migration and invasion of trophoblast cells, and under-expression of MALAT-1 during early placentation may be involved in the pathogenesis of preeclampsia. PMID:26722461

  17. 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 of...

  18. Mechanisms of angiotensin II-mediated activation of aldosterone synthase in H295R human adrenocortical and rat adrenal glomerulosa cells

    OpenAIRE

    Szekeres, Mária; Turu, Gábor; Orient, Anna; Szalai, Bence; Süpeki, Katinka; Cserz?, Miklós; Várnai, Péter; Hunyady, László

    2009-01-01

    Abstract In adrenal zona glomerulosa cells angiotensin II (Ang II) is a key regulator of steroidogenesis. Our purpose was to compare the mechanisms of Ang II-induced changes in the expression level of early transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1) genes, and the CYP11B2 gene encoding aldosterone synthase in H295R human adrenocortical tumor cells and in primary rat adrenal glomerulosa cells. Real-time PCR studies have demonstrated that Ang II increased the expression le...

  19. 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 on...

  20. High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells.

    OpenAIRE

    Todoroki, S.; Goto, S.; Urata, Y; Komatsu, K.; Sumikawa, K.; T. Ogura; Matsuda, I.; Kondo, T.

    1998-01-01

    Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6)...

  1. In Vitro Activity of a New Oral Glucan Synthase Inhibitor (MK-3118) Tested against Aspergillus spp. by CLSI and EUCAST Broth Microdilution Methods

    OpenAIRE

    Pfaller, Michael A.; Messer, Shawn A.; Motyl, Mary R.; Ronald N Jones; Castanheira, Mariana

    2013-01-01

    MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ?4 ?g/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC90)/MIC90 values (?g/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fum...

  2. The cellulose synthase gene of Dictyostelium

    OpenAIRE

    Blanton, Richard L.; Fuller, Danny; Iranfar, Negin; Grimson, Mark J.; Loomis, William F.

    2000-01-01

    Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum. We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development. Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again. No spores or stalk ce...

  3. 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, thu...

  4. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  5. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  6. 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

  7. 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.

  8. Helper component-proteinase enhances the activity of 1-deoxy-D -xylulose-5-phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y-infected tobacco.

    Science.gov (United States)

    Li, Heng; Ma, Dongyuan; Jin, Yongsheng; Tu, Yayi; Liu, Liping; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2015-10-01

    Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships. PMID:25736930

  9. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Science.gov (United States)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  10. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Science.gov (United States)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  11. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  12. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

    Directory of Open Access Journals (Sweden)

    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  13. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes): isolated isoforms and kinetics properties

    Scientific Electronic Library Online (English)

    Maria Regina de, Aquino-Silva; Maria Luiza Barcellos, Schwantes; Flavia Simone, Munin; Arno Rudi, Schwantes; Silvana Pereira dos, Santos.

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were [...] detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  14. Mitochondrial malate dehydrogenase (Mor-1) in the mouse: linkage to chromosome 5 markers

    Energy Technology Data Exchange (ETDEWEB)

    Womack, J.E.; Hawes, N.L.; Soares, E.R.; Roderick, T.H.

    1975-01-01

    Malate dehydogenase is present in most mammalian tissues in both supernatant and mitochondrial forms. Although genetic variation for the supernatant form has not been observed in the mouse, electrophoretic variants caused by alleles at the mitochondrial locus (Mor-1) have been previously described. We have located this locus 11.0 +- 2.9 cM from the ..beta..-glucuronidase structural gene, Gus, on chromosome 5. The gene order is Hm--Pgm-1--rd--bf--Gus--Mor-1. Thus Mor-1 is presently the most distal marker on chromosome 5. Three different nuclear loci for mitochondrial enzymes (Mod-2, Got-2, and Mor-1) have now been mapped in the mouse, all on different chromosomes.

  15. Improved production of propionic acid in Propionibacterium jensenii via combinational overexpression of glycerol dehydrogenase and malate dehydrogenase from Klebsiella pneumoniae.

    Science.gov (United States)

    Liu, Long; Zhuge, Xin; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2015-04-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA. PMID:25595755

  16. Evaluation of the Reproductive Toxicity, Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Zhang, Weijie; Zhao, Ting; Mao, Guanghua; Wang, Wei; Wu, Xueshan; Zhou, Zhaoxiang; Huang, Jing; Bao, Yongtuan; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    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 reproductive toxicity of chromium malate in Sprague-Dawley rats and then inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, and lipid metabolism. The results showed that no pathological, toxic feces and urine changes were observed in clinical signs of parental and fetal rats in chromium malate groups. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of chromium malate groups have no significant change compared with control group and chromium picolinate group. The serum and organ contents of Cr in chromium malate groups have no significant change when compared with control group. No measurable damage on liver, brain, kidney, and testis/uterus of chromium malate groups was found. No significant change in body mass, absolute and relative organ weights, and hematological and biochemical changes of rats were observed compared with the control and chromium picolinate groups. The results indicated that supplements with chromium malate does not cause obvious damage and has no obvious effect on glycometabolism, glycometabolism-related enzyme, and lipid metabolism on female and male rats. The results of this study suggested that chromium malate is safe for human consumption and has the potential for application as a functional food ingredient and dietary supplement. PMID:25876088

  17. 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.

  18. In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

    Science.gov (United States)

    Lucas, Simon; Heim, Ralf; Ries, Christina; Schewe, Katarzyna E; Birk, Barbara; Hartmann, Rolf W

    2008-12-25

    Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats. PMID:19049427

  19. 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.

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Fr?czyk, Tomasz; Ruman, Tomasz

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

  1. A rapid, radiometric assay for sucrose synthase

    International Nuclear Information System (INIS)

    Investigations of sucrose synthase in maize root tips have required development of a means to circumvent the rapid decline of activity observed after extraction dialysis and either synthetic or degradative assays. Several protease inhibitors were tested; although PMSF increased initial activity, no inhibitor prevented the drop in activity with time. Western blot analysis indicated that activity decline was not associated with protein degradation. Therefore, a procedure was developed which (1) shortened extraction-to-assay period from ca. 24 hours to 7 minutes, (2) simplified previous assays and (3) reduced the amount of tissue required. Extract was desalted with spun columns and the 14C-UDPG product recovered with DEAE ion exchange paper. The minute quantities of product recovered can be concealed by the presence of trace impurities in the 14C-sucrose utilized. DEAE ion exchange paper was used to remove interfering radio-labelled compounds from the 14C-sucrose prior to assay

  2. Spermidine synthase as affected by osmotic stress in oat leaves

    International Nuclear Information System (INIS)

    Osmotically-induced putrescine (Put) accumulation in cereals could result not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of spermidine synthase, the enzyme which catalyzes the transformation of Put to spermidine (Spd). To test the latter possibility, they evaluated Spd synthase activity in oat leaves as affected by osmotic stress. They developed a new assay for Spd synthase activity by adding S-adenosylmethionine, C14-Put and pyridoxal phosphate to the assay mixture. Incorporation of the C14-label into Spd can be detected after 45 min of incubation at 370C. Labelled Spd is separated from labelled Put or spermine by elution with HCl in Dowex 50 W-H+ columns. In peeled oat leaves floated in the dark over 0.6 M sorbitol in 1mM PO4 buffer (pH 5.8) for 6 and 136 h. Spd synthase activity is reduced by 24 and 53%, respectively, as compared with controls. The results suggest that the activity of this enzyme is inhibited by osmotic stress, and could partially account for the accumulation of Put

  3. Search for genes involved in the synthesis of poly(L-malate) in the plasmodium of Physarum polycephalum

    OpenAIRE

    Pinchai, Nadthanun

    2005-01-01

    The accellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleate amoebae and multinucleate syncytial plasmodia. These two cell types differ in cellular organization, behaviour and gene expression. Plasmodium distinguishes from other stages of the life cycle by the production of an unusual polyester, ß-poly(L-malate) (PMLA). The polymer is concentrated in the nuclei and has been proposed to function as storage molecule and a mobile matrix for nucle...

  4. Inhibition of the malate-aspartate shuttle by pre-ischaemic aminooxyacetate loading of the heart induces cardioprotection

    DEFF Research Database (Denmark)

    Støttrup, Nicolaj; Løfgren, Bo; Birkler, Rune Dupont; Nielsen, J. M.; Wang, Lixing; Caldarone, Christopher Anthony; Kristiansen, Steen Buus; Contractor, Hussain; Johannsen, Mogens; Bøtker, Hans Erik; Nielsen, Torsten Toftegaard

    2010-01-01

    AIMS: Preserved mitochondrial function is essential for protection against ischaemia-reperfusion (IR) injury. The malate-aspartate (MA) shuttle constitutes the principal pathway for transport of reducing cytosolic equivalents for mitochondrial oxidation. We hypothesized that a transient shut-down of the MA-shuttle by aminooxyacetate (AOA) during ischaemia and early reperfusion modulates IR injury by mechanisms comparable to ischaemic preconditioning (IPC). METHODS AND RESULTS: Isolated perfused ...

  5. Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potato plants.

    Science.gov (United States)

    Lehmann, Marco M; Rinne, Katja T; Blessing, Carola; Siegwolf, Rolf T W; Buchmann, Nina; Werner, Roland A

    2015-09-01

    Dissimilation of carbon sources during plant respiration in support of metabolic processes results in the continuous release of CO2. The carbon isotopic composition of leaf dark-respired CO2 (i.e. ? (13) C R ) shows daily enrichments up to 14.8‰ under different environmental conditions. However, the reasons for this (13)C enrichment in leaf dark-respired CO2 are not fully understood, since daily changes in ?(13)C of putative leaf respiratory carbon sources (? (13) C RS ) are not yet clear. Thus, we exposed potato plants (Solanum tuberosum) to different temperature and soil moisture treatments. We determined ? (13) C R with an in-tube incubation technique and ? (13) C RS with compound-specific isotope analysis during a daily cycle. The highest ? (13) C RS values were found in the organic acid malate under different environmental conditions, showing less negative values compared to ? (13) C R (up to 5.2‰) and compared to ? (13) C RS of soluble carbohydrates, citrate and starch (up to 8.8‰). Moreover, linear relationships between ? (13) C R and ? (13) C RS among different putative carbon sources were strongest for malate during daytime (r(2)=0.69, P?0.001) and nighttime (r(2)=0.36, P?0.001) under all environmental conditions. A multiple linear regression analysis revealed ? (13) C RS of malate as the most important carbon source influencing ? (13) C R . Thus, our results strongly indicate malate as a key carbon source of (13)C enriched dark-respired CO2 in potato plants, probably driven by an anapleurotic flux replenishing intermediates of the Krebs cycle. PMID:26139821

  6. Superior aluminium (Al) tolerance of Stylosanthes is achieved mainly by malate synthesis through an Al-enhanced malic enzyme, SgME1.

    Science.gov (United States)

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

    2014-04-01

    Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism. A novel malic enzyme, SgME1, was identified from the Al-tolerant genotype TPRC2001-1 after 72 h Al exposure by two-dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean (Phaseolus vulgaris) hairy roots. Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001-1, as indicated by 5.8-fold higher root malate concentrations and approximately two-fold higher Al concentrations in roots and root symplasts of TPRC2001-1 than those of the Al-sensitive genotype Fine-stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001-1. Gene and protein expression of SgME1 was only enhanced in TPRC2001-1 after 72 h Al exposure. Overexpressing SgME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation. These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al-enhanced malate synthesis, functionally controlled by SgME1. PMID:24325195

  7. Identification of poly G bound to thymidylate synthase.

    Science.gov (United States)

    Thorndike, J; Kisliuk, R L

    1986-09-14

    Thymidylate synthase activity is increased in some methotrexate-resistant strains of Streptococcus faecium. The purified enzyme is associated with a polynucleotide which is not removed by dialysis. This polynucleotide contains one mole each of purine ribose and phosphate per mole base. Phosphate analyses after incubation with digestive enzymes indicate a tetranucleotide with one terminal phosphate. The constituent nucleosides are recovered quantitatively in a specific assay for guanosine. On HPLC, they are inseparable from authentic guanosine and the UV spectrum after HPLC is identical to that of guanosine. We conclude that poly G (GpGpGpGp) is bound to thymidylate synthase. PMID:3094514

  8. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    Ahmad, Zulfiqar; Thomas F. Laughlin; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null cont...

  9. Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

    Science.gov (United States)

    Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

    2015-11-25

    Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3? (GSK-3?) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3? inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3?. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3? inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3? inhibitors as new tools in the development of new treatments for mood disorders. PMID:26486317

  10. Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.

    Science.gov (United States)

    Ohnuma, S; Nakazawa, T; Hemmi, H; Hallberg, A M; Koyama, T; Ogura, K; Nishino, T

    1996-04-26

    Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP. PMID:8626566

  11. Kinetic simulation of malate-aspartate and citrate-pyruvate shuttles in association with Krebs cycle.

    Science.gov (United States)

    Korla, Kalyani; Vadlakonda, Lakshmipathi; Mitra, Chanchal K

    2015-11-01

    In the present work, we have kinetically simulated two mitochondrial shuttles, malate-aspartate shuttle (used for transferring reducing equivalents) and citrate-pyruvate shuttle (used for transferring carbon skeletons). However, the functions of these shuttles are not limited to the points mentioned above, and they can be used in different arrangements to meet different cellular requirements. Both the shuttles are intricately associated with Krebs cycle through the metabolites involved. The study of this system of shuttles and Krebs cycle explores the response of the system in different metabolic environments. Here, we have simulated these subsets individually and then combined them to study the interactions among them and to bring out the dynamics of these pathways in focus. Four antiports and a pyruvate pump were modelled along with the metabolic reactions on both sides of the inner mitochondrial membrane. Michaelis-Menten approach was extended for deriving rate equations of every component of the system. Kinetic simulation was carried out using ordinary differential equation solver in GNU Octave. It was observed that all the components attained steady state, sooner or later, depending on the system conditions. Progress curves and phase plots were plotted to understand the steady state behaviour of the metabolites involved. A comparative analysis between experimental and simulated data show fair agreement thus validating the usefulness and applicability of the model. PMID:25559761

  12. Mechanical and thermal properties of biodegradable hydroxyapatite/poly(sorbitol sebacate malate composites

    Directory of Open Access Journals (Sweden)

    Weng Hong Tham

    2013-02-01

    Full Text Available In this project, novel hydroxyapatite (HAp/poly(sorbitol sebacate malate (PSSM composites for potential application in soft tissue engineering were developed. The composites consist of the biodegradable polyester prepared from sorbitol,sebacic acid, malic acid and various amount of HAp (5, 10, and 15 wt%. Effects of different weight percents of HAp on theproperties of the composites were studied. Fourier transform infrared spectroscopy was performed to analyze chemical interactions between HAp/PSSM. Tensile tests and differential scanning calorimetry were conducted to evaluate the mechanicaland thermal properties of HAp/PSSM composites. Tensile testing on HAp/PSSM composites showed that their mechanicalproperties improved with increasing concentration of HAp. The Young’s modulus and tensile strength of the compositesranged from 16.20±1.73 to 23.96±2.56 MPa and 626.96±81.04 to 1,026.46±105.12 MPa, respectively. The glass transition temperature of all samples was slightly higher than room temperature.

  13. 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.

  14. Genetic control and development expression of malate dehydrogenase in Apis mellifera.

    Science.gov (United States)

    Contel, E P; Mestriner, M A; Martins, E

    1977-10-01

    Starch gel electrophoresis of extracts of Apis mellifera indicates that genetic variability exists for the enzyme cytoplasmic malate dehydrogenase (E.C. 1.1.1.37). Analysis of individuals throughout development indicates that the isozyme patterns are identical for larvae and adults and suggests a dimeric structure for the molecule. The isozyme pattern observed in pupae is more complex than that of larvae and adults may be due to an additional pupal-specific MDH gene being expressed or to an epigenetic modification of the isozymes. Forty-three colonies with artificially inseminated queens were used to study the Mendelian pattern of inheritance. The data revealed that the MDH isozymes are encoded by three alleles, Mdh-1A, Mdh-1B, and Mdh-1C. The frequency of the Mdh-1 alleles is different in two analyzed subspecies, A. m. adansonii (African bees) and A. m. ligustica (Italian bees), with Mdh-1A and Mdh-1B in the African bees being 0-768 and 0.202, respectively. For the Italian bees, these frequencies are 0.136 and 0:154, respectively. PMID:588235

  15. Growth, crystal structure and thermal properties of calcium bis(malate) dihydrate

    Energy Technology Data Exchange (ETDEWEB)

    Jini, T. [Department of Physics, St. Berchmans College, Changanassery 686 101, Kerala (India); Saban, K.V. [Department of Physics, St. Berchmans College, Changanassery 686 101, Kerala (India); Varghese, G. [Department of Physics, St. Berchmans College, Changanassery 686 101, Kerala (India); Naveen, S. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Sridhar, M.A. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India)]. E-mail: mas@physics.uni-mysore.ac.in; Prasad, J.S. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India)

    2007-05-16

    A new coordination compound crystal of calcium with malic acid is prepared by gel aided solution growth. Single crystal X-ray diffraction studies revealed that the structural formula of the compound is Ca(C{sub 4}H{sub 4}O{sub 5}){sub 2}.2H{sub 2}O. It crystallizes in the monoclinic system with space group C2/c, Z = 4, with unit cell parameters a = 15.916(9) A, b = 5.886(3) A, c = 13.046(6) A and {beta} = 90.678(4){sup o}. Data were collected by oscillation method and full-matrix least squares refinement was applied to the model converging to final R indices R {sub 1} = 0.0416 and {omega}R {sub 2} = 0.1255. Compound forms a layer-type polymeric structure, stabilized by intermolecular hydrogen bonding. Ca{sup 2+} is eight-fold coordinated. Malate is coordinated to Ca{sup 2+} tridendate-bidendate through two carboxylates and monodendate through oxygen atom of the hydroxyl group. Thermal behavior investigated using TG and DTA studies is in conformity with the proposed structure.

  16. 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.

  17. Growth, crystal structure and thermal properties of calcium bis(malate) dihydrate

    International Nuclear Information System (INIS)

    A new coordination compound crystal of calcium with malic acid is prepared by gel aided solution growth. Single crystal X-ray diffraction studies revealed that the structural formula of the compound is Ca(C4H4O5)2.2H2O. It crystallizes in the monoclinic system with space group C2/c, Z = 4, with unit cell parameters a = 15.916(9) A, b = 5.886(3) A, c = 13.046(6) A and ? = 90.678(4)o. Data were collected by oscillation method and full-matrix least squares refinement was applied to the model converging to final R indices R 1 = 0.0416 and ?R 2 = 0.1255. Compound forms a layer-type polymeric structure, stabilized by intermolecular hydrogen bonding. Ca2+ is eight-fold coordinated. Malate is coordinated to Ca2+ tridendate-bidendate through two carboxylates and monodendate through oxygen atom of the hydroxyl group. Thermal behavior investigated using TG and DTA studies is in conformity with the proposed structure

  18. 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

  19. Intrinsic uncoupling in the ATP synthase of Escherichia coli.

    Science.gov (United States)

    D'Alessandro, Manuela; Turina, Paola; Melandri, B Andrea

    2008-12-01

    The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi. PMID:18952048

  20. A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase.

    Science.gov (United States)

    Wilderman, P Ross; Peters, Reuben J

    2007-12-26

    Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to "short circuit" the complex cyclization reaction normally catalyzed by ent-kaurene synthases to instead produce ent-pimaradiene. Here we report that the complex cyclization/rearrangement reaction catalyzed by abietadiene synthase can be similarly cut short to produce pimaradienes by an analogous Ser for Ala change, albeit with a slight shift in active site location to accommodate the difference in substrate stereochemistry. This result has mechanistic implications for enzymatic catalysis of abietadiene cyclization, and terpene synthases more broadly. Furthermore, these defined single residue switches may be useful in engineering product outcome in diterpene synthases more generally. PMID:18052062

  1. Expression, crystallization and structure elucidation of ?-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of ?-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of ?-terpinene synthase (TPS). Purified ?-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to ?-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65?Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata. PMID:26750479

  2. Genome-wide screening of salt tolerant genes by activation-tagging using dedifferentiated calli of Arabidopsis and its application to finding gene for Myo-inositol-1-p-synthase.

    Science.gov (United States)

    Ahmad, Aftab; Niwa, Yasuo; Goto, Shingo; Kobayashi, Kyoko; Shimizu, Masanori; Ito, Sohei; Usui, Yumiko; Nakayama, Tsutomu; Kobayashi, Hirokazu

    2015-01-01

    Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis. PMID:25978457

  3. Genome-Wide Screening of Salt Tolerant Genes by Activation-Tagging Using Dedifferentiated Calli of Arabidopsis and Its Application to Finding Gene for Myo-Inositol-1-P-Synthase

    Science.gov (United States)

    Ahmad, Aftab; Niwa, Yasuo; Goto, Shingo; Kobayashi, Kyoko; Shimizu, Masanori; Ito, Sohei; Usui, Yumiko; Nakayama, Tsutomu; Kobayashi, Hirokazu

    2015-01-01

    Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis. PMID:25978457

  4. Discovery of 4-Aryl-5,6,7,8-tetrahydroisoquinolines as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys.

    Science.gov (United States)

    Martin, Rainer E; Aebi, Johannes D; Hornsperger, Benoit; Krebs, Hans-Jakob; Kuhn, Bernd; Kuglstatter, Andreas; Alker, André M; Märki, Hans Peter; Müller, Stephan; Burger, Dominique; Ottaviani, Giorgio; Riboulet, William; Verry, Philippe; Tan, Xuefei; Amrein, Kurt; Mayweg, Alexander V

    2015-10-22

    Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg(-1). This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man. PMID:26403853

  5. Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants.

    OpenAIRE

    SATO, T.; Theologis, A

    1989-01-01

    Ethylene is the plant hormone that controls several features of plant growth and development. The rate-limiting step in its synthesis is the formation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from S-adenosylmethionine (AdoMet), catalyzed by ACC synthase. We have isolated a complementary DNA sequence encoding ACC synthase from zucchini (Cucurbita) fruits. The biological activity of the clone was confirmed by the ability of the cloned sequence to direct ACC synthase...

  6. Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle.

    OpenAIRE

    Mitchell, J.A.; Sheng, H.; Förstermann, U.; Murad, F.

    1991-01-01

    Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat ...

  7. Candesartan ameliorates acute myocardial infarction in rats through inducible nitric oxide synthase, nuclear factor??B, monocyte chemoattractant protein?1, activator protein?1 and restoration of heat shock protein 72.

    Science.gov (United States)

    Lin, Xuefeng; Wu, Min; Liu, Bo; Wang, Junkui; Guan, Gongchang; Ma, Aiqun; Zhang, Yong

    2015-12-01

    Candesartan, an angiotensin II type 1 receptor antagonist, has a variety of biological activities, including antioxidant, anti?inflammatory and anticancer activities, with specific pharmacological effects. The present study investigated the mechanisms and protective effect of candesartan on acute myocardial infarction in rats. Male Wistar rats (8?week?old) were induced as a model of acute myocardial infarction and treated with candesartan (0.25 mg/kg) for 2 weeks. The present study first measured the activities of casein kinase (CK), the MB isoenzyme of creatine kinase (CK?MB) and lactate dehydrogenase (LDH), the level of cardiac troponin T (cTnT) and infarct size. Subsequently, western blot analysis was performed to analyze the protein expression levels of inducible nitric oxide synthase (iNOS) and heat shock protein 72 (HSP72) in the rats. An enzyme linked immunosorbent assay was used to detect iNOS and nuclear factor??B (NF??B) activity. In addition, gene expression levels of monocyte chemotactic protein?1 (MCP?1) and activating protein?1 (AP?1) were determined using reverse transcription?quantitative polymerase chain reaction analysis. Finally, the activities of caspase?3 and caspase?9 were examined using colorimetric assay kits. In the serum of the rat model of acute myocardial infarction, candesartan significantly increased the activities of CK, CK?MB and LDH, and the level of cTnT. The infarction size was perfected by candesartan treatment. Candesartan significantly reduced the protein expression and activity of iNOS, the activity of NF??B p65, and the gene expression levels of MCP?1 and AP?1 in the rat model of acute myocardial infarction. Candesartan increased the protein expression of HSP?72 in the acute myocardial infarction rat model. However, candesartan did not effect the levels of caspase?3 or caspase?9 in the rat model of acute myocardial infarction. These results suggested that candesartan ameliorates acute myocardial infarction in rats through iNOS, NF??B, MCP?1 and AP?1, and the restoration of HSP72. PMID:26499133

  8. 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 λgt10 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 contains the putative catalytic site common to other human phosphoribosyltransferases

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

    DEFF Research Database (Denmark)

    Gaster, Michael; Petersen, Ingrid; Højlund, Kurt; Poulsen, Pernille; Beck-Nielsen, Henning

    2002-01-01

    (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The...... present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic...... and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal....

  10. Functional reconstitution of cellulose synthase in Escherichia coli.

    Science.gov (United States)

    Imai, Tomoya; Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji

    2014-11-10

    Cellulose is a high molecular weight polysaccharide of ?1 ? 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials. PMID:25285473

  11. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K; Prats, C; Hirshman, M F; Dufresne, S D; Sakamoto, K; Vilardo, P G; Steele, M; Kim, J H; Jing, S L; Goodyear, L J; DePaoli-Roach, A A

    2001-01-01

    was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A....... (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of...... contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly...

  12. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    OpenAIRE

    Ternes, Philipp; Brouwers, Jos F H M; Van Den Dikkenberg, Joep; Holthuis, Joost C M

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the S...

  13. Increased hippocampal nitric oxide synthase activity and stress responsiveness after imipramine discontinuation: role of 5HT 2A/C-receptors

    DEFF Research Database (Denmark)

    Harvey, Brian H; Retief, Renché; Korff, Ané; Wegener, Gregers

    dependence of any biobehavioral changes following IMI withdrawal on 5HT(2A/C) receptor-mediated events was studied using the 5HT(2A/C) receptor antagonist, ritanserin (RIT; 4 mg/kg/day ip x 7 days), administered alone or during IMI withdrawal. IMI significantly inhibited the situational stress response to...... NOS hyper-function during IMI withdrawal, although alone it increased NOS activity. Antidepressant discontinuation therefore increases stress responsiveness together with disinhibition of hippocampal NOS through a mechanism involving 5HT(2A/C) receptor activation. The resulting increased nitrergic...

  14. 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; Ploug, Kenneth Beri; Hay-Schmidt, Anders; Jansen-Olesen, Inger; Gupta, Saurabh; Olesen, Jes

    2014-01-01

    NOS 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...

  15. Upregulation of Cysteine Synthase and Cystathionine ?-Synthase Contributes to Leishmania braziliensis Survival under Oxidative Stress.

    Science.gov (United States)

    Romero, Ibeth; Téllez, Jair; Romanha, Alvaro José; Steindel, Mario; Grisard, Edmundo Carlos

    2015-08-01

    Cysteine metabolism is considered essential for the crucial maintenance of a reducing environment in trypanosomatids due to its importance as a precursor of trypanothione biosynthesis. Expression, activity, functional rescue, and overexpression of cysteine synthase (CS) and cystathionine ?-synthase (C?S) were evaluated in Leishmania braziliensis promastigotes and intracellular amastigotes under in vitro stress conditions induced by hydrogen peroxide (H2O2), S-nitroso-N-acetylpenicillamine, or antimonial compounds. Our results demonstrate a stage-specific increase in the levels of protein expression and activity of L. braziliensis CS (LbrCS) and L. braziliensis C?S (LbrC?S), resulting in an increment of total thiol levels in response to both oxidative and nitrosative stress. The rescue of the CS activity in Trypanosoma rangeli, a trypanosome that does not perform cysteine biosynthesis de novo, resulted in increased rates of survival of epimastigotes expressing the LbrCS under stress conditions compared to those of wild-type parasites. We also found that the ability of L. braziliensis promastigotes and amastigotes overexpressing LbrCS and LbrC?S to resist oxidative stress was significantly enhanced compared to that of nontransfected cells, resulting in a phenotype far more resistant to treatment with the pentavalent form of Sb in vitro. In conclusion, the upregulation of protein expression and increment of the levels of LbrCS and LbrC?S activity alter parasite resistance to antimonials and may influence the efficacy of antimony treatment of New World leishmaniasis. PMID:26033728

  16. Alteration of Ceramide Synthase 6/C16-Ceramide Induces Activating Transcription Factor 6-mediated Endoplasmic Reticulum (ER) Stress and Apoptosis via Perturbation of Cellular Ca2+ and ER/Golgi Membrane Network*

    Science.gov (United States)

    Senkal, Can E.; Ponnusamy, Suriyan; Manevich, Yefim; Meyers-Needham, Marisa; Saddoughi, Sahar A.; Mukhopadyay, Archana; Dent, Paul; Bielawski, Jacek; Ogretmen, Besim

    2011-01-01

    Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1–6 (CerS1–6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C16-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C16-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca2+ from the ER stores ([Ca2+]ER), which resulted in the fragmentation of Golgi membranes in response to CerS6/C16-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca2+ chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C16-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C16-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas. PMID:22013072

  17. Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp. / Atividade da sintetase da glutamina e sintase do glutamato em relação a fixação de nitrogênio em Lotus spp.

    Scientific Electronic Library Online (English)

    SUSANA, GONNET; PEDRO, DÍAZ.

    Full Text Available Plantas de Lotus corniculatus, L. tenuis, L. pedunculatus e L. subbiflorus foram inoculadas com Mesorhizobium loti cepa NZP2037 e mantidas numa câmara de crescimento. A massa seca da planta (MS), massa fresca dos nódulos (MF), atividade de nitrogenase, atividades de sintetase de glutamina (GS) e sin [...] tase de glutamato (GOGAT), bem como o teor de leghemoglobina e de aminoácidos no caule foram avaliados 28 dias após inoculação. A maior MS das plantas foi encontrada em L. tenuis e a maior MF de nódulos foi encontrada em L. pedunculatus. Atividade de nitrogenase em L. tenuis, L. pedunculatus e L. subbiflorus foi seis vezes a atividade em L. corniculatus. As atividades de GS e GOGAT nos nódulos não mostraram o mesmo padrão. As maiores atividades de GS e GOGAT foram encontradas nos nódulos de L. tenuis associadas com a alta atividade de nitrogenase, resultados compativeis com sua alta MS. As quatro espécies de Lotus foram comparadas e nenhuma correlação entre os parâmetros de fixação de nitrogênio e enzimas de assimilação de amonia foi encontrada, mas a razão GS/GOGAT tem uma correlação positiva e significativa (r²=0.82**) com o teor de aminoácidos nos caules. Abstract in english Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) [...] activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of nodules was measured in L. pedunculatus. Nitrogenase activity in L. tenuis, L. pedunculatus and L. subbiflorus was six fold the activity in L. corniculatus. Nodule GS and GOGAT activities did not follow this same pattern. L. tenuis had the highest values of GS and GOGAT activities in the nodule, and a high nitrogenase activity which is consistent with its high plant DW. The four species of Lotus were compared and no correlation between nitrogen fixation parameters and ammonia assimilation enzymes was found, but the GS/GOGAT ratio has a positive and significant correlation (r²=0.82**) with the amino acid content in stems.

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

    OpenAIRE

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

    2014-01-01

    •CN-Cbl dodecylamine, a derivative of cyanocobalamin, was absorbed by C. elegans.•CN-Cbl dodecylamine decreased activities of cobalamin-dependent enzymes.•CN-Cbl dodecylamine induced cobalamin deficiency in C. elegans.•CN-Cbl dodecylamine acts as an inhibitor of cobalamin-dependent enzymes.

  19. Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

    Indian Academy of Sciences (India)

    Sam Annie-JeyachristYn; Arumugam Geetha; Rajagopal Surendran

    2008-03-01

    Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis. In the present investigation, we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis. The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver. Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis. Platelet aggregation was also measured in the presence of the eNOS inhibitor, diphenylene iodinium chloride (DIC). The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects. During the course of platelet aggregation, concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples, whereas the elevation was not significant in platelets of patients with cirrhosis. A parallel increase was observed in the levels of NO and NOS activity. In the presence of the eNOS inhibitor, platelet aggregation was enhanced and accompanied by an elevated calcium level. The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS. Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis. The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance, and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.

  20. Domain loss has independently occurred multiple times in plant terpene synthase evolution.

    Science.gov (United States)

    Hillwig, Matthew L; Xu, Meimei; Toyomasu, Tomonobu; Tiernan, Mollie S; Wei, Gao; Cui, Guanghong; Huang, Luqi; Peters, Reuben J

    2011-12-01

    The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the ? domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/?' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/?' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/?' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family. PMID:21999670

  1. Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factor during Ethylene Induction in Arabidopsis

    OpenAIRE

    Li, Guojing; Meng, Xiangzong; Wang, Ruigang; Mao, Guohong; Han, Ling; Liu, Yidong; Zhang, Shuqun

    2012-01-01

    Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea–induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS ...

  2. A functional cellulose synthase from ascidian epidermis

    OpenAIRE

    Matthysse, Ann G; Deschet, Karine; Williams, Melanie; Marry, Mazz; Alan R. White; Smith, William C.

    2004-01-01

    Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase ami...

  3. 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.

  4. microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus

    DEFF Research Database (Denmark)

    Leucci, Eleonora; Patella, Francesca; Waage, Johannes; Holmstrøm, Kim; Lindow, Morten; Porse, Bo; Kauppinen, Sakari; Lund, Anders H.

    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 ...

  5. Effects of fatty acids on cardioprotection by pre-ischaemic inhibition of the malate-aspartate shuttle

    DEFF Research Database (Denmark)

    Dalgas, Christian; Povlsen, Jonas Agerlund; Løfgren, Bo; Erichsen, Sune Brinck; Bøtker, Hans Erik

    2012-01-01

    1. The malate-aspartate shuttle (MAS) is the main pathway for balancing extra- and intramitochondrial glucose metabolism. Pre-ischaemic shutdown of the MAS by aminooxyacetate (AOA) mimics ischaemic preconditioning (IPC) in rat glucose-perfused hearts. The aim of the present study was to determine the effects of fatty acids (FA) on cardioprotection by pre-ischaemic inhibition of the MAS. 2. Isolated rat hearts were divided into four groups (control; pre-ischaemic AOA (0.2 mmol/L); IPC; and AOA + ...

  6. Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

    Directory of Open Access Journals (Sweden)

    S.R. Martins

    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.

  7. 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.

  8. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning

    OpenAIRE

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J.; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M.

    2014-01-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H+-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H+-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H+-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the a...

  9. Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

    Science.gov (United States)

    Luzak, Boguslawa; Boncler, Magdalena; Rywaniak, Joanna; Dudzinska, Dominika; Rozalski, Marek; Krajewska, Urszula; Balcerczak, Ewa; Podsedek, Anna; Redzynia, Malgorzata; Watala, Cezary

    2014-12-01

    The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 ?g/ml, and up to 33 % for 15 ?g/ml, p BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 ?g/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium. PMID:25407137

  10. Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

    Science.gov (United States)

    Sparacino-Watkins, Courtney E; Tejero, Jesús; Sun, Bin; Gauthier, Marc C; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T

    2014-04-11

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  11. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    Science.gov (United States)

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  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. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes Um2552 in E. coli, makes the 50S subunit less stable at 1 mM Mg++ (Bügl et al. 2000) and inhibits subunit joining (Caldas et al. 2000), but, in this case, it is not yet known whether the effects are due to the lack of 2'-O-methylation or to the absence of the enzyme itself. Is there any role for the psi residues themselves? First, as noted above, the 3 psi made by RluD which cluster in the end-loop of helix 69 are highly conserved, with one being universal (Fig. 2B). In the 70S-tRNA structure (Yusupov et al. 2001), the loop of this helix containing the psi supports the anticodon arm of A-site tRNA near its juncture with the amino acid arm. The middle of helix 69 does the same thing for P-site tRNA. Unfortunately, the resolution is not yet sufficient to provide a more precise alignment of the psi residues with the other structural elements of the tRNA-ribosome complex so that one cannot yet determine what role, if any, is played by the N-1 H that distinguishes psi from U. Second, and more generally, some psi residues in the LSU appear to be near the site of peptide-bond formation or tRNA binding but not actually at it (Fig. 2B) (Nissen et al. 2000; Yusupov et al. 2001). For example, position 2492 is commonly psi and is only six residues away from A2486, the A postulated to catalyze peptide-bond formation. Position 2589 is psi in all the eukaryotes and is next to 2588, which base-pairs with the C75 of A-site tRNA. Residue 2620, which interacts with the A76 of A-site-bound tRNA, is a psi or is next to a psi in eukaryotes and Archaea, and is five residues away from psi 2580 in E. coli. A2637, which is between the two CCA ends of P- and A-site tRNA, is near psi 2639, psi 2640, and psi 2641, found in a number of organisms. Residue 2529, which contacts the backbone of A-site tRNA residues 74-76, is near psi 2527 psi 2528 in H. marismortui. Residues 2505-2507, which contact A-site tRNA residues 50-53, are near psi 2509 in higher eukaryotes, and residues 2517-2519 in contact with A-site tRNA residues 64-65 are within 1-3 nucleotides of psi 2520 in higher eukaryotes and psi 2514 in H. marismortui. A way to rationalize this might be to invoke the concept suggested in the Introduction that psi acts as a molecular glue to hold loose elements in a more rigid configuration. It may well be that this is more important near the site of peptide-bond formation and tRNA binding, accounting for the preponderance of psi in this vicinity. What might be the role of all the other psi in eukaryotes? One can only surmise that cells, having once acquired the ability to make psi with guide RNAs, took advantage of the system to inexpensively place psi wherever an undesirable loose region was found. It might be that in some of these cases, psi performs the role played by proteins in other regions, namely that of holding the rRNA in its proper configuration. Confirmation of this hypothesis will have to await structural determination of eukaryotic ribosomes. PMID:12762017

  14. 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.

  15. Mutation in cysteine bridge domain of the gamma-subunit affects light regulation of the ATP synthase in Arabidopsis

    Science.gov (United States)

    The chloroplast ATP synthase functions to synthesize ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine bridge in CF...

  16. New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase.

    Science.gov (United States)

    Lohou, Elodie; Sopkova-de Oliveira Santos, Jana; Schumann-Bard, Pascale; Boulouard, Michel; Stiebing, Silvia; Rault, Sylvain; Collot, Valérie

    2012-09-01

    Taking into account the potency of 4- and 7-nitro and haloindazoles as nNOS inhibitors previously reported in the literature by our team, a multidisciplinary study, described in this article, has recently been carried out to elucidate their binding mode in the enzyme active site. Firstly, nitrogenous fastening points on the indazole building block have been investigated referring to molecular modeling hypotheses and thanks to the in vitro biological evaluation of N(1)- and N(2)-methyl and ethyl-4-substituted indazoles on nNOS. Secondly, we attempted to confirm the importance of the substitution in position 4 or 7 by a hydrogen bond acceptor group thanks to the synthesis and the in vitro biological evaluation of a new analogous 4-substituted derivative, the 4-cyanoindazole. Finally, by opposition to previous hypotheses describing NH function in position 1 of the indazole as a key fastening point, the present work speaks in favour of a crucial role of nitrogen in position 2. PMID:22831803

  17. Immunolocalization of nitric oxide synthase (NOS) isoforms in ovarian follicles of the catfish, Clarias batrachus and its relation with ovarian activity.

    Science.gov (United States)

    Singh, Vinay Kumar; Lal, Bechan

    2015-09-01

    Nitric oxide, a gaseous molecule, is produced during the conversion of arginine to citrulline by the action of NOS isoforms (eNOS, iNOS or nNOS). Role of NO in regulation of mammalian reproduction is well established; however, practically no report is available on fishes. Hence, in the present study, expression of all three isoforms of NOS was worked out in the ovary of Clarias batrachus immunohistochemically during different phases of its reproductive cycle and its relation with ovarian activities. No immunoreactivity of eNOS was observed in the ovary of C. batrachus during the late-quiescence and early-recrudescence phases. While during the recrudescence phase (April and May) it expressed intensely in thecal and granulosa cells of the oocyte-II and III, but immune-intensity decreased in the late-recrudescence and spawning phases (June and July). Similar pattern of immunoprecipitation was also observed in case of iNOS. However, the immunoreactivity pattern of nNOS was quite varied, it expressed moderately only in the nucleus and cytoplasm of perinuclear and oocyte-I stages during late-quiescence phase. While during the early recrudescence phase, the expression of nNOS disappeared completely from the nucleus and cytoplasm, rather it expressed intensely in the thecal and granulosa cells, which declined in the late-recrudescence and spawning phases. Moderate immunoreactivity of iNOS could also be localized in the zona radiata of ovulated oocyte. The intense NOS immunoreactivity in the thecal and granulosa cells coincided with increased levels of ovarian NO and 17?-estradiol content. They exhibited statistically significant positive correlation amongst themselves, suggesting the involvement of ovarian NOS/NO system in oogenesis and steroidogenesis in the catfish. PMID:24755185

  18. Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

    Science.gov (United States)

    Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W V; Sivaraman, J

    2015-11-01

    ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ?20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

  19. 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.

  20. 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.

  1. 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.

  2. Isoenzyme composition of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase of rat brain at various times after external 1 Gy ?-irradiation

    International Nuclear Information System (INIS)

    The influence of external single ?-irradiation with a dose of 1 Gy on the isoenzyme composition of lactate dehydrogenase, malate dehydrogenase, sterase and acid phosphatase in the cytoplasm of rat brain cells was investigated. Irradiation was shown to cause differently directed changes in the ratio of the isoenzymes under study at different times after exposure. The isoenzyme spectrum of lactate dehydrogenase and malate dehydrogenase was shown to be normalized on day 30 after irradiation, wheras the isoform composition of esterase and acid phosphatase was not stabilized at that time

  3. microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus

    DEFF Research Database (Denmark)

    Leucci, Eleonora; Patella, Francesca; Waage, Johannes; Holmstrøm, Kim; Lindow, Morten; Porse, Bo; Kauppinen, Sakari; Lund, Anders H.

    2013-01-01

    -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....

  4. Properties of Succinyl-Coenzyme A:l-Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

    OpenAIRE

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E.; Fuchs, Georg

    2006-01-01

    The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by l-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:l-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding gen...

  5. Bacillus subtilis YxkJ Is a Secondary Transporter of the 2-Hydroxycarboxylate Transporter Family That Transports l-Malate and Citrate

    OpenAIRE

    Krom, Bastiaan P; Aardema, Ronald; Lolkema, Juke S.

    2001-01-01

    The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two, yxkJ, which codes for a transporter protein named CimHbs. The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E. coli and the malate-negative phenotype of the E. coli strain JRG4008, which is defective in malate uptake. Subse...

  6. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    DEFF Research Database (Denmark)

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.; Almo, Steven C.

    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 compl...

  7. Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians

    DEFF Research Database (Denmark)

    Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh; Kohli, Harbir Singh; Sud, Kamal; Bhansali, Anil; Khullar, Madhu

    2008-01-01

    Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, including diabetic nephropathy. Endothelial-derived nitric oxide synthase (eNOS) gene polymorphisms affect eNOS activity and are associated with endothelial dysfunction. We evaluated the association of the constitutive endothelial nitric oxide synthase gene (eNOS) polymorphisms with type 2 diabetic nephropathy. We genotyped three polymorphisms of eNOS (Two SNPs: -786T > C, 894G > T and one 27-bp repeat pol...

  8. Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

    OpenAIRE

    Escartin, Frédéric; Skouloubris, Stéphane; Liebl, Ursula; Myllykallio, Hannu

    2008-01-01

    We have investigated the hitherto unexplored possibility that differences in the catalytic efficiencies of thymidylate synthases ThyX and ThyA, enzymes that produce the essential DNA precursor dTMP, have influenced prokaryotic genome evolution. We demonstrate that DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10-fold decreased compared with species that contain the catalytically more efficient ThyA. Our statistical studies of >400 genomes ind...

  9. 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...

  10. 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.

  11. 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.

  12. Overexpression of the malate-aspartate NADH shuttle member Aralar1 in the clonal beta-cell line BRIN-BD11 enhances amino-acid-stimulated insulin secretion and cell metabolism.

    Science.gov (United States)

    Bender, Katrin; Maechler, Pierre; McClenaghan, Neville H; Flatt, Peter R; Newsholme, Philip

    2009-11-01

    In the present study, we have investigated the effects of the transduction with recombinant adenovirus AdCA-Aralar1 (aspartate-glutamate carrier 1) on the metabolism, function and secretory properties of the glucose- and amino-acid-responsive clonal insulin-secreting cell line BRIN-BD11. Aralar1 overexpression increased long-term (24 h) and acute (20 min) glucose- and amino-acid-stimulated insulin secretion, cellular glucose metabolism, L-alanine and L-glutamine consumption, cellular ATP and glutamate concentrations, and stimulated glutamate release. However, cellular triacylglycerol and glycogen contents were decreased as was lactate production. These findings indicate that increased malate-aspartate shuttle activity positively shifted beta-cell metabolism, thereby increasing glycolysis capacity, stimulus-secretion coupling and, ultimately, enhancing insulin secretion. We conclude that Aralar1 is a key metabolic control site in insulin-secreting cells. PMID:19344310

  13. Posttranscriptional silencing of the lncRNA MALAT1 by miR-217 inhibits the epithelial-mesenchymal transition via enhancer of zeste homolog 2 in the malignant transformation of HBE cells induced by cigarette smoke extract.

    Science.gov (United States)

    Lu, Lu; Luo, Fei; Liu, Yi; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-12-01

    Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial-mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis. PMID:26415832

  14. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    OpenAIRE

    R. GUTIERREZ; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D W

    2009-01-01

    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide...

  15. 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

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

    DEFF Research Database (Denmark)

    Vestergaard, H; Lund, S; Bjørbaek, C; Pedersen, O

    1995-01-01

    metabolism (p=0.02) was demonstrated in teh gliclazide-treated patients when compared to pre-treatment values. In biopsies obtained from vastus lateralis muscle during insulin infusion, the half-maximal activation of glycogen synthase was achieved at a significantly lower concentration of the allosteric...... activator glucose 6-phosphate (p=0.01). However, despite significant increases in both insulin-stimulated non-oxidative glucose metabolism and muscle glycogen synthase activation in gliclazide-treated patients no changes were found in levels of glycogen synthase mRNA or immunoreactive protein in muscle. In...

  17. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    DEFF Research Database (Denmark)

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.; Almo, Steven C.

    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...... 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...

  18. Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Aimy Sebastian

    2015-10-01

    Full Text Available Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO osteoblasts and wildtype (WT osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

  19. Novel inhibitors of nitric oxide synthase with antioxidant properties.

    Science.gov (United States)

    Salerno, Loredana; Modica, Maria N; Romeo, Giuseppe; Pittalà, Valeria; Siracusa, Maria A; Amato, Maria E; Acquaviva, Rosaria; Di Giacomo, Claudia; Sorrenti, Valeria

    2012-03-01

    We previously described a series of imidazole-based inhibitors substituted at N-1 with an arylethanone chain as interesting inhibitors of neuronal nitric oxide synthase (nNOS), endowed with good selectivity vs endothelial nitric oxide synthase (eNOS). As a follow up of these studies, several analogs characterized by the presence of substituted imidazoles or other mono or bicyclic nitrogen-containing heterocycles instead of simple imidazole were synthesized, and their biological evaluation as in vitro inhibitors of both nNOS and eNOS is described herein. Most of these compounds showed improved nNOS and eNOS inhibitory activity with respect to reference inhibitors. Selected compounds were also tested to analyze their antioxidant properties. Some of them displayed good capacity to scavenge free radicals and ability to reduce lipid peroxidation. PMID:22280820

  20. 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.

  1. Granulocyte-macrophage colony-stimulating factor activates the transcription of nuclear factor kappa B and induces the expression of nitric oxide synthase in a skin dendritic cell line

    OpenAIRE

    Cruz, M Teresa; Carlos B. Duarte; Gonçalo, Margarida; Figueiredo, Américo; Carvalho, Arsélio P.; Lopes, M Celeste

    2001-01-01

    Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM-CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal-skin-derived dendritic cell line (FSDC) and, consequently, increases the nitrite production ...

  2. A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels

    OpenAIRE

    Chung J Sook

    2008-01-01

    Abstract Trehalose in ectoderms functions in energy metabolism and protection in extreme environmental conditions. We structurally characterized trehalose 6-phosphate synthase (TPS) from hemocytes of the blue crab, Callinectes sapidus. C. sapidus Hemo TPS (CasHemoTPS), like insect TPS, encodes both TPS and trehalose phosphate phosphatase domains. Trehalose seems to be a major sugar, as it shows higher levels than does glucose in hemocytes and hemolymph. Increases in HemoTPS expression, TPS en...

  3. 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)

  4. 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)

  5. 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

  6. 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 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 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

  7. Concerted versus stepwise mechanism in thymidylate synthase.

    Science.gov (United States)

    Islam, Zahidul; Strutzenberg, Timothy S; Gurevic, Ilya; Kohen, Amnon

    2014-07-16

    Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design. PMID:24949852

  8. Effects of Supplemental Citrulline-Malate Ingestion on Blood Lactate, Cardiovascular Dynamics, and Resistance Exercise Performance in Trained Males.

    Science.gov (United States)

    Wax, Benjamin; Kavazis, Andreas N; Luckett, William

    2016-05-01

    Citrulline-malate (CM) has been proposed to provide an ergogenic effect during resistance exercise; however, there is a paucity of research investigating these claims. Therefore, we investigated the impact that CM supplementation would have on repeated bouts of resistance exercise. Fourteen resistance-trained males participated in a randomized, counterbalanced, double-blind study. Subjects were randomly assigned to placebo (PL) or CM (8 g) and performed three sets each of chin-ups, reverse chin-ups, and push-ups to failure. One week later, subjects ingested the other supplement and performed the same protocol. Blood lactate (BLa), heart rate (HR), and blood pressure (BP) were measured preexercise, with BLa measured a second time immediately following the last set, while HR and BP were measured 5 and 10 min postexercise. Citrulline-malate ingestion significantly increased the amount of repetitions performed for each exercise (chin-ups: PL = 28.4 ± 7.1, CM = 32.2 ± 5.6, p = .003; reverse chin-ups: PL = 26.6 ± 5.6, CM = 32.1 ± 7.1, p = .017; push-ups: PL = 89.1 ± 37.4, CM = 97.7 ± 36.1, p Blood lactate data indicated a time effect (p < .001), but no treatment differences (p = .935). Systolic BP data did not show differences for time (p = .078) or treatment (p = .119). Diastolic BP data did not show differences for time (p = .069), but indicated treatment differences (p = .014) for subjects ingesting CM. Collectively, these findings suggests that CM increased upper-body resistance performance in trained college-age males. PMID:25674699

  9. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Some affected individuals have changes in skin coloring (pigmentation), including dark freckle-like spots on the arms ... shortage of GM3 synthase and changes in skin pigmentation is also unknown. Read more about the ST3GAL5 ...

  10. Cellulose Synthase Complexes: Composition and Regulation

    OpenAIRE

    Lei, Lei; Li, Shundai; Gu, Ying

    2012-01-01

    Live cell imaging has greatly advanced our knowledge on the molecular mechanism by which cellulose is deposited. Both the actin and microtubule cytoskeleton are involved in assuring the proper distribution, organization, and dynamics of cellulose synthase complexes (CSCs). This review is an update on the most recent progress on the characterization of the composition, regulation, and trafficking of CSCs. With the newly identified cellulose synthase interactive protein 1 (CSI1) on hand, we beg...

  11. Terpene synthases are widely distributed in bacteria

    OpenAIRE

    Yamada, Yuuki; Kuzuyama, Tomohisa; KOMATSU, MAMORU; SHIN-YA, KAZUO; OMURA, SATOSHI; CANE, DAVID E.; IKEDA, HARUO

    2014-01-01

    Terpenes are generally considered to be plant or fungal metabolites, although a small number of odoriferous terpenes of bacterial origin have been known for many years. Recently, extensive bacterial genome sequencing and bioinformatic analysis of deduced bacterial proteins using a profile based on a hidden Markov model have revealed 262 distinct predicted terpene synthases. Although many of these presumptive terpene synthase genes seem to be silent in their parent microorganisms, controlled e...

  12. Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot1[OPEN

    Science.gov (United States)

    Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

    2015-01-01

    Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. PMID:26157114

  13. Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot.

    Science.gov (United States)

    Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

    2015-11-01

    Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. PMID:26157114

  14. 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

  15. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  16. 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

  17. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  18. 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

  19. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.).

    Science.gov (United States)

    Zheng, Desen; Hrazdina, Geza

    2008-02-15

    Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3' region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product. PMID:18068110

  20. The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose

    OpenAIRE

    Yonekura, Madoka; Aoki, Naohiro; Hirose, Tatsuro; Onai, Kiyoshi; Ishiura, Masahiro; Okamura, Masaki; Ohsugi, Ryu; Ohto, Chikara

    2013-01-01

    Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transge...

  1. 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

  2. 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

  3. Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens.

    Science.gov (United States)

    Hayashi, Ken-Ichiro; Kawaide, Hiroshi; Notomi, Miho; Sakigi, Yuka; Matsuo, Akihiko; Nozaki, Hiroshi

    2006-11-13

    ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene). PMID:17064690

  4. Molecular evolution of dihydrouridine synthases

    Directory of Open Access Journals (Sweden)

    Kasprzak Joanna M

    2012-06-01

    Full Text Available Abstract Background Dihydrouridine (D is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS. DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as “predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family”. Results To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model. Conclusions We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.

  5. 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

  6. Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production.

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A; Britton, Robert; Bohlmann, Jörg

    2012-04-01

    The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

  7. Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

    2012-01-01

    The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

  8. Effects of pressure and osmolytes on the allosteric equilibria of Salmonella typhimurium tryptophan synthase.

    Science.gov (United States)

    Phillips, Robert S; Wang, Alexandre Kim; Marchal, Stephane; Lange, Reinhard

    2012-11-20

    Osmolytes are common constituents of bacteria that may be produced or accumulate at high concentrations, up to 1 M, when cells are subjected to stresses like ionic strength and temperature. However, the effects of osmolytes on the allosteric properties of bacterial enzymes have rarely been examined. We have studied the effects of osmolytes and hydrostatic pressure on the allosteric equilibria of Salmonella typhimurium tryptophan (Trp) synthase. Trp synthase is a well-studied multienzyme complex with activity tightly regulated by allosteric interactions between the ?- and ?-subunits. Trp synthase activity is affected by a wide range of physical parameters, including monovalent cations, pH, ligands, solvents, and hydrostatic pressure. Osmolytes, including betaine, taurine, sucrose, and polyethylene glycol, activate Trp synthase 2-3-fold in the absence of monovalent cations, indicating that osmolytes can stabilize the active closed conformation. However, in the presence of monovalent cations, osmolytes have only minor effects on activity and allosteric equilibria, but 1 M betaine stabilizes the Trp synthase-Ser-indoline complex against apparent pressure-induced subunit dissociation. Na(+) and K(+) are more effective at shifting the ?-aminoacrylate-indoline quinonoid equilibrium toward the quinonoid side, with a K(Q) of 8-10, than NH(4)(+)(K(Q) ~ 2). Furthermore, pressure-jump experiments show that the mechanism of indoline reaction to form a quinonoid complex may be different for the NH(4)(+) enzyme than the Na(+) and K(+) forms. These results show that osmolytes have subtle but significant effects on the allosteric properties of Trp synthase, and these effects may be important in vivo. PMID:23088292

  9. Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Tomomi [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Oikawa, Tadao; Muraoka, Ikuo [Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Soda, Kenji [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Hata, Yasuo, E-mail: hata@scl.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2007-11-01

    A psychrophilic malate dehydrogenase from the novel Antarctic bacterium F. frigidimaris KUC-1 was crystallized using the hanging-drop vapour-diffusion method. The crystals contained one tetrameric molecule per asymmetric unit. The best crystal diffracted to 1.8 Å resolution. Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 Å. It contains one tetrameric molecule in the asymmetric unit.

  10. 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 (NDA)

    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 ...

  11. Purification and characterization of prostaglandin F synthase from bovine liver.

    Science.gov (United States)

    Chen, L Y; Watanabe, K; Hayaishi, O

    1992-07-01

    Prostaglandin D2 11-ketoreductase activity of bovine liver was purified 340-fold to apparent homogeneity. The purified enzyme was a monomeric protein with a molecular weight of about 36 kDa, and had a broad substrate specificity for porstaglandins D1, D2, D3, and H2, and various carbonyl compounds (e.g., phenanthrenequinone and nitrobenzaldehyde, etc.). Prostaglandin D2 was reduced to 9 alpha,11 beta-prostaglandin F2 and prostaglandin H2 to prostaglandin F2 alpha with NADPH as a cofactor. Phenanthrenequinone competitively inhibited the reduction of prostaglandin D2, while it did not inhibit that of prostaglandin H2. Moreover, chloride ion stimulated the reduction of prostaglandin D2 and carbonyl compounds, while it had no effect on that of prostaglandin H2. Besides, the enzyme was inhibited by flavonoids (e.g., quercetin) that inhibit carbonyl reductase, but was not inhibited by barbital and sorbinil, which are the inhibitors of aldehyde and aldose reductases, respectively. These results indicate that the bovine liver enzyme has two different active sites, i.e., one for prostaglandin D2 and carbonyl compounds and the other for prostaglandin H2, and appears to be a kind of carbonyl reductase like bovine lung prostaglandin F synthase (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O., 1985, J. Biol. Chem. 260, 7035-7041). However, the bovine liver enzyme was different from prostaglandin F synthase of bovine lung with regard to the Km value for prostaglandin D2 (10 microM for the liver enzyme and 120 microM for the lung enzyme), the sensitivity to chloride ion (threefold greater activation for the liver enzyme) and the inhibition by CuSO4 and HgCl2 (two orders of magnitude more resistant in the case of the liver enzyme). These results suggest that the bovine liver enzyme is a subtype of bovine lung prostaglandin F synthase. PMID:1605628

  12. 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 le...

  13. Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells

    International Nuclear Information System (INIS)

    Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase and thymidylate synthase when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence of enhanced expression of the thymidylate synthase cycle

  14. Implication of citrate, malate and histidine in the accumulation and transport of nickel in Mesembryanthemum crystallinum and Brassica juncea.

    Science.gov (United States)

    Amari, Taoufik; Lutts, Stanley; Taamali, Manel; Lucchini, Giorgio; Sacchi, Gian Attilio; Abdelly, Chedly; Ghnaya, Tahar

    2016-04-01

    Citrate, malate and histidine have been involved in many processes including metal tolerance and accumulation in plants. These molecules have been frequently reported to be the potential nickel chelators, which most likely facilitate metal transport through xylem. In this context, we assess here, the relationship between organics acids and histidine content and nickel accumulation in Mesembryanthemum crystallinum and Brassica juncea grown in hydroponic media added with 25, 50 and 100µM NiCl2. Results showed that M. crystallinum is relatively more tolerant to Ni toxicity than B. juncea. For both species, xylem transport rate of Ni increased with increasing Ni supply. A positive correlation was established between nickel and citrate concentrations in the xylem sap. In the shoot of B. juncea, citric and malic acids concentrations were significantly higher than in the shoot of M. crystallinum. Also, the shoots and roots of B. juncea accumulated much more histidine. In contrast, a higher root citrate concentration was observed in M. crystallinum. These findings suggest a specific involvement of malic and citric acid in Ni translocation and accumulation in M. crystallinum and B. juncea. The high citrate and histidine accumulation especially at 100µM NiCl2, in the roots of M. crystallinum might be among the important factors associated with the tolerance of this halophyte to toxic Ni levels. PMID:26745003

  15. Multiple isotope effects as a probe of the tartrate dehydrogenase-catalysed oxidative decarboxylation of D-malate

    International Nuclear Information System (INIS)

    A change in the dinucleotide reactant from NAD+ to more oxidizing APAD+ in the maleic enzyme reaction results in a change in the mechanism of oxidative decarboxylation of malate from stepwise to concerted. In order to determine w heather this is a phenomenon general to metal ion dependent ?-hydroxy acid oxidative decarboxylases, tartrate dehydrogenase, which catalyses a reaction diastereotopic to maleic enzyme, was studied using the technique of multiple isotope effects. A primary deuterium isotope effect of 1.41 on V/Kmaleate was measured as well as a primary 13C-isotope effect of 1.0096. A decrease in the measured value of the 13C-isotope effect to 1.0078 is consistent with a stepwise mechanism, as observed in maleic enzyme. The 13C-isotope effect with thioNAD+ also decreases from a value of 1.0053 using D-maleate to 1.0009 using D-maleate-2-D, consistent with stepwise oxidative decarboxylation with this alternative dinucleotide substrate. The data suggest that the change from a stepwise to a concerted mechanism with maleic enzyme may be a unique phenomenon. (author)

  16. Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene from Chromobacterium violaceum

    OpenAIRE

    Kolibachuk, Dana; Miller, Andrea; Dennis, Douglas

    1999-01-01

    The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaCCv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaCCv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaACv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when gr...

  17. Synthesis of N-(Methoxycarbonylthienylmethylthioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Michael D. Threadgill

    2010-04-01

    Full Text Available Two isomeric N-(methoxycarbonylthienylmethylthioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS.

  18. Affinity of Cystathionine ?-Synthase for Pyridoxal 5?-Phosphate in Cultured Cells: A MECHANISM FOR PYRIDOXINE-RESPONSIVE HOMOCYSTINURIA

    OpenAIRE

    Lipson, Mark H.; Kraus, Jan; Rosenberg, Leon E.

    1980-01-01

    Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine ?-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating...

  19. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation.

    Science.gov (United States)

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle

    2014-02-15

    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities. PMID:24338369

  20. Bacillus PHA Synthase III C Gene showed Regulatory Functions: An In-silico Analysis

    Directory of Open Access Journals (Sweden)

    Gargi N. Edkie

    2014-01-01

    Full Text Available PHA synthase IIIC gene of 1114 bp isolated from Bacillus is AT rich. Scratch Protein Predictor analysis suggest that the protein is globular in nature and predicted 5 disulphide bridges. GRAVY index suggests that the protein is hydrophobic in nature. SOPMA analysis indicate that the predicted protein possess 44.71% alpha-helices with transmembrane domains, possibly play a role in cell recognition and signalling. Prediction of the position of helices using EXPASY tool suggest that Bacillus PHA synthase IIIC has two major helices with lengthy NORS region. Prosite- prediction analysis suggests that Bacillus PHA synthase IIIC has potential N-glycosylation, myristylation, protein kinase C and casein kinase II type phosphorylation domains. These are responsible for general metabolic activities associated with the protein and most of them are located in NORS region. Sequence alignment of PHA synthase IIIC using ClustalW program and MEGA4.0 program showed 96% identity with Bacillus megaterium PHA synthase IIIC gene.

  1. Cyclopropane fatty acid synthase from Oenococcus oeni: expression in Lactococcus lactis subsp. cremoris and biochemical characterization.

    Science.gov (United States)

    To, Thi Mai Huong; Grandvalet, Cosette; Alexandre, Hervé; Tourdot-Maréchal, Raphaëlle

    2015-11-01

    Bacterial cyclopropane fatty acid synthases (CFA synthases) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the double bond of a lipid chain, thereby forming a cyclopropane ring. CFAs contribute to resistance to acidity, dryness, and osmotic imbalance in many bacteria. This work describes the first biochemical characterization of a lactic acid bacterium CFA synthase. We have overexpressed Oenococcus oeni CFA synthase in E. coli in order to purify the enzyme. The optimum cyclopropanation activity was obtained at pH 5.6 and 35.8 °C. The high K m (AdoMet) value obtained (2.26 mM) demonstrates the low affinity of O. oeni enzyme toward the L. lactis subsp. cremoris unsaturated phospholipids. These results explain the partial complementation of the L. lactis subsp. cremoris cfa mutant by the O. oeni cfa gene and suggest a probable substrate specificity of the O. oeni enzyme. The current study reveals an essential hypothesis about the specificity of O. oeni CFA synthase which could play a key function in the acid tolerance mechanisms of this enological bacterium. PMID:26294376

  2. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    International Nuclear Information System (INIS)

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy ? and originally identified CoA synthase, CoASy ?. The transcript specific for CoASy ? was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy ?. In contrast to CoASy ?, which shows ubiquitous expression, CoASy ? is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  3. Identification of a novel CoA synthase isoform, which is primarily expressed in the brain.

    Science.gov (United States)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy beta and originally identified CoA synthase, CoASy alpha. The transcript specific for CoASy beta was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy beta. In contrast to CoASy alpha, which shows ubiquitous expression, CoASy beta is primarily expressed in the brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in the brain, requires further elucidation. PMID:16460672

  4. Producing dicarboxylic acids using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  5. Loop residues and catalysis in OMP synthase

    DEFF Research Database (Denmark)

    Wang, Gary P.; Hansen, Michael Riis; Grubmeyer, Charles

    2012-01-01

    Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100?109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 104-fold decrease...

  6. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    Directory of Open Access Journals (Sweden)

    Hsueh-Fen Juan

    2011-08-01

    Full Text Available Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs.

  7. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells.

    OpenAIRE

    Wu, K. K.; Sanduja, R; Tsai, A. L.; Ferhanoglu, B.; Loose-Mitchell, D S

    1991-01-01

    Prostaglandin H (PGH) synthase (EC 1.14.99.1) 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 (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-i...

  8. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica; Svensson, Birte

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity fo...

  9. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms......+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit the B. caldolyticus enzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP. Ki values are determined as 113 and 490 microm for ADP and GDP, respectively. At low concentrations ADP inhibition is...

  10. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s a...

  11. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS).

    Science.gov (United States)

    Zhan, Xin; Bach, Søren Spanner; Hansen, Nikolaj Lervad; Lunde, Christina; Simonsen, Henrik Toft

    2015-11-01

    The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis. PMID:26248039

  12. Identification of novel isoprene synthases through genome mining and expression in Escherichia coli.

    Science.gov (United States)

    Ilmén, Marja; Oja, Merja; Huuskonen, Anne; Lee, Sangmin; Ruohonen, Laura; Jung, Simon

    2015-09-01

    Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls. PMID:26275749

  13. 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 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.

  14. Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

    Science.gov (United States)

    Bartee, David; Morris, Francine; Al-Khouja, Amer; Freel Meyers, Caren L

    2015-08-17

    1-Deoxy-D-xylulose 5-phosphate (DX