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Sample records for acetolactate synthase inhibitor

  1. MULTI-ANALYTE CHEMISTRY METHODS FOR PESTICIDES WHICH ARE ACETOLACTATE SYNTHASE (ALS) INHIBITORS IN SOIL

    Science.gov (United States)

    A joint EPA/state/industry working group has developed several multi-analyte methods to analyze soils for low ppb (parts per billion) levels of herbicides (such as sulfonylureas, imidazolinones, and sulfonamides) that are acetolactate synthase (ALS) inhibitors and may cause phyto...

  2. ECOLOGICAL FITNESS OF ACETOLACTATE SYNTHASE INHIBITOR–RESISTANT AND –SUSCEPTIBLE DOWNY BROME (BROMUS TECTORUM) BIOTYPES

    Science.gov (United States)

    Studies were conducted to determine the relative fitness and competitive ability of an acetolactate synthase (ALS) inhibitor–resistant (R) downy brome biotype compared with a susceptible (S) biotype. In previous research, the mechanism of resistance was determined to be an altered ALS enzyme. Seed g...

  3. Effect of Acetolactate Synthase Inhibitor Herbicides on Upland Rice (Oryza Sativa Linn.) Cultivars

    OpenAIRE

    Fabiano André Petter; Alan Mario Zuffo; Leandro Pereira Pacheco

    2013-01-01

    This study aimed to evaluate the selectivity of the acetolactate synthase (ALS) inhibitor recommended herbicides for upland rice cultivars on different developmental stages. The experiment was conducted in the field, in Nova Xavantina-MT, Mato Grosso, Brazil, from season 2009/2010. The experimental design was the one of randomized blocks in factorial scheme, composed by the herbicide treatments penoxsulam (36 g ha-1); bispyribac-sodium (50 g ha-1); pyrazosulfuron-ethyl (20 g ha-1) and weeded ...

  4. Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

    Science.gov (United States)

    Richie, Daryl L; Thompson, Katherine V; Studer, Christian; Prindle, Vivian C; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N Rao; Tallarico, John A; Ryder, Neil S; Hoepfner, Dominic

    2013-05-01

    High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. PMID:23478965

  5. The current state of resistance to acetohydroxyacid/acetolactate synthase inhibitors

    Science.gov (United States)

    The acetohydroxyacid/acetolactate synthase (ALS) inhibiting herbicides are used for weed management in multiple crop and non-crop situations. Herbicides with this mechanism of action were introduced in the early 1980s and quickly came to dominate many cropping situations due to their broad spectrum...

  6. Influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation, regulation and organisation of photosynthesis in Solanum nigrum

    OpenAIRE

    Riethmuller-Haage, I.C.P.; Bastiaans, L.; Harbinson, J.; kempenaar, C.; Kropff, M. J.

    2006-01-01

    The influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation of the photosynthetic apparatus was examined on 4-weeks-old climate chamber-grown Solanum nigrum plant. To have an indication on the relative performance of the photosynthetic apparatus of ALS-treated plants, the level of carbon dioxide (CO2) fixation, the relative quantum efficiency of photosystem I (¿PSI) or photosystem II (¿PSII) electron transport and leaf chlorophyll content were assessed for both co...

  7. Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II.

    OpenAIRE

    Dyk, T. K.; Larossa, R. A.

    1986-01-01

    Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium. Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with Tn10. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The Tn10 insertion in strain SMS409 was mapped by conjugation and transduction to the region b...

  8. Multiple resistance of acetolactate synthase and protoporphyrinogen oxidase inhibitors in Euphorbia heterophylla biotypes.

    Science.gov (United States)

    Trezzi, Michelangelo M; Felippi, C L; Mattei, D; Silva, H L; Nunes, A L; Debastiani, C; Vidal, R A; Marques, A

    2005-01-01

    Resistance to acetolactate synthase (ALS)-inhibiting herbicides in Brazil has been documented for six species. The probability to select biotypes of Euphorbia heterophylla (EPPHL) with multiple resistance increases in the same order of magnitude as the use of other herbicides belonging to only one mechanism of action. The objectives of this work were to evaluate the distribution of resistant populations (R) in the states of the Parana and Santa Catarina; to determine the existence of populations of EPHHL with multiple resistance to ALS and PROTOX inhibitors, and to confirm the occurrence of cross resistance to compounds of these mechanisms of action. Seeds of EPHHL of areas with suspected resistance had been sampled in 97 places during 2003. In the greenhouse experiment samples of each population were sprayed with imazethapyr or fomesafen, at only one rate. To identify the resistant ones they were sprayed with different levels of the herbicides imazethapyr and fomesafen. Later they were sprayed with diverse herbicides of the same mechanisms of action to confirm the multiple/cross resistance. There is widespread distribution in the region of populations with resistance to ALS inhibitors. Some biotypes demonstrated resistance to herbicides from the two mechanisms of action. The resistance factor (FR), or the relation of resistance between R and susceptible biotypes, confirms the existence of two biotypes of EPHHL with cross resistance to several herbicides inhibitors of ALS and PROTOX. PMID:15656167

  9. Biology, management and biochemical/genetic characterization of weed biotypes resistant to acetolactate synthase inhibitor herbicides

    Directory of Open Access Journals (Sweden)

    Monquero Patrícia Andrea

    2003-01-01

    Full Text Available Bidens pilosa and Amaranthus quitensis are major weeds infesting soybean [Glycine max L (Merrill] fields in Brazil and Argentina. The repetitive use of acetolactate synthase (ALS EC 4.1.3.18 inhibiting herbicides in São Gabriel do Oeste, MS, Brazil and in the provinces of Córdoba and Tucumã, Argentina, has selected for resistant (R biotypes of these weeds. Research work was developed to study the management, growth, biochemistry, and genetics of these R weed biotypes. In a field experiment it was found that chlorimuron-ethyl and imazethapyr at recommended rates (both ALS inhibitor herbicides, did not control R B. pilosa, but the alternative lactofen, fomesafen and bentazon were effective, either sprayed alone or mixed with the ALS inhibitor herbicides. Greenhouse studies confirmed the cross-resistance of both R biotypes to the imidazolinone and sulfonylurea herbicides, and these alternative herbicides, when sprayed alone or mixed with the ALS inhibitor, efficiently controlled both R and S populations. A growth analysis of the R and S biotypes of these weeds, under non-competitive conditions, indicated that there is no adaptive cost to the R biotypes (pleiotropic effect. A quick bioassay using ALS and ketoacid reductoisomerase (KARI inhibitors showed that the resistance of the R biotypes to herbicides is related to a lack of sensitivity of the ALS enzyme to the herbicides. On the other hand, the sequencing of the gene that codifies the ALS resistance in R A. quitensis did not present any mutation in the A Domain region, suggesting that other positions of the gene that confer insensitivity of the ALS to sulfonylurea and imidazolinone herbicides could have mutated.

  10. Effect of Acetolactate Synthase Inhibitor Herbicides on Upland Rice (Oryza Sativa Linn. Cultivars

    Directory of Open Access Journals (Sweden)

    Fabiano André Petter

    2013-09-01

    Full Text Available This study aimed to evaluate the selectivity of the acetolactate synthase (ALS inhibitor recommended herbicides for upland rice cultivars on different developmental stages. The experiment was conducted in the field, in Nova Xavantina-MT, Mato Grosso, Brazil, from season 2009/2010. The experimental design was the one of randomized blocks in factorial scheme, composed by the herbicide treatments penoxsulam (36 g ha-1; bispyribac-sodium (50 g ha-1; pyrazosulfuron-ethyl (20 g ha-1 and weeded control. Herbicides were applied at three times: 15, 30 and 45 days after emergence (DAE with four replications in two upland rice cultivars: BRS Pepita and BRS Monarca. At 7, 14 and 28 days after application (DAA the following assessments were performed: phytotoxicity to the crop, plant height, dry weight, number of panicles m-2, grains per panicle-1 and productivity. The highest levels of phytotoxicity were observed in plants treated with bispyribac-sodium applied at 15 and 30 DAE. The rice plants were able to recover as for height influenced by herbicides from 14 DAA. The dry weight of plants was not affected by herbicide application. Since they have not reduced the grain productivity, the herbicides tested, when applied at 30 DAE, have showed their potential to be used only in BRS Monarca cultivar.

  11. Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.).

    Science.gov (United States)

    Liu, Weitang; Bi, Yaling; Li, Lingxu; Yuan, Guohui; Du, Long; Wang, Jinxin

    2013-09-01

    Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I50 value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene. PMID:25149235

  12. Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

    OpenAIRE

    Richie, Daryl L.; Thompson, Katherine V.; Studer, Christian; Prindle, Vivian C.; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A.; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Ju?rg; Schuierer, Sven; Hartmann, Nicole

    2013-01-01

    High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the ...

  13. Influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation, regulation and organisation of photosynthesis in Solanum nigrum.

    Science.gov (United States)

    Riethmuller-Haage, Ingrid; Bastiaans, Lammert; Harbinson, Jeremy; Kempenaar, Corné; Kropff, Martin J

    2006-06-01

    The influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation of the photosynthetic apparatus was examined on 4-weeks-old climate chamber-grown Solanum nigrum plant. To have an indication on the relative performance of the photosynthetic apparatus of ALS-treated plants, the level of carbon dioxide (CO(2)) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) or photosystem II (Phi(PSII)) electron transport and leaf chlorophyll content were assessed for both control and treated plants at 2, 4 and 7 days after application of the herbicide. Results indicated a progressive inhibition of the level of CO(2) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) and II (Phi(PSII)) electron transport and the leaf chlorophyll content already 2 days after application of the herbicide. The linear relationship between the photosystem I and II was unaltered by herbicidal treatment and was sustained under conditions where large changes in pigment composition of the leaves occurred. It appears that the stress-induced loss of leaf chlorophyll is not a catastrophic process but rather is the consequence of a well-organised breakdown of components. Under photorespiratory and non-photorespiratory conditions, the relationship between the index of electron transport flow through photosystem I and II and the rate of CO(2) fixation is altered so that electron transport becomes less efficient at driving CO(2) fixation. PMID:16691366

  14. ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl.

    OpenAIRE

    Larossa, R. A.; Smulski, D. R.

    1984-01-01

    The herbicide sulfometuron methyl is a potent inhibitor of the branched-chain amino acid biosynthetic enzyme acetolactate synthase (ALS) isolated from bacteria, fungi, and plants. However, it did not prevent growth of wild-type Salmonella typhimurium LT2 or Escherichia coli K-12. These species each contain two acetolactate synthase isozymes. Growth of S. typhimurium and E. coli mutants lacking ALS I was prevented by the herbicide, suggesting that activity of the remaining ALS isoenzyme (II or...

  15. Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon.

    Science.gov (United States)

    Iwakami, Satoshi; Endo, Masaki; Saika, Hiroaki; Okuno, Junichi; Nakamura, Naoki; Yokoyama, Masao; Watanabe, Hiroaki; Toki, Seiichi; Uchino, Akira; Inamura, Tatsuya

    2014-04-23

    Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon. PMID:24760819

  16. Bioensaio rápido de determinação da sensibilidade da acetolactato sintase (ALS) a herbicidas inibidores / Rapid bioassay to determine the sensitivity of acetolactate synthase (ALS) to inhibitor herbicides

    Scientific Electronic Library Online (English)

    Patrícia Andrea, Monqueiro; Pedro Jacob, Christoffoleti.

    2001-03-01

    Full Text Available Foi avaliada a atividade da acetolactato sintase (ALS), em plantas resistentes e suscetíveis de B. pilosa e A. quitensis após a aplicação de herbicidas inibidores da ALS. O método baseia-se na utilização do ácido ciclopropanodicarboxílico (CPCA) para inibir a cetoácido reductoisomerase (KARI), enzim [...] a que catalisa a reação seguinte do acetolactato na cadeia de biossíntese dos aminoácidos valina, leucina e isoleucina, provocando assim, o acúmulo de acetolactato, que na presença de um ácido forte forma acetoína. A base para a distinção entre os biotipos resistentes e suscetíveis é a quantidade de acetoína formada, que será maior nos biotipos em que a enzima ALS não sofreu inibição, ou seja, nos biotipos resistentes. A quantificação da acetoína acumulada ocorreu através da formação de um complexo colorido vermelho, devido a reação entre acetoína, creatina e naftol, cuja densidade ótica a 530 nm é proporcional à concentração do acetolactato formado na reação. Sendo assim, foi desenvolvido um ensaio utilizando este método após a aplicação dos herbicidas chlorimuron-ethyl e imazethapyr nos biotipos R e S de Bidens pilosa, Amaranthus quitensis no estádio de dois pares de folhas. O bioensaio demonstrou que a enzima ALS dos biotipos resistentes é insensível aos herbicidas inibidores da ALS e que este tipo de bioensaio é uma forma rápida e eficaz de diferenciação entre biotipos resistentes e suscetíveis. Abstract in english In order to compare the acetolactate synthase (ALS) activity of resistant and susceptible biotypes of Bidens pilosa and Amaranthus quitensis to ALS inhibitor herbicides, a method based on ciclopronocarboxilic acid (CPCA) to inhibit the enzyme ketoacidredutoisomerase (KARI) is used. This enzyme catal [...] yzes the reaction after acetolactate in the biosynthesis reaction chain of the aminoacids valine, leucine and isoleucine. In the presence of a KARI inhibitor, carbon from pyruvate flows through the branched chain aminoacid biosynthetic pathway and accumulates in acetolactate, which in the presence of sulfuric acid can be converted to acetoin. The base to distinguish between the resistant and susceptible biotypes is the amount of acetoin formed, which will be much higher in the biotype where the ALS was not inhibited by the herbicide. If acetoin is mixed with naphtol and creatine the solution will develop a reddish color, so that it is possible to quantify indirectly the sensitivity of the ALS to the herbicide by the color of the solution formed. An experiment was carried out with suspected resistant biotypes of Bidens pilosa and Amaranthus quitensis using this method after spraying the plants at the two pair leaf stage with chlorimuron-ethyl and imazethapyr. The ALS of the resistant biotype has insensitivity to ALS inhibitor herbicides.

  17. Resistência de amendoim-bravo aos herbicidas inibidores da enzima acetolactato sintase / Wild poinsettia resistance to acetolactate synthase inhibitor herbicides

    Scientific Electronic Library Online (English)

    Ribas A., Vidal; Aldo, Merotto Jr..

    1999-12-01

    Full Text Available O controle contínuo de plantas daninhas através da aplicação de herbicidas que apresentam atividade em um único local de ação nas plantas favorece a seleção de biótipos resistentes a estes herbicidas, em certas espécies vegetais. Quatro experimentos foram conduzidos em condições casa-de-vegetação, n [...] a Faculdade de Agronomia da Universidade Federal do Rio Grande do Sul, com os objetivos de avaliar a ocorrência de resistência aos herbicidas inibidores da enzima acetolactato sintase (ALS) em vários biótipos de leiteiro ou amendoim-bravo (Euphorbia heterophylla EPHHL) e avaliar a ocorrência de resistência múltipla a herbicidas com atividade em outros locais de ação. Biótipo oriundo de Passo Fundo foi resistente ao imazethapyr, enquanto biótipo oriundo de Porto Alegre foi suscetível. O biótipo de Passo Fundo apresentou resistência cruzada aos herbicidas imidazolinonas: imazapyr, imazaquin e imazethapyr; sulfoniluréias: chlorimuron, nicosulfuron e metsulfuron; e sulfonanilida: flumetsulan. Este biótipo não foi resistente aos herbicidas com os seguintes mecanismos de ação: inibidores de EPSPs, mimetizadores de auxina, inibidores dos fotossistemas I e II e inibidores de PROTOX. A confirmação de resistência aos inibidores de ALS em biótipos oriundos de Nãome-Toque, Passo Fundo e Rio Pardo sugere ampla dispersão no Rio Grande do Sul de resistência de E. heterophylla aos herbicidas deste mecanismo de ação. Abstract in english The continuous weed control with herbicides of only one site of action selects biotypes resistant to these herbicides. Four experiments were conducted in greenhouse of UFRGS, Brazil, to confirm the occurence of wild poinsettia (Euphorbia heterophylla) biotypes resistance to herbicides inhibitors of [...] acetholactate synthase (ALS), and to determine whether there was cross resistance to herbicides with other site of action. A biotype from Passo Fundo -RS was resistant to imazethapyr, whereas a biotype from Porto Alegre -RS was susceptible to this compound. The biotype from Passo Fundo was resistant to the following ALS-inhibitors: imazapyr, imazaquin, imazethapyr, chlorimuron, nicosulfuron, metsulfuron e flumetsulan. This biotype was not resistant to herbicides from the following modes of action: EPSPs inhibitors, auxin agonists, fotossystems I and II inhibitors, and PROTOX inhibitors. The confirmation of resistance to ALS inhibitors in biotypes from Não-me-Toque, Passo Fundo and Rio Pardo suggests a wide spread of wild poinsettia resistance to compounds of this mode of action in the Rio Grande do Sul state.

  18. Resistência de amendoim-bravo aos herbicidas inibidores da enzima acetolactato sintase Wild poinsettia resistance to acetolactate synthase inhibitor herbicides

    Directory of Open Access Journals (Sweden)

    Ribas A. Vidal

    1999-12-01

    Full Text Available O controle contínuo de plantas daninhas através da aplicação de herbicidas que apresentam atividade em um único local de ação nas plantas favorece a seleção de biótipos resistentes a estes herbicidas, em certas espécies vegetais. Quatro experimentos foram conduzidos em condições casa-de-vegetação, na Faculdade de Agronomia da Universidade Federal do Rio Grande do Sul, com os objetivos de avaliar a ocorrência de resistência aos herbicidas inibidores da enzima acetolactato sintase (ALS em vários biótipos de leiteiro ou amendoim-bravo (Euphorbia heterophylla EPHHL e avaliar a ocorrência de resistência múltipla a herbicidas com atividade em outros locais de ação. Biótipo oriundo de Passo Fundo foi resistente ao imazethapyr, enquanto biótipo oriundo de Porto Alegre foi suscetível. O biótipo de Passo Fundo apresentou resistência cruzada aos herbicidas imidazolinonas: imazapyr, imazaquin e imazethapyr; sulfoniluréias: chlorimuron, nicosulfuron e metsulfuron; e sulfonanilida: flumetsulan. Este biótipo não foi resistente aos herbicidas com os seguintes mecanismos de ação: inibidores de EPSPs, mimetizadores de auxina, inibidores dos fotossistemas I e II e inibidores de PROTOX. A confirmação de resistência aos inibidores de ALS em biótipos oriundos de Nãome-Toque, Passo Fundo e Rio Pardo sugere ampla dispersão no Rio Grande do Sul de resistência de E. heterophylla aos herbicidas deste mecanismo de ação.The continuous weed control with herbicides of only one site of action selects biotypes resistant to these herbicides. Four experiments were conducted in greenhouse of UFRGS, Brazil, to confirm the occurence of wild poinsettia (Euphorbia heterophylla biotypes resistance to herbicides inhibitors of acetholactate synthase (ALS, and to determine whether there was cross resistance to herbicides with other site of action. A biotype from Passo Fundo -RS was resistant to imazethapyr, whereas a biotype from Porto Alegre -RS was susceptible to this compound. The biotype from Passo Fundo was resistant to the following ALS-inhibitors: imazapyr, imazaquin, imazethapyr, chlorimuron, nicosulfuron, metsulfuron e flumetsulan. This biotype was not resistant to herbicides from the following modes of action: EPSPs inhibitors, auxin agonists, fotossystems I and II inhibitors, and PROTOX inhibitors. The confirmation of resistance to ALS inhibitors in biotypes from Não-me-Toque, Passo Fundo and Rio Pardo suggests a wide spread of wild poinsettia resistance to compounds of this mode of action in the Rio Grande do Sul state.

  19. Reference genes to study herbicide stress response in Lolium sp.: up-regulation of P450 genes in plants resistant to acetolactate-synthase inhibitors.

    Science.gov (United States)

    Duhoux, Arnaud; Délye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

  20. A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

    Science.gov (United States)

    Liu, Weitang; Yuan, Guohui; Du, Long; Guo, Wenlei; Li, Lingxu; Bi, Yaling; Wang, Jinxin

    2015-01-01

    Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB,?> 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP,?> 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation. PMID:25619909

  1. New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

    International Nuclear Information System (INIS)

    The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyrvuate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, and initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [14C]chlorsulfuron and [14C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency

  2. Functional expression of plant acetolactate synthase genes in Escherichia coli

    OpenAIRE

    Smith, Julie K.; Schloss, John V.; Mazur, Barbara J.

    1989-01-01

    Acetolactate synthase (ALS; EC 4.1.3.18) is the first common enzyme in the biosynthetic pathways leading to leucine, isoleucine, and valine. It is the target enzyme for three classes of structurally unrelated herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. A cloned ALS gene from the small cruciferous plant Arabidopsis thaliana has been fused to bacterial transcription/translation signals and the resulting plasmid has been used to transform Escherichia coli. The...

  3. Absorption and translocation of imazethapyr as a mechanism responsible for resistance of Euphorbia heterophylla L. biotypes to acetolactate synthase (ALS inhibitors

    Directory of Open Access Journals (Sweden)

    Plaza Guido A.

    2006-12-01

    Full Text Available

    The effect of weeds on reduction of agricultural production is estimated between 30% and 50%. Imazethapyr is a herbicide of imidazolinone group that inhibits activity of enzyme acetolactate synthase (ALS, the first common enzyme in the biosynthetic pathway of valine, leucine, and isoleucine. Euphorbia heterophylla is common specie in soybean fields of Brazil. The study reports about a population of Euphorbia heterophylla resistant to imazethapyr. The objectives of the present work were to quantify the level of sensitivity to this herbicide in imazethapyr-resistant and -susceptible E.  heterophylla populations evaluate the role of differential penetration into leaves as determining plant resistance to imazethapyr, and compare the waxy cells of R and S populations. The R population had a lower penetration rate compared with that of S population during the six first hours of incubation with the herbicide. Further studies indicated that R population was not different from S population in terms of translocation, metabolism, or target site (ALS enzyme of imazethapyr action. Analysis of the leaf cuticle surface by scanning electron microscopy revealed higher wax density in the leaf cuticles of population R than that in S population. Thus, it is suggested that R population is resistant  to imazethapyr because increased wax content of its cuticle permits less penetration of herbicide into the plant.

  4. Detailed structure-function correlations of Bacillus subtilis acetolactate synthase.

    Science.gov (United States)

    Sommer, Bettina; von Moeller, Holger; Haack, Martina; Qoura, Farah; Langner, Clemens; Bourenkov, Gleb; Garbe, Daniel; Loll, Bernhard; Brück, Thomas

    2015-01-01

    Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues. PMID:25393087

  5. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl

    OpenAIRE

    Yadav, Narendra; Mcdevitt, Raymond E.; Benard, Susan; Falco, S. Carl

    1986-01-01

    Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from Escherichia coli and Saccharomyces cerevisiae. The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucle...

  6. Toxic accumulation of alpha-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium.

    OpenAIRE

    Larossa, R. A.; Dyk, T. K.; Smulski, D. R.

    1987-01-01

    Biochemical and genetic analyses of the bacterium Salmonella typhimurium suggest that accumulation of alpha-ketobutyrate partially mediates the herbicidal activity of acetolactate synthase inhibitors. Growth inhibition of wild-type bacteria by the herbicide sulfometuron methyl was prevented by supplementing the medium with isoleucine, an allosteric inhibitor of threonine deaminase-catalyzed synthesis of alpha-ketobutyrate. In contrast, isoleucine did not rescue the growth of a mutant containi...

  7. Biology, management and biochemical/genetic characterization of weed biotypes resistant to acetolactate synthase inhibitor herbicides / Biologia, manejo e caracterização bioquímica e genética de biótipos resistentes aos herbicidas inibidores da acetolactato sintase

    Scientific Electronic Library Online (English)

    Patrícia Andrea, Monquero; Pedro Jacob, Christoffoleti; Helaine, Carrer.

    Full Text Available Bidens pilosa e Amaranthus quitensis são as principais plantas daninhas infestantes na cultura de soja [Glycine max L (Merrill)] no Brasil e Argentina, respectivamente. O uso repetitivo de herbicidas inibidores da acetolactato sintase (ALS EC 4.1.3.18) em São Gabriel do Oeste (MS - Brasil) e nas pro [...] víncias de Córdoba e Tucumã (Argentina), selecionaram biótipos resistentes (R) destas plantas daninhas. Esta pesquisa foi desenvolvida para estudar o manejo, crescimento, a bioquímica e genética destes biótipos resistentes. Em um experimento de campo concluiu-se que chlorimuron-ethyl e imazethapyr (inibidores da ALS), aplicados nas doses recomendadas, não controlaram o biótipo R de B. pilosa, mas os herbicidas alternativos lactofen, fomesafen e bentazon foram eficientes quando aplicados sozinhos ou em mistura com os herbicidas inibidores da ALS. Estudos em casa-de-vegetação confirmaram a resistência cruzada para os biótipos de ambas espécies aos herbicidas dos grupos químicos das imidazolinonas e sulfuniluréias e os herbicidas alternativos sozinhos ou em mistura com os inibidores da ALS controlaram eficientemente populações resistentes e suscetíveis. Análises de crescimento dos biótipos R e S destas plantas daninhas em condições não competitivas mostraram que não existe um custo adaptativo para os biótipos R (efeitos pleiotrópicos). O bioensaio rápido usando inibidores da ALS e ketoacid reductoisomerase (KARI) indicaram que a resistência decorre da insensibilidade da enzima ALS aos herbicidas. Por outro lado, o seqüenciamento do gene que codifica a ALS em R A. quitensis não mostrou mutação no Domínio A, sugerindo que outras posições do gene poderiam estar sofrendo mutações que conferem a insensibilidade da ALS a sulfuniluréias e imidazolinonas. Abstract in english Bidens pilosa and Amaranthus quitensis are major weeds infesting soybean [Glycine max L (Merrill)] fields in Brazil and Argentina. The repetitive use of acetolactate synthase (ALS EC 4.1.3.18) inhibiting herbicides in São Gabriel do Oeste, MS, Brazil and in the provinces of Córdoba and Tucumã, Argen [...] tina, has selected for resistant (R) biotypes of these weeds. Research work was developed to study the management, growth, biochemistry, and genetics of these R weed biotypes. In a field experiment it was found that chlorimuron-ethyl and imazethapyr at recommended rates (both ALS inhibitor herbicides), did not control R B. pilosa, but the alternative lactofen, fomesafen and bentazon were effective, either sprayed alone or mixed with the ALS inhibitor herbicides. Greenhouse studies confirmed the cross-resistance of both R biotypes to the imidazolinone and sulfonylurea herbicides, and these alternative herbicides, when sprayed alone or mixed with the ALS inhibitor, efficiently controlled both R and S populations. A growth analysis of the R and S biotypes of these weeds, under non-competitive conditions, indicated that there is no adaptive cost to the R biotypes (pleiotropic effect). A quick bioassay using ALS and ketoacid reductoisomerase (KARI) inhibitors showed that the resistance of the R biotypes to herbicides is related to a lack of sensitivity of the ALS enzyme to the herbicides. On the other hand, the sequencing of the gene that codifies the ALS resistance in R A. quitensis did not present any mutation in the A Domain region, suggesting that other positions of the gene that confer insensitivity of the ALS to sulfonylurea and imidazolinone herbicides could have mutated.

  8. Manejo de Bidens subalternans resistente aos herbicidas inibidores da acetolactato sintase / Management of Bidens subalternans resistant to acetolactate synthase inhibitor herbicides

    Scientific Electronic Library Online (English)

    D.L.P., Gazziero; C.E.C., Prete; M., Sumiya.

    2003-08-01

    Full Text Available A extensão das áreas com seleção de populações de plantas daninhas resistentes a herbicidas tem aumentado rapidamente no Brasil nos últimos anos, sendo citado como causa principal desta seleção a recomendação inadequada de produtos. Com o objetivo de avaliar a eficácia de controle de plantas daninha [...] s através de herbicidas, com diferentes mecanismos de ação, sobre plantas de Bidens subalternans, foi conduzido o presente trabalho, que envolveu um experimento de casa de vegetação e dois de campo, com as culturas de milho e soja. A pesquisa foi realizada a partir de populações de plantas de Bidens subalternans com suspeita de resistência aos herbicidas inibidores da ALS encontradas em área de produção comercial nas quais ocorriam falhas de controle através desses herbicidas. Os resultados permitiram confirmar a seleção de populações resistentes aos herbicidas inibidores da acetolactato sintase (ALS) e encontrar alternativas para o manejo destas populações, por meio do uso de produtos com mecanismo de ação diferenciado, tanto para a cultura da soja quanto para a do milho. Produtos inibidores da protoporfirinogênio oxidase (PROTOX), da fotossíntese e da divisão celular, aplicados isoladamente ou em misturas, controlaram adequadamente o biótipo resistente. Abstract in english The acreage with herbicide resistant weed populations has rapidly increased in Brazil in recent years. Inadequate herbicide recommendation is pointed as the main cause of this problem. This study aimed to evaluate Bidens subalternans control efficacy through herbicides with alternative mechanisms of [...] action, consisting of a greenhouse and two field experiments, with corn and soybean crops. A Bidens subalternans population suspected to be resistant to ALS inhibitor herbicides, found in a commercial crop area, was used in the experiments. The results confirmed beggartick resistance to ALS inhibitor herbicides. Management alternatives found for this weed include herbicides recommended for soybean and corn with differentiated mechanism of action: protoporphyrinogen oxidase (PROTOX) inhibitors, mitotic disrupters and photosynthesis inhibitor herbicides, applied alone or in tank mixture.

  9. Manejo de Bidens subalternans resistente aos herbicidas inibidores da acetolactato sintase Management of Bidens subalternans resistant to acetolactate synthase inhibitor herbicides

    Directory of Open Access Journals (Sweden)

    D.L.P. Gazziero

    2003-08-01

    Full Text Available A extensão das áreas com seleção de populações de plantas daninhas resistentes a herbicidas tem aumentado rapidamente no Brasil nos últimos anos, sendo citado como causa principal desta seleção a recomendação inadequada de produtos. Com o objetivo de avaliar a eficácia de controle de plantas daninhas através de herbicidas, com diferentes mecanismos de ação, sobre plantas de Bidens subalternans, foi conduzido o presente trabalho, que envolveu um experimento de casa de vegetação e dois de campo, com as culturas de milho e soja. A pesquisa foi realizada a partir de populações de plantas de Bidens subalternans com suspeita de resistência aos herbicidas inibidores da ALS encontradas em área de produção comercial nas quais ocorriam falhas de controle através desses herbicidas. Os resultados permitiram confirmar a seleção de populações resistentes aos herbicidas inibidores da acetolactato sintase (ALS e encontrar alternativas para o manejo destas populações, por meio do uso de produtos com mecanismo de ação diferenciado, tanto para a cultura da soja quanto para a do milho. Produtos inibidores da protoporfirinogênio oxidase (PROTOX, da fotossíntese e da divisão celular, aplicados isoladamente ou em misturas, controlaram adequadamente o biótipo resistente.The acreage with herbicide resistant weed populations has rapidly increased in Brazil in recent years. Inadequate herbicide recommendation is pointed as the main cause of this problem. This study aimed to evaluate Bidens subalternans control efficacy through herbicides with alternative mechanisms of action, consisting of a greenhouse and two field experiments, with corn and soybean crops. A Bidens subalternans population suspected to be resistant to ALS inhibitor herbicides, found in a commercial crop area, was used in the experiments. The results confirmed beggartick resistance to ALS inhibitor herbicides. Management alternatives found for this weed include herbicides recommended for soybean and corn with differentiated mechanism of action: protoporphyrinogen oxidase (PROTOX inhibitors, mitotic disrupters and photosynthesis inhibitor herbicides, applied alone or in tank mixture.

  10. IMIDAZOLINONE-RESISTANT WHEAT ACETOLACTATE SYNTHASE IN VIVO RESPONSE TO IMAZAMOX

    Science.gov (United States)

    Several experiments were conducted to evaluate the utility of an in vivo acetolactate synthase (ALS) assay for comparing sensitivity to imazamox among imidazolinone-resistant wheat cultivars/lines. Ten single-gene imidazolinone-resistant winter wheat cultivars/lines, one two-gene and four single-gen...

  11. Resistance of Amaranthus retroflexus to acetolactate synthase inhibitor herbicides in Brazil / Resistência de Amaranthus retroflexus a herbicidas inibidores da enzima acetolactato sintase no Brasil

    Scientific Electronic Library Online (English)

    A.C., Francischini; J., Constantin; R.S., Oliveira Jr.; G., Santos; L.H.M., Franchini; D.F., Biffe.

    2014-06-01

    Full Text Available Quando em competição com a cultura do algodoeiro, Amaranthus retroflexus é capaz de promover grande perda de produtividade. Devido à limitada disponibilidade de herbicidas seletivos para controle em pós-emergência dessa espécie daninha, algumas moléculas têm sido usadas por safras seguidas, o que po [...] de ter levado à seleção de biótipos resistentes. Biótipos de A. retroflexus coletados das principais regiões produtoras de algodão do Brasil foram submetidos a ensaios de dose-resposta, por meio da aplicação de doses dos herbicidas trifloxysulfuron-sodium e pyrithiobac­sodium equivalentes a 0, ¼, ½, 1, 2 e 4 vezes a dose recomendada. Foi confirmada a ocorrência de biótipos de A. retroflexus resistentes aos herbicidas inibidores da enzima ALS. O biótipo MS 2, oriundo do Mato Grosso do Sul, apresentou resistência cruzada ao trifloxysulfuron-sodium e ao pyrithiobac-sodium, ao passo que o biótipo MS 1 mostrou resistência apenas ao trifloxysulfuron­sodium. Da mesma maneira, foram confirmados casos de resistência nos biótipos coletados no Estado de Goiás (GO 3, GO 4 e GO 6) aos herbicidas trifloxysulfuron-sodium e ao pyrithiobac-sodium, demonstrando resistência singular e cruzada. Um biótipo oriundo do Mato Grosso (MT 13) não apresentou resistência aos herbicidas inibidores da ALS testados. Abstract in english When in competition with cotton, Amaranthus retroflexus can cause high yield losses. Due to the limited availability of selective herbicides registered for post emergence control of this weed, the same herbicides have been used repeated times over the last few years, which may have selected resistan [...] t biotypes. Biotypes of A. retroflexus collected from the main areas of cotton cultivation in Brazil were submitted to dose-response trials, by applying the herbicides trifloxysulfuron-sodium and pyrithiobac-sodium in doses equivalent to 0, ¼, ½, 1, 2 and 4 times the recommended rates. Resistance to ALS inhibitors was confirmed in biotypes of A. retroflexus. Biotype MS 2 from Mato Grosso do Sul, was cross-resistant to both trifloxysulfuron-sodium and pyrithiobac-sodium, while biotype MS 1 was resistant to trifloxysulfuron-sodium only. Likewise, singular and cross resistance was also confirmed in biotypes from Goiás (GO 3, GO 4 and GO 6), in relation to trifloxysulfuron­sodium and pyrithiobac-sodium. One biotype from Mato Grosso (MT 13) was not resistant to any of the ALS inhibitors evaluated in this work.

  12. Protein trans-Splicing To Produce Herbicide-Resistant Acetolactate Synthase

    OpenAIRE

    Sun, Luo; Ghosh, Inca; Paulus, Henry; Xu, Ming-qun

    2001-01-01

    Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichi...

  13. Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase.

    OpenAIRE

    Falco, S. C.; Dumas, K. S.; Livak, K. J.

    1985-01-01

    We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS). ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl. The coding sequence for the ILV2 polypeptide contains 2061 base pairs. Comparison of deduced amino acid sequences indicates considerable conservation between the yeast protein and the large subunits of the E. coli ALS II and A...

  14. Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli?

    OpenAIRE

    Atsumi, Shota; Li, Zhen; Liao, James C.

    2009-01-01

    A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylatio...

  15. Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp.

    OpenAIRE

    Xing, R. Y.; Whitman, W. B.

    1987-01-01

    The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive...

  16. Down-regulation of acetolactate synthase compromises Ol-1- mediated resistance to powdery mildew in tomato

    OpenAIRE

    Gao, D.; Huibers, R. P.; Loonen, A. E. H. M.; Visser, R. G. F.; Wolters, A. M. A.; Bai, Y.

    2014-01-01

    Background - In a cDNA-AFLP analysis comparing transcript levels between powdery mildew (Oidium neolycopersici)-susceptible tomato cultivar Moneymaker (MM) and near isogenic lines (NILs) carrying resistance gene Ol-1 or Ol-4, a transcript-derived fragment (TDF) M11E69-195 was found to be present in NIL-Ol-1 but absent in MM and NIL-Ol-4. This TDF shows homology to acetolactate synthase (ALS). ALS is a key enzyme in the biosynthesis of branched-chain amino acids valine, leucine and isoleucine,...

  17. Establishing the Geographical Distribution and Level of Acetolactate Synthase Resistance of Palmer Amaranth (Amaranthus palmeri) Accessions in Georgia

    Science.gov (United States)

    Palmer amaranth resisitance to acetolactate synthase inhibiting (ALS) herbicides was first identified in Georgia in 2000. Since then, complaints from farmers have increased concerning failure of ALS-herbicides in controlling Palmer amaranth in peanut. Efficacy of ALS-herbicides can be compromised ...

  18. Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions.

    OpenAIRE

    Platteeuw, C.; Hugenholtz, J.; Starrenburg, M.; Alen-boerrigter, I.; Vos, W. M.

    1995-01-01

    The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. la...

  19. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    International Nuclear Information System (INIS)

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS

  20. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung [Hanyang Univ., Seoul (Korea, Republic of); Koo, Bonsung [Rural Development Administration, Suwon (Korea, Republic of)

    2013-02-15

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS.

  1. The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

    Directory of Open Access Journals (Sweden)

    Nanaka Kikuchi

    2012-03-01

    Full Text Available Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT and neomycin phosphotransferase II gene (NPT II. Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS from rice (W548L/S627IOsALS as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry. Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 ?M of bispyribac-sodium (BS as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP based detection procedures.

  2. Control of Glyphosate and Acetolactate Synthase Resistant Common Ragweed (Ambrosia artemisiifolia L. in Soybean (Glycine max L. with Preplant Herbicides

    Directory of Open Access Journals (Sweden)

    Annemarie C. Van Wely

    2014-12-01

    Full Text Available A population of common ragweed in Ontario was confirmed to be resistant to glyphosate in 2011. Group 2 [acetolactate synthase (ALS inhibitors] resistant common ragweed was first confirmed in Ontario in 2000. Previously, glyphosate provided excellent control of common ragweed in glyphosate resistant soybean but with the confirmation of glyphosate resistant (GR common ragweed, alternative herbicides need to be evaluated. Eight field trials with preplant herbicides were completed over two years (2013 and 2014 in fields with confirmed GR common ragweed. Tank-mixes of glyphosate and linuron or metribuzin provided 88% - 99% and 86% - 98% control 4 weeks after application (WAA and 80% - 92% and 80% - 95% control 8 WAA, respectively. However, these herbicides also had among the highest environmental impact of the herbicides tested. Based on the results of these studies, GR common ragweed can be controlled with residual herbicides when applied preemergence in soybean. Currently, there are no post emergence herbicides that provide adequate control of GR common ragweed, therefore, preemergence herbicides with residual are essential for full season control.

  3. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase

    OpenAIRE

    Vyver, Christell; Conradie, Tobie; Kossmann, Jens; Lloyd, James

    2013-01-01

    Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbi...

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

    OpenAIRE

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

    2006-01-01

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

  5. Enhanced production of 2,3-butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae.

    Science.gov (United States)

    Guo, Xue-Wu; Zhang, Yun-Hui; Cao, Chun-Hong; Shen, Tong; Wu, Ming-Yue; Chen, Ye-Fu; Zhang, Cui-Ying; Xiao, Dong-Guang

    2014-01-01

    Mutants with overexpression of ?-acetolactate synthase (ALS), ?-acetolactate decarboxylase, and acetoin reductase (AR), either individually or in combination, were constructed to improve 2,3-butanediol (2,3-BD) production in Klebsiella pneumoniae. The recombinant strains were characterized in terms of the enzyme activity, 2,3-BD yield, and expression levels. The recombinant K. pneumoniae strain (KG-rs) that overexpressed both ALS and AR showed an improved 2,3-BD yield. When cultured in the media with five different carbon sources (glucose, galactose, fructose, sucrose, and lactose), the mutant exhibited higher 2,3-BD productivity and production than the parental strain in all the tested carbon sources except for lactose. The 2,3-BD production of KG-rs in a batch fermentation with glucose as the carbon source was 12% higher than that of the parental strain. PMID:24527770

  6. Identification of cofactor and herbicide binding domains in acetolactate synthase by bromopyruvate modification

    International Nuclear Information System (INIS)

    Bromopyruvate is an affinity label for acetolactate synthase isozyme II from Salmonella typhimurium (ALSII). The concentration of bromopyruvate giving half-maximal inactivation is 0.1 mM, and the maximal rate of inactivation is 0.56 hr-1. Inactivation with [14C]bromopyruvate is associated with the incorporation of 4 molecules of reagent per active site lost. Two cysteinyl residues are modified extremely rapidly, with no loss of enzymatic activity, as judged by quenching the reaction with thiol after its initial phase. Inactivation is a consequence of the additional two moles of reagent incorporated per mole of protomer. The additional incorporation is divided between one major and two minor sites of modification. Substantial protection against inactivation is afforded by FAD, with virtually complete protection provided by a mixture of FAD and thiamine pyrophosphate (TPP). The major site of modification, protected by FAD, is cysteinyl residue number67, based upon amino acid sequence analysis of the purified tryptic peptide that encompasses this site. The remaining site of modification, protected by TPP, is associated with cysteinyl residue number44. Both sites of modification are afforded protection by the sulfonylurea herbicide sulfometuron methyl (SM). Although inactivation by bromopyruvate exhibits rate saturation, indicating binding as a prerequisite to inactivation, neither pyruvate nor ?-ketobutyrate prevent modification of the enzyme by br prevent modification of the enzyme by bromopyruvate. Thus, it would appear that the bromopyruvate binding site is not the site normally occupied by substrate

  7. Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

    Science.gov (United States)

    Lee, Sang-Choon; Kim, Jinheung; La, Im-Joung; Kim, Soon-Kil; Yoon, Moon-Young

    2013-01-10

    The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60 kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent K(m) for pyruvate was calculated to be 1.37 mM, while the K(c) for thiamin diphosphate (ThDP) and Mg(2+) were found to be 0.031 ?M and 1.27 mM, respectively. Negligible absorbance at 450 nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol. PMID:23199739

  8. Differential sensitivity of locally naturalized Panicum species to 4-hydroxyphenyl pyruvate dioxygenase and acetolactate synthase-inhibiting herbicides

    Directory of Open Access Journals (Sweden)

    De Cauwer, Benny

    2014-02-01

    Full Text Available One of the possible reasons for the expansion of the alien panicoid grasses Panicum schinzii (Transvaal millet, Panicum dichotomiflorum (Fall panicum and Panicum capillare (Witchgrass in maize fields in Belgium might be a lower sensitivity to post-emergence herbicides acting against panicoid grasses, in particular those inhibiting 4-hydroxyphenyl pyruvate dioxygenase (HPPD and acetolactate synthase (ALS. Dose-response pot experiments were conducted in the greenhouse to evaluate the effectiveness of five HPPD-inhibiting herbicides (sulcotrione, mesotrione, isoxaflutole, topramezone, tembotrione and two ALS-inhibiting herbicides (nicosulfuron, foramsulfuron for controlling naturalized Belgian populations of P. schinzii, P. dichotomiflorum and P. capillare. In another dose-response pot experiment, sensitivity of five local P. dichotomiflorum populations to HPPD-inhibitors and nicosulfuron was investigated. Finally, the influence of growth stage at time of herbicide application on efficacy of topramezone and nicosulfuron for Panicum control was evaluated. Large interspecific differences in sensitivity to HPPD-inhibiting herbicides were observed. Panicum schinzii was sensitive (i.e., required a three-fold lower dose than maximum authorized field dose to achieve 90% reduction in biomass to tembotrione but moderately sensitive (i.e. required maximum field dose to topramezone and poorly sensitive (i.e. required three-fold higher dose than maximum field dose to mesotrione and sulcotrione. However, P. dichotomiflorum, a species that morphologically closely resembles P. schinzii, was sensitive to mesotrione and topramezone but moderately sensitive to tembotrione. Panicum capillare was sensitive to sulcotrione and topramezone, moderately sensitive to tembotrione and poorly sensitive to mesotrione. All Panicum species were sensitive to low doses of nicosulfuron and foramsulfuron. Naturalized Panicum dichotomiflorum populations exhibited differential herbicide sensitivity profiles. All species tested showed a progressive decrease in sensitivity to topramezone and nicosulfuron with seedling age. A satisfactory post-emergence control of Panicum species in the field will require appropriate choice of herbicide and dose, as well as a more timely application (i.e. before weeds reach the four leaves stage.

  9. Resistência de plantas aos herbicidas inibidores da acetolactato sintase / Plant resistance to acetolactate synthase-inhibiting herbicides

    Scientific Electronic Library Online (English)

    M.A., Rizzardi; R.A., Vidal; N.G., Fleck; D., Agostinetto.

    2002-04-01

    Full Text Available A resistência de plantas aos herbicidas é conseqüência, na maioria das vezes, de mutação ou da preexistência de genes que conferem resistência à população. No caso dos herbicidas inibidores da acetolactato sintase (ALS) ocorreram casos de resistência tanto em plantas daninhas quanto em culturas. Ess [...] a revisão foi realizada com o objetivo de discutir aspectos bioquímicos, genéticos e moleculares da resistência de plantas aos herbicidas inibidores da ALS, sendo destacados também os efeitos na ecofisiologia das plantas daninhas e em mutações que conferem resistência em plantas daninhas e a possibilidade de utilizá-las para o desenvolvimento de culturas resistentes aos inibidores da ALS. Em plantas daninhas, a resistência aos herbicidas inibidores da ALS resulta de uma ou mais mutações no gene que codifica a ALS; quando a herança desse gene é monogênica, ele possui característica dominante a semidominante. As substituições em uma única seqüência nucleotídica ocasionam alteração na ALS, conferindo resistência aos herbicidas inibidores dessa enzima. Embora o biótipo resistente apresente alteração genética e enzimática quando comparado com biótipo suscetível, o comportamento ecofisiológico dos biótipos resistentes e suscetíveis é similar. Essa característica tem implicações muito importantes no estabelecimento das populações resistentes. Já foram desenvolvidos cultivares resistentes para diversas culturas, incluindo arroz e milho, as quais variam no nível de resistência aos diferentes grupos químicos de herbicidas inibidores da ALS. Abstract in english Herbicide resistance in plants arises mostly through mutation or pre-existence of genes that confer resistance to the population. When using herbicides inhibitors of the acetolactate synthase (ALS), resistance has occurred in weeds as well as in crops. This literature review was conducted to discuss [...] biochemical, genetic, and molecular aspects of plant resistance to ALS inhibitors, its effects on weed ecophysiology and mutations which confer resistance to weeds, as well as the possibilities to develop resistant crops to ALS inhibitors. In weeds, resistance to ALS-inhibiting herbicides results from one or more mutations in the gene that codifies the ALS, which possesses dominant or semi-dominant characteristics when resistance is codified by one gene. Substitutions on a single nucleotide sequence cause alterations in the ALS, conferring resistance to herbicides inhibitors of this enzyme. Although the resistant biotype presents genetic and enzymatic alteration, when compared to the susceptible biotype, the ecophysiological behaviour of resistant and susceptible biotypes is similar. Resistant cultivars have already been developed in various crops, including rice and corn, which vary in their level of resistance to different chemical groups of ALS-inhibiting herbicides.

  10. Resistência de plantas aos herbicidas inibidores da acetolactato sintase Plant resistance to acetolactate synthase-inhibiting herbicides

    Directory of Open Access Journals (Sweden)

    M.A. Rizzardi

    2002-04-01

    Full Text Available A resistência de plantas aos herbicidas é conseqüência, na maioria das vezes, de mutação ou da preexistência de genes que conferem resistência à população. No caso dos herbicidas inibidores da acetolactato sintase (ALS ocorreram casos de resistência tanto em plantas daninhas quanto em culturas. Essa revisão foi realizada com o objetivo de discutir aspectos bioquímicos, genéticos e moleculares da resistência de plantas aos herbicidas inibidores da ALS, sendo destacados também os efeitos na ecofisiologia das plantas daninhas e em mutações que conferem resistência em plantas daninhas e a possibilidade de utilizá-las para o desenvolvimento de culturas resistentes aos inibidores da ALS. Em plantas daninhas, a resistência aos herbicidas inibidores da ALS resulta de uma ou mais mutações no gene que codifica a ALS; quando a herança desse gene é monogênica, ele possui característica dominante a semidominante. As substituições em uma única seqüência nucleotídica ocasionam alteração na ALS, conferindo resistência aos herbicidas inibidores dessa enzima. Embora o biótipo resistente apresente alteração genética e enzimática quando comparado com biótipo suscetível, o comportamento ecofisiológico dos biótipos resistentes e suscetíveis é similar. Essa característica tem implicações muito importantes no estabelecimento das populações resistentes. Já foram desenvolvidos cultivares resistentes para diversas culturas, incluindo arroz e milho, as quais variam no nível de resistência aos diferentes grupos químicos de herbicidas inibidores da ALS.Herbicide resistance in plants arises mostly through mutation or pre-existence of genes that confer resistance to the population. When using herbicides inhibitors of the acetolactate synthase (ALS, resistance has occurred in weeds as well as in crops. This literature review was conducted to discuss biochemical, genetic, and molecular aspects of plant resistance to ALS inhibitors, its effects on weed ecophysiology and mutations which confer resistance to weeds, as well as the possibilities to develop resistant crops to ALS inhibitors. In weeds, resistance to ALS-inhibiting herbicides results from one or more mutations in the gene that codifies the ALS, which possesses dominant or semi-dominant characteristics when resistance is codified by one gene. Substitutions on a single nucleotide sequence cause alterations in the ALS, conferring resistance to herbicides inhibitors of this enzyme. Although the resistant biotype presents genetic and enzymatic alteration, when compared to the susceptible biotype, the ecophysiological behaviour of resistant and susceptible biotypes is similar. Resistant cultivars have already been developed in various crops, including rice and corn, which vary in their level of resistance to different chemical groups of ALS-inhibiting herbicides.

  11. Downy Brome (Bromus tectorum L.) and Broadleaf Weed Control in Winter Wheat with Acetolactate Synthase-Inhibiting Herbicides

    OpenAIRE

    Geier, Patrick W.; Stahlman, Phillip W.; Reddy, Seshadri S.

    2013-01-01

    A study was conducted for three seasons in northwest Kansas, USA to evaluate acetolactate synthase (ALS)-inhibiting herbicides for downy brome (Bromus tectorum L.) and winter annual broadleaf weed control in winter wheat. Herbicides included pyroxsulam at 18.4 g ai ha?1, propoxycarbazone-Na at 44 g ai ha?1, premixed propoxycarbazone-Na & mesosulfuron-methyl at 27 g ai ha?1, and sulfosulfuron at 35 g ai ha?1. The herbicides were applied postemergence in fall and spring seasons. Ave...

  12. Resistência de Bidens subalternans aos herbicidas inibidores da enzima acetolactato sintase utilizados na cultura da soja Resistance of Bidens subalternans to the acetolactate synthase inhibitor herbicides used in soybean crop

    Directory of Open Access Journals (Sweden)

    G.A. Gelmini

    2002-08-01

    Full Text Available O uso contínuo e prolongado de produtos com o mesmo mecanismo de ação pode provocar a manifestação de biótipos resistentes. Para verificar possíveis novos casos de resistência, bem como alternativas para prevenção e manejo, foram coletadas sementes de Bidens subalternans na região de São Gabriel D' Oeste-MS, em plantas que sobreviveram a tratamentos em que inibidores da ALS foram sistematicamente utilizados. Em experimento conduzido em vasos em casa de vegetação, o biótipo com histórico de resistente foi comparado ao suscetível quando submetido aos diversos herbicidas com diferentes mecanismos de ação usados em pós-emergência, os quais foram aplicados nas doses de zero, uma, duas, quatro e oito vezes a recomendada. Decorridos 20 dias, foram avaliadas a porcentagem de controle e a produção da fitomassa verde, visando estabelecimento de curvas de dose-resposta e obtenção dos fatores de resistência. O biótipo oriundo de área com histórico de aplicações repetidas de inibidores da ALS apresentou elevado nível de resistência aos herbicidas chlorimuron-ethyl e imazethapyr, demonstrando ser portador de resistência cruzada aos inibidores da ALS dos grupos das sulfoniluréias e imidazolinonas. Entretanto, esse biótipo foi eficientemente controlado pelos herbicidas fomesafen, lactofen, bentazon, glufosinato de amônio e glyphosate.The continuous and prolonged use of products with the same mechanism of action can provoke the manifestation of resistant biotypes. In horder to verify possible new cases, as well as alternatives for prevention and control, seeds of Bidens subalternans were collected at São Gabriel D' Oeste (MS region at plants that survived continuous treatments which sistematically ALS inhibitors. Through an experiment performed in pots inside a greenhouse, a resistant biotype was compared to a susceptible one when submitted to herbicides with different mechanisms of action and applied at post emergence. These herbicides were applied at doses zero, one, two, four and eight times the recommended dosage. Twenty days after, the control and the green weight production were analysed aiming to get the dose-response curves as well as the resistance factor. The biotype from the area with repeated application of ALS inhibitors showed a high level of resistance to chlorimuron-ethyl and imazethapyr, demonstrating therefore to be a carrier of crossed resistance to the ALS inhibitors of the sulfonilurea and imidazolinona groups. However, this biotype was controlled by fomesafen, lactofen, bentazon, ammonium glufosinate and glyphosate.

  13. Resistência de Bidens subalternans aos herbicidas inibidores da enzima acetolactato sintase utilizados na cultura da soja / Resistance of Bidens subalternans to the acetolactate synthase inhibitor herbicides used in soybean crop

    Scientific Electronic Library Online (English)

    G.A., Gelmini; R., Victória Filho; M.C.S.S., Novo; M.L., Adoryan.

    2002-08-01

    Full Text Available O uso contínuo e prolongado de produtos com o mesmo mecanismo de ação pode provocar a manifestação de biótipos resistentes. Para verificar possíveis novos casos de resistência, bem como alternativas para prevenção e manejo, foram coletadas sementes de Bidens subalternans na região de São Gabriel D' [...] Oeste-MS, em plantas que sobreviveram a tratamentos em que inibidores da ALS foram sistematicamente utilizados. Em experimento conduzido em vasos em casa de vegetação, o biótipo com histórico de resistente foi comparado ao suscetível quando submetido aos diversos herbicidas com diferentes mecanismos de ação usados em pós-emergência, os quais foram aplicados nas doses de zero, uma, duas, quatro e oito vezes a recomendada. Decorridos 20 dias, foram avaliadas a porcentagem de controle e a produção da fitomassa verde, visando estabelecimento de curvas de dose-resposta e obtenção dos fatores de resistência. O biótipo oriundo de área com histórico de aplicações repetidas de inibidores da ALS apresentou elevado nível de resistência aos herbicidas chlorimuron-ethyl e imazethapyr, demonstrando ser portador de resistência cruzada aos inibidores da ALS dos grupos das sulfoniluréias e imidazolinonas. Entretanto, esse biótipo foi eficientemente controlado pelos herbicidas fomesafen, lactofen, bentazon, glufosinato de amônio e glyphosate. Abstract in english The continuous and prolonged use of products with the same mechanism of action can provoke the manifestation of resistant biotypes. In horder to verify possible new cases, as well as alternatives for prevention and control, seeds of Bidens subalternans were collected at São Gabriel D' Oeste (MS) reg [...] ion at plants that survived continuous treatments which sistematically ALS inhibitors. Through an experiment performed in pots inside a greenhouse, a resistant biotype was compared to a susceptible one when submitted to herbicides with different mechanisms of action and applied at post emergence. These herbicides were applied at doses zero, one, two, four and eight times the recommended dosage. Twenty days after, the control and the green weight production were analysed aiming to get the dose-response curves as well as the resistance factor. The biotype from the area with repeated application of ALS inhibitors showed a high level of resistance to chlorimuron-ethyl and imazethapyr, demonstrating therefore to be a carrier of crossed resistance to the ALS inhibitors of the sulfonilurea and imidazolinona groups. However, this biotype was controlled by fomesafen, lactofen, bentazon, ammonium glufosinate and glyphosate.

  14. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase.

    Science.gov (United States)

    van der Vyver, Christell; Conradie, Tobie; Kossmann, Jens; Lloyd, James

    2013-04-01

    Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 ?g/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 ?g/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide. PMID:23543883

  15. Evolution and diversity of the mechanisms endowing resistance to herbicides inhibiting acetolactate-synthase (ALS) in corn poppy (Papaver rhoeas L.).

    Science.gov (United States)

    Délye, Christophe; Pernin, Fanny; Scarabel, Laura

    2011-02-01

    We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg???, His???, Leu???, Ser???, Thr??? and Leu??? were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms. PMID:21421378

  16. Acetolactate synthases MoIlv2 and MoIlv6 are required for infection-related morphogenesis in Magnaporthe oryzae.

    Science.gov (United States)

    Du, Yan; Zhang, Haifeng; Hong, Li; Wang, Jiamei; Zheng, Xiaobo; Zhang, Zhengguang

    2013-12-01

    Amino acids are important components in the metabolism of a variety of pathogens, plants and animals. Acetolactate synthase (ALS) catalyses the first common step in leucine, isoleucine and valine biosynthesis, and is the target of several classes of inhibitors. Here, MoIlv2, an orthologue of the Saccharomyces cerevisiae?ALS catalytic subunit Ilv2, and MoIlv6, an orthologue of the S.?cerevisiae?ALS regulatory subunit Ilv6, were identified. To characterize MoILV2 and MoILV6 functions, we generated the deletion mutants ?Moilv2 and ?Moilv6. Phenotypic analysis showed that both mutants were auxotrophic for leucine, isoleucine and valine, and were defective in conidial morphogenesis, appressorial penetration and pathogenicity. Further studies suggested that MoIlv2 and MoIlv6 play a critical role in maintaining the balance of intracellular amino acid levels. MoIlv2 and MoIlv6 are both localized to the mitochondria and the signal peptide of MoIlv6 is critical for its localization. In summary, our evidence indicates that MoIlv2 plays a crucial role in isoleucine and valine biosynthesis, whereas MoIlv6 contributes to isoleucine and leucine biosynthesis; both genes are required for fungal pathogenicity. This study indicates the potential of targeting branched-chain amino acid biosynthesis for anti-rice blast management. PMID:23782532

  17. Constricted Flux through the Branched-Chain Amino Acid Biosynthetic Enzyme Acetolactate Synthase Triggers Elevated Expression of Genes Regulated by rpoS and Internal Acidification

    OpenAIRE

    Dyk, Tina K.; Ayers, Brenda L.; Morgan, Robin W.; Larossa, Robert A.

    1998-01-01

    The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism, Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random E. coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in act...

  18. Herbicide-Resistant Mutations in Acetolactate Synthase Can Reduce Feedback Inhibition and Lead to Accumulation of Branched-Chain Amino Acids

    OpenAIRE

    Shuichi Yanagisawa; Tsutomu Shimizu; Tamaki Fujimori; Seiichi Toki; Masaki Endo

    2013-01-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., vali...

  19. Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2?) mutants is influenced by the carbon source and rapamycin

    OpenAIRE

    Kingsbury, Joanne M.; Mccusker, John H.

    2010-01-01

    The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2? mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal dr...

  20. Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

    Science.gov (United States)

    Lee, Sang-Choon; Jung, In-Pil; Baig, Irshad Ahmed; Chien, Pham Ngoc; La, Im-Joung; Yoon, Moon-Young

    2015-01-01

    Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS. PMID:25128823

  1. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Science.gov (United States)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  2. Downy Brome (Bromus tectorum L. and Broadleaf Weed Control in Winter Wheat with Acetolactate Synthase-Inhibiting Herbicides

    Directory of Open Access Journals (Sweden)

    Patrick W. Geier

    2013-04-01

    Full Text Available A study was conducted for three seasons in northwest Kansas, USA to evaluate acetolactate synthase (ALS-inhibiting herbicides for downy brome (Bromus tectorum L. and winter annual broadleaf weed control in winter wheat. Herbicides included pyroxsulam at 18.4 g ai ha?1, propoxycarbazone-Na at 44 g ai ha?1, premixed propoxycarbazone-Na & mesosulfuron-methyl at 27 g ai ha?1, and sulfosulfuron at 35 g ai ha?1. The herbicides were applied postemergence in fall and spring seasons. Averaged over time of application, no herbicide controlled downy brome more than 78% in any year. When downy brome densities were high, control was less than 60%. Pyroxsulam controlled downy brome greater than or similar to other herbicides tested. Flixweed (Descurainia sophia L., blue mustard [Chorispora tenella (Pallas DC.], and henbit (Lamium amplexicaule L. control did not differ among herbicide treatments. All herbicides tested controlled flixweed and blue mustard at least 87% and 94%, respectively. However, none of the herbicides controlled henbit more than 73%. Fall herbicide applications improved weed control compared to early spring applications; improvement ranged from 3% to 31% depending on the weed species. Henbit control was greatly decreased by delaying herbicide applications until spring compared to fall applications (49% vs. 80% control. Herbicide injury was observed in only two instances. The injury was ?13% with no difference between herbicides and the injury did not impact final plant height or grain yield.

  3. Absorption and translocation of imazethapyr as a mechanism responsible for resistance of Euphorbia heterophylla L. biotypes to acetolactate synthase (ALS) inhibitors / Absorción y translocación de imazetapir como mecanismo responsable de la resistencia a inhibidores de la acetolactato sintasa (ALS) en biotipos de Euphorbia heterophylla L.

    Scientific Electronic Library Online (English)

    Guido A., Plaza; María Dolores, Osuna; Rafael, De Prado; Antonio, Heredia.

    2006-07-01

    Full Text Available El efecto de las malas hierbas en la disminución de la producción agrícola está considerado entre 30% y 50%. Imazetapir es un herbicida que actúa sobre la enzima acetolactato sintasa (ALS), primera enzima común en la ruta biosintética de la valina, leucina e isoleucina. Euphorbia heterophylla es una [...] especie común en los campos de soya del Brasil. Actualmente se reporta una población resistente a imazetapir, herbicida perteneciente al grupo de las imidazolinonas. El objetivo de los ensayos de absorción y translocación fue estudiar las posibles diferencias de penetración foliar y movimiento del 14Cimazetapir en dos biotipos de E. heterophylla L. En el biotipo resistente, se registró una menor absorción durante las primeras 6 h después del tratamiento, tendencia que se diluye en los siguientes tiempos de evaluación. Las tendencias de los valores de translocación fueron similares durante las evaluaciones realizadas. Los resultados de los análisis de química de ceras no arrojaron diferencias entre la composición cuticular entre los biotipos; sin embargo, los estudios de microscopía electrónica de la hoja sí muestran diferencias en la morfología y la cantidad de ceras cuniculares, factores que determinan el comportamiento resistente del biotipo R. Abstract in english The effect of weeds on reduction of agricultural production is estimated between 30% and 50%. Imazethapyr is a herbicide of imidazolinone group that inhibits activity of enzyme acetolactate synthase (ALS), the first common enzyme in the biosynthetic pathway of valine, leucine, and isoleucine. Euphor [...] bia heterophylla is common specie in soybean fields of Brazil. The study reports about a population of Euphorbia heterophylla resistant to imazethapyr. The objectives of the present work were to quantify the level of sensitivity to this herbicide in imazethapyr-resistant and -susceptible E. heterophylla populations evaluate the role of differential penetration into leaves as determining plant resistance to imazethapyr, and compare the waxy cells of R and S populations. The R population had a lower penetration rate compared with that of S population during the six first hours of incubation with the herbicide. Further studies indicated that R population was not different from S population in terms of translocation, metabolism, or target site (ALS enzyme) of imazethapyr action. Analysis of the leaf cuticle surface by scanning electron microscopy revealed higher wax density in the leaf cuticles of population R than that in S population. Thus, it is suggested that R population is resistant to imazethapyr because increased wax content of its cuticle permits less penetration of herbicide into the plant.

  4. Physiological and molecular basis of acetolactate synthase-inhibiting herbicide resistance in barnyardgrass (Echinochloa crus-galli).

    Science.gov (United States)

    Riar, Dilpreet S; Norsworthy, Jason K; Srivastava, Vibha; Nandula, Vijay; Bond, Jason A; Scott, Robert C

    2013-01-16

    Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala??? to Val and Ala??? to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of ¹?C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. ¹?C-Penoxsulam translocation out of the treated leaf (?2%) was similar among all biotypes. ¹?C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. ¹?C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1. PMID:23237199

  5. Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

    OpenAIRE

    Marugg, J. D.; Goelling, D.; Stahl, U.; Ledeboer, A. M.; Toonen, M. Y.; Verhue, W. M.; Verrips, C. T.

    1994-01-01

    The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for ...

  6. Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

    Science.gov (United States)

    Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

    2013-09-01

    Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes. PMID:25149243

  7. Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

    OpenAIRE

    Snoep, J. L.; Teixeira Mattos, M. J.; Starrenburg, M. J.; Hugenholtz, J.

    1992-01-01

    The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruva...

  8. A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis.

    Science.gov (United States)

    Saika, Hiroaki; Horita, Junko; Taguchi-Shiobara, Fumio; Nonaka, Satoko; Nishizawa-Yokoi, Ayako; Iwakami, Satoshi; Hori, Kiyosumi; Matsumoto, Takashi; Tanaka, Tsuyoshi; Itoh, Takeshi; Yano, Masahiro; Kaku, Koichiro; Shimizu, Tsutomu; Toki, Seiichi

    2014-11-01

    Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species. PMID:24406793

  9. Resistência do girassol a herbicidas inibidores da enzima acetolactato sintase / Sunflower resistance to acetolactate synthase-inhibiting herbicides

    Scientific Electronic Library Online (English)

    Alexandre Magno, Brighenti.

    2012-06-01

    Full Text Available O girassol é bastante sensível a herbicidas aplicados em pós-emergência da cultura, com o objetivo de controlar espécies daninhas de folhas largas. Diante disto, foram desenvolvidos genótipos resistentes a herbicidas do grupo químico das imidazolinonas. Este trabalho objetivou avaliar a seletividade [...] de herbicidas dos grupos químicos das imidazolinonas e sulfonilureias, aplicados sobre plantas de girassol (Tera 8003 e Tera 8011) resistentes aos inibidores da enzima acetolactato sintase (ALS). Experimentos foram conduzidos em área experimental da Embrapa Gado de Leite, nos municípios de Coronel Pacheco (MG) e Valença (RJ). O delineamento experimental foi em blocos casualizados, com quatro repetições. Os tratamentos foram: testemunha capinada, imazapyr 25 g i.a. ha-1 e 50 g i.a. ha-1, imazethapyr 70 g i.a. ha-1 e 100 g i.a. ha-1, nicosulfuron 20 g i.a. ha-1 e 32 g i.a. ha-1 e chlorimuron 7,5 g i.a. ha-1 + 0,05% v/v de óleo mineral. Foi avaliada a percentagem de fitotoxicidade, teor de clorofila (índice SPAD), altura de plantas, produção e percentagem de matéria seca e produtividade. As doses de 70 g i.a. ha-1 e 100 g i.a. ha-1 de imazethapyr foram as mais seletivas, a dose de 20 g i.a. ha-1 do nicosulfuron apresentou tolerância moderada e os tratamentos com imazapyr e chlorimuron foram aqueles que causaram maior injúria, para ambos os híbridos de girassol. Abstract in english Sunflower is very sensitive to herbicides applied in post-emergence to control broad-leaf weeds. Researchers have developed herbicide-resistant genotypes to imidazolinone herbicides. This study aimed to evaluate the selectivity of imidazolinone and sulfonylurea herbicides applied on sunflower plants [...] (Tera 8003 and Tera 8011) resistant to acetolactate synthase-inhibiting herbicides. The experiments were conducted at Embrapa Gado de Leite, in Coronel Pacheco, Minas Gerais State, and Valença, Rio de Janeiro State, Brazil. The experimental design was randomized complete blocks, with four replications. The treatments consisted of hoed control, imazapyr 25 g a.i. ha-1 and 50 g a.i. ha-1, imazethapyr 70 g a.i. ha-1 and 100 g a.i. ha-1, nicosulfuron 20 g a.i. ha-1 and 32 g a.i. ha-1, and chlorimuron 7.5 g a.i. ha-1 + 0.05% v/v of mineral oil. The crop injury percentage, chlorophyll content (SPAD index), plant height, dry matter production and percentage, and yield were evaluated. The imazethapyr doses (70 g a.i. ha-1 and 100 g a.i. ha-1) were the most selective ones, the nicosulfuron dose (20 g a.i. ha-1) showed moderate tolerance, and imazapyr and chlorimuron caused greater injury, for both sunflower hybrids.

  10. Herbicide-Resistant Mutations in Acetolactate Synthase Can Reduce Feedback Inhibition and Lead to Accumulation of Branched-Chain Amino Acids

    Directory of Open Access Journals (Sweden)

    Shuichi Yanagisawa

    2013-05-01

    Full Text Available The branched-chain amino acids (BCAAs valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

  11. Enhancement of 1,3-propanediol production by expression of pyruvate decarboxylase and aldehyde dehydrogenase from Zymomonas mobilis in the acetolactate-synthase-deficient mutant of Klebsiella pneumoniae.

    Science.gov (United States)

    Lee, Sung-Mok; Hong, Won-Kyung; Heo, Sun-Yeon; Park, Jang Min; Jung, You Ree; Oh, Baek-Rock; Joe, Min-Ho; Seo, Jeong-Woo; Kim, Chul Ho

    2014-08-01

    The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol. PMID:24841211

  12. Expression of flavonoid 3',5'-hydroxylase and acetolactate synthase genes in transgenic carnation: assessing the safety of a nonfood plant.

    Science.gov (United States)

    Chandler, Stephen F; Senior, Michael; Nakamura, Noriko; Tsuda, Shinzo; Tanaka, Yoshikazu

    2013-12-01

    For 16 years, genetically modified flowers of carnation ( Dianthus caryophyllus ) have been sold to the floristry industry. The transgenic carnation carries a herbicide tolerance gene (a mutant gene encoding acetolactate synthase (ALS)) and has been modified to produce delphinidin-based anthocyanins in flowers, which conventionally bred carnation cannot produce. The modified flower color has been achieved by introduction of a gene encoding flavonoid 3',5'-hydroxylase (F3'5'H). Transgenic carnation flowers are produced in South America and are primarily distributed to North America, Europe, and Japan. Although a nonfood crop, the release of the genetically modified carnation varieties required an environmental risk impact assessment and an assessment of the potential for any increased risk of harm to human or animal health compared to conventionally bred carnation. The results of the health safety assessment and the experimental studies that accompanied them are described in this review. The conclusion from the assessments has been that the release of genetically modified carnation varieties which express F3'5'H and ALS genes and which accumulate delphinidin-based anthocyanins do not pose an increased risk of harm to human or animal health. PMID:23646984

  13. Fungal degradation of an acetolactate synthase (ALS) inhibitor pyrazosulfuron-ethyl in soil.

    Science.gov (United States)

    Sondhia, Shobha; Waseem, Uzma; Varma, R K

    2013-11-01

    Owing to reported phytotoxicity of some sulfonylurea class of herbicides in number of sensitive crops and higher persistence in soil, present study was conducted to isolate and identify pyrazosulfuron-ethyl degrading fungi from soil of rice field. Penicillium chrysogenum and Aspergillus niger, were isolated and identified from rhizospere soil of rice field, as potent pyrazosulfuron-ethyl degrading fungi. Degradation of pyrazosulfuron-ethyl by P. chrysogenum and A. niger, yielded transformation products/metabolites which were identified and characterized by LC/MS/MS. The rate of dissipation of pyrazosulfuron-ethyl was found higher in soil of rice field and soil inoculated with P. chrysogenum. This showed important route of degradation of pyrazosulfuron-ethyl by microbes apart from chemical degradation. PMID:23993642

  14. 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.

  15. Inhibitors of glycogen synthase 3 kinase

    Science.gov (United States)

    Schultz, Peter (Oakland, CA); Ring, David B. (Palo Alto, CA); Harrison, Stephen D. (Berkeley, CA); Bray, Andrew M. (Victoria, AU)

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  16. Enhancement of vascular targeting by inhibitors of nitric oxide synthase

    International Nuclear Information System (INIS)

    Purpose: This study investigates the enhancement of the vascular targeting activity of the tubulin-binding agent combretastatin A4 phosphate (CA4P) by various inhibitors of nitric oxide synthases. Methods and Materials: The syngeneic tumors CaNT and SaS growing in CBA mice were used for this study. Reduction in perfused vascular volume was measured by injection of Hoechst 33342 24 h after drug administration. Necrosis (hematoxylin and eosin stain) was assessed also at 24 h after treatment. Combretastatin A4 phosphate was synthesized by a modification of the published procedure and the nitric oxide synthase inhibitors L-NNA, L-NMMA, L-NIO, L-NIL, S-MTC, S-EIT, AMP, AMT, and L-TC, obtained from commercial sources. Results: A statistically significant augmentation of the reduction in perfused vascular volume by CA4P in the CaNT tumor was observed with L-NNA, AMP, and AMT. An increase in CA4P-induced necrosis in the same tumor achieved significance with L-NNA, L-NMMA, L-NIL, and AMT. CA4P induced little necrosis in the SaS tumor, but combination with the inhibitors L-NNA, L-NMMA, L-NIO, S-EIT, and L-TC was effective. Conclusions: Augmentation of CA4P activity by nitric oxide synthase inhibitors of different structural classes supports a nitric oxide-related mechanism for this effect. L-NNA was the most effective inhibitor studied

  17. Resistência cruzada da losna-branca (Parthenium hysterophorus) aos herbicidas inibidores da enzima acetolactato sintase / Ragweed parthenium (Parthenium hysterophorus) cross-resistance to acetolactate synthase inhibiting herbicides

    Scientific Electronic Library Online (English)

    D.L.P., Gazziero; A.M., Brighenti; E., Voll.

    Full Text Available A aplicação de um mesmo herbicida, ou de herbicidas com o mesmo mecanismo de ação, durante anos consecutivos, numa mesma área, pode resultar na seleção de biótipos de plantas daninhas resistentes a herbicidas. O objetivo deste trabalho foi confirmar a resistência de um biótipo da planta daninha losn [...] a-branca (Parthenium hysterophorus) aos herbicidas inibidores da enzima acetolactato sintase (ALS), proveniente de uma propriedade rural no município de Mandaguari, norte do Estado do Paraná. Plantas com suspeita de resistência foram tratadas com diversos herbicidas e doses e comparadas com plantas de uma população suscetível. Os tratamentos foram as doses recomendadas dos herbicidas, duas e quatro vezes superiores à dose recomendada. Os produtos e as doses aplicadas foram cloransulam-methyl a 0,0; 33,6; 67,2; e 134,4 g i.a. ha-1 mais o adjuvante Agral a 0,2% v/v, chlorimuron-ethyl a 0,0; 20,0; 40,0; e 80,0 g i.a. ha-1, imazethapyr a 0,0; 100,0; 200,0; e 400,0 g i.a. ha-1 e iodosulfuron-methyl-sodium mais foramsulfuron a 0,0; 3,0 + 45,0 g i.a. ha-1 (150,0 g p.c. ha¹); 6,0 + 90,0 g i.a. ha-1 (300,0 g p.c. ha-1); e 12,0 + 180,0 g i.a. ha-1 (600,0 g p.c. ha-1). Foi acres centado um tratamento com o herbicida 2,4-D na dose de 536,0 g e.a. ha-1. As curvas de doseresposta do biótipo resistente foram inferiores às do biótipo suscetível em todas as doses e herbicidas estudados. O biótipo de losna-branca foi confirmado como resistente aos herbicidas inibidores da ALS. A ocorrência de resistência cruzada foi observada em relação aos herbicidas pertencentes aos grupos químicos das imidazolinonas (imazethapyr), triazolopirimidinas (cloransulam-methyl) e sulfoniluréias (chlorimuron-ethyl e iodosulfuron-methyl-sodium mais foramsulfuron). O herbicida 2,4-D, apresentou alto índice de controle de ambos os biótipos de losna-branca avaliados, confirmando que esse mecanismo de ação do herbicida é uma importante alternativa para manejar áreas com problemas de resistência. Abstract in english Weed control using herbicide application is a common agricultural practice. However, the application of the same herbicide or herbicides with the same mechanism of action, for consecutive years, in the same area, can result in the selection of herbicide resistant biotypes. The aim of this work was t [...] o confirm the resistance of a ragweed (Parthenium hysterophorus) biotype to acetolactate synthase (ALS) inhibiting herbicides. The plants were collected on a farm in Mandaguari, north of Parana State, Brazil. Plants with suspicious resistance were treated with several herbicides and rates and compared with those of a susceptible population. The herbicide treatments were established considering the recommended rates, double and four times higher than the recommended rate as follows: cloransulam-methyl 0.0, 33.6, 67.2 and 134.4 g a.i. ha-1 plus adjuvant 0.2% v/v, chlorimuron-ethyl 0.0, 20.0, 40.0 and 80.0 g a.i., imazethapyr 0.0, 100.0, 200.0 and 400.0 g a.i. ha-1, iodosulfuron-methyl-sodium plus foramsulfuron 0.0, 3.0 + 45.0 ga.i. ha-1 (150.0 g c.p. ha-1), 6.0 + 90.0 g a.i. ha-1 (300.0 g c.p. ha-1) and 12.0 + 180.0 g a.i. ha¹ (600.0 g c.p. ha-1). In addition, a treatment with 2,4-D (536.0 g a.e. ha¹) was applied. Resistant plant dose-response curves presented lower values when compared to the susceptible population, in all rates and herbicides studied. The ragweed biotype was confirmed as resistant to the ALS inhibiting herbicides. Cross-resistance was observed with herbicides belonging to the chemical groups of imidazolinones (imazethapyr), triazolopyrimidines (cloransulam-methyl), sulfonylureas (chlorimuron-ethyl and iodosulfuron-methyl-sodium plus foramsulfuron). 2,4-D has a different mechanism of action, presenting high values of control, and thus being a management alternative in areas with ragweed resistant population.

  18. Resistência cruzada da losna-branca (Parthenium hysterophorus aos herbicidas inibidores da enzima acetolactato sintase Ragweed parthenium (Parthenium hysterophorus cross-resistance to acetolactate synthase inhibiting herbicides

    Directory of Open Access Journals (Sweden)

    D.L.P. Gazziero

    2006-01-01

    Full Text Available A aplicação de um mesmo herbicida, ou de herbicidas com o mesmo mecanismo de ação, durante anos consecutivos, numa mesma área, pode resultar na seleção de biótipos de plantas daninhas resistentes a herbicidas. O objetivo deste trabalho foi confirmar a resistência de um biótipo da planta daninha losna-branca (Parthenium hysterophorus aos herbicidas inibidores da enzima acetolactato sintase (ALS, proveniente de uma propriedade rural no município de Mandaguari, norte do Estado do Paraná. Plantas com suspeita de resistência foram tratadas com diversos herbicidas e doses e comparadas com plantas de uma população suscetível. Os tratamentos foram as doses recomendadas dos herbicidas, duas e quatro vezes superiores à dose recomendada. Os produtos e as doses aplicadas foram cloransulam-methyl a 0,0; 33,6; 67,2; e 134,4 g i.a. ha-1 mais o adjuvante Agral a 0,2% v/v, chlorimuron-ethyl a 0,0; 20,0; 40,0; e 80,0 g i.a. ha-1, imazethapyr a 0,0; 100,0; 200,0; e 400,0 g i.a. ha-1 e iodosulfuron-methyl-sodium mais foramsulfuron a 0,0; 3,0 + 45,0 g i.a. ha-1 (150,0 g p.c. ha¹; 6,0 + 90,0 g i.a. ha-1 (300,0 g p.c. ha-1; e 12,0 + 180,0 g i.a. ha-1 (600,0 g p.c. ha-1. Foi acres centado um tratamento com o herbicida 2,4-D na dose de 536,0 g e.a. ha-1. As curvas de doseresposta do biótipo resistente foram inferiores às do biótipo suscetível em todas as doses e herbicidas estudados. O biótipo de losna-branca foi confirmado como resistente aos herbicidas inibidores da ALS. A ocorrência de resistência cruzada foi observada em relação aos herbicidas pertencentes aos grupos químicos das imidazolinonas (imazethapyr, triazolopirimidinas (cloransulam-methyl e sulfoniluréias (chlorimuron-ethyl e iodosulfuron-methyl-sodium mais foramsulfuron. O herbicida 2,4-D, apresentou alto índice de controle de ambos os biótipos de losna-branca avaliados, confirmando que esse mecanismo de ação do herbicida é uma importante alternativa para manejar áreas com problemas de resistência.Weed control using herbicide application is a common agricultural practice. However, the application of the same herbicide or herbicides with the same mechanism of action, for consecutive years, in the same area, can result in the selection of herbicide resistant biotypes. The aim of this work was to confirm the resistance of a ragweed (Parthenium hysterophorus biotype to acetolactate synthase (ALS inhibiting herbicides. The plants were collected on a farm in Mandaguari, north of Parana State, Brazil. Plants with suspicious resistance were treated with several herbicides and rates and compared with those of a susceptible population. The herbicide treatments were established considering the recommended rates, double and four times higher than the recommended rate as follows: cloransulam-methyl 0.0, 33.6, 67.2 and 134.4 g a.i. ha-1 plus adjuvant 0.2% v/v, chlorimuron-ethyl 0.0, 20.0, 40.0 and 80.0 g a.i., imazethapyr 0.0, 100.0, 200.0 and 400.0 g a.i. ha-1, iodosulfuron-methyl-sodium plus foramsulfuron 0.0, 3.0 + 45.0 ga.i. ha-1 (150.0 g c.p. ha-1, 6.0 + 90.0 g a.i. ha-1 (300.0 g c.p. ha-1 and 12.0 + 180.0 g a.i. ha¹ (600.0 g c.p. ha-1. In addition, a treatment with 2,4-D (536.0 g a.e. ha¹ was applied. Resistant plant dose-response curves presented lower values when compared to the susceptible population, in all rates and herbicides studied. The ragweed biotype was confirmed as resistant to the ALS inhibiting herbicides. Cross-resistance was observed with herbicides belonging to the chemical groups of imidazolinones (imazethapyr, triazolopyrimidines (cloransulam-methyl, sulfonylureas (chlorimuron-ethyl and iodosulfuron-methyl-sodium plus foramsulfuron. 2,4-D has a different mechanism of action, presenting high values of control, and thus being a management alternative in areas with ragweed resistant population.

  19. 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)

  20. 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)

  1. Inhibitors to Polyhydroxyalkanoate (PHA) Synthases: Synthesis, Molecular Docking, and Implications

    Science.gov (United States)

    Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered as an ideal alternative to nonbiodegradable synthetic plastics. However, study of PhaC has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty along with lack of a structure has become the main hurdle to understand and engineer PhaCs for economical PHA production. Here we reported the synthesis of two carbadethia CoA analogs, sT-CH2-CoA 26a and sTet-CH2-CoA 26b as well as sT-aldehyde 29 as new PhaC inhibitors. Study of these analogs with PhaECAv revealed that 26a/b and 29 are competitive and mixed inhibitors, respectively. It was observed that CoA moiety and PHA chain extension can increase binding affinity, which is consistent with the docking study. Estimation from Kic of 26a/b predicts that a CoA analog attached with an octameric-HB chain may facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

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

    International Nuclear Information System (INIS)

    Acetohyroxyacid synthease I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. The authors confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site

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

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, P.M.; Eoyang, L.

    1987-06-01

    Acetohyroxyacid synthease I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo(2-/sup 14/C)pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of /sup 14/C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. The authors confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.

  4. Structure-based inhibitor discovery of Helicobacter pylori dehydroquinate synthase.

    Science.gov (United States)

    Liu, Jai-Shin; Cheng, Wen-Chi; Wang, Hung-Jung; Chen, Yen-Cheng; Wang, Wen-Ching

    2008-08-15

    Dehydroquinate synthase (DHQS) is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme that converts 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Since it catalyzes the second key step in the shikimate pathway, which is crucial for the aromatic amino acid metabolism in bacteria, fungi, and plants, but not in mammals, DHQS is a potential target for new antimicrobial agents, anti-parasitic agents and herbicides. The crystal structure of Helicobacter pylori DHQS (HpDHQS) complexed with NAD has been determined at 2.4-A resolution and was found to possess an N-terminal Rossmann-fold domain and a C-terminal alpha-helical domain. Structural comparison reveals that the binary complex adopts an open-state conformation and shares conserved residues in the binding pocket. Virtual docking of compounds into the active site of the HpDHQS structure using the GOLD docking program led to the identification of several inhibitors. The most active compound had an IC(50) value of 61 microM, which may serve as a lead for potent inhibitors. PMID:18503755

  5. Resistência de amendoim-bravo aos herbicidas inibidores da enzima acetolactato sintase Wild poinsettia resistance to acetolactate synthase inhibitor herbicides

    OpenAIRE

    Vidal, Ribas A.; Aldo Merotto Jr.

    1999-01-01

    O controle contínuo de plantas daninhas através da aplicação de herbicidas que apresentam atividade em um único local de ação nas plantas favorece a seleção de biótipos resistentes a estes herbicidas, em certas espécies vegetais. Quatro experimentos foram conduzidos em condições casa-de-vegetação, na Faculdade de Agronomia da Universidade Federal do Rio Grande do Sul, com os objetivos de avaliar a ocorrência de resistência aos herbicidas inibidores da enzima acetolactato sint...

  6. Caracterização genética de Euphorbia heterophylla resistente a herbicidas inibidores da acetolactato sintase Genetic characterization of Euphorbia heterophylla resistant to acetolactate synthase-inhibiting herbicides

    Directory of Open Access Journals (Sweden)

    Larissa Macedo Winkler

    2003-09-01

    Full Text Available O aumento do número de plantas daninhas resistentes aos herbicidas inibidores da enzima acetolactato sintase é um tema abordado com freqüência por produtores e comunidade científica. No Brasil, nove espécies já foram documentadas por apresentarem tal problema. O objetivo deste trabalho foi determinar a diversidade genética de populações de leiteira (Euphorbia heterophylla L. resistentes aos herbicidas inibidores da enzima acetolactato sintase. Quarenta populações de plantas oriundas de sementes coletadas em áreas do Estado do Rio Grande do Sul, Brasil, com suspeita de resistência, foram selecionadas, a partir da aplicação prévia de herbicidas com este mecanismo de ação em casa de vegetação. Vinte plantas de cada população serviram de amostra para a extração de DNA. Trinta marcadores de polimorfismo de DNA amplificado ao acaso (RAPD foram selecionados, cada um com 10 oligonucleotídeos de seqüência arbitrária. Na análise de agrupamento, cujo coeficiente médio de similaridade foi de 40%, as populações foram separadas em sete grupos. As populações dos municípios de Pontão, Augusto Pestana e Não-me-Toque foram consideradas geneticamente diferentes. Há variabilidade genética relacionada à resistência do herbicida entre as populações de E. heterophylla que ocorrem no planalto do Estado do Rio Grande do Sul.The increase of the number of weed plants resistant to enzyme acetolactate sintase (ALS-inhibiting herbicides of is a subject frequently discussed by farmers and scientific community. In Brazil, nine species were registered with such problem. The objective of this work was to determine the genetic diversity of wild poinsettia (Euphorbia heterophylla L. ALS-resistant populations. Forty populations deriving from seeds collected in areas of the State of Rio Grande do Sul, Brazil, with resistance suspicion, were selected from the previous application of herbicides in greenhouse. Twenty plants of each population were sampled for DNA extraction. Analysis of 30 random amplified polymorphic DNA (RAPD markers were performed. Each marker had 10 oligonucleotide of arbitrary sequence. On the grouping analysis, the overall coefficient of similarity was 40% and the populations were separated in seven groups. The populations of the counties of Pontão, Augusto Pestana and Não-me-Toque were genetically different. There is genetic variability related to herbicide resistence among E. heterophylla populations from plateaus of the State of Rio Grande do Sul.

  7. Caracterização genética de Euphorbia heterophylla resistente a herbicidas inibidores da acetolactato sintase / Genetic characterization of Euphorbia heterophylla resistant to acetolactate synthase-inhibiting herbicides

    Scientific Electronic Library Online (English)

    Larissa Macedo, Winkler; Ribas Antônio, Vidal; José Fernandes, Barbosa Neto.

    1067-10-01

    Full Text Available O aumento do número de plantas daninhas resistentes aos herbicidas inibidores da enzima acetolactato sintase é um tema abordado com freqüência por produtores e comunidade científica. No Brasil, nove espécies já foram documentadas por apresentarem tal problema. O objetivo deste trabalho foi determina [...] r a diversidade genética de populações de leiteira (Euphorbia heterophylla L.) resistentes aos herbicidas inibidores da enzima acetolactato sintase. Quarenta populações de plantas oriundas de sementes coletadas em áreas do Estado do Rio Grande do Sul, Brasil, com suspeita de resistência, foram selecionadas, a partir da aplicação prévia de herbicidas com este mecanismo de ação em casa de vegetação. Vinte plantas de cada população serviram de amostra para a extração de DNA. Trinta marcadores de polimorfismo de DNA amplificado ao acaso (RAPD) foram selecionados, cada um com 10 oligonucleotídeos de seqüência arbitrária. Na análise de agrupamento, cujo coeficiente médio de similaridade foi de 40%, as populações foram separadas em sete grupos. As populações dos municípios de Pontão, Augusto Pestana e Não-me-Toque foram consideradas geneticamente diferentes. Há variabilidade genética relacionada à resistência do herbicida entre as populações de E. heterophylla que ocorrem no planalto do Estado do Rio Grande do Sul. Abstract in english The increase of the number of weed plants resistant to enzyme acetolactate sintase (ALS)-inhibiting herbicides of is a subject frequently discussed by farmers and scientific community. In Brazil, nine species were registered with such problem. The objective of this work was to determine the genetic [...] diversity of wild poinsettia (Euphorbia heterophylla L.) ALS-resistant populations. Forty populations deriving from seeds collected in areas of the State of Rio Grande do Sul, Brazil, with resistance suspicion, were selected from the previous application of herbicides in greenhouse. Twenty plants of each population were sampled for DNA extraction. Analysis of 30 random amplified polymorphic DNA (RAPD) markers were performed. Each marker had 10 oligonucleotide of arbitrary sequence. On the grouping analysis, the overall coefficient of similarity was 40% and the populations were separated in seven groups. The populations of the counties of Pontão, Augusto Pestana and Não-me-Toque were genetically different. There is genetic variability related to herbicide resistence among E. heterophylla populations from plateaus of the State of Rio Grande do Sul.

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

    OpenAIRE

    Silverman, P. M.; Eoyang, L.

    1987-01-01

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

  9. Fatty acid synthase inhibitors isolated from Punica granatum L.

    Scientific Electronic Library Online (English)

    He-Zhong, Jiang; Qing-Yun, Ma; Hui-Jin, Fan; Wen-Juan, Liang; Sheng-Zhuo, Huang; Hao-Fu, Dai; Peng-Cheng, Wang; Xiao-Feng, Ma; You-Xing, Zhao.

    2012-05-01

    Full Text Available Este trabalho tem por objetivo o isolamento de inibidores da enzima ácido graxo sintase (FAS) a partir de acetato de etila proveniente de extratos de cascas de frutas da Punica granatum L. A investigação química guiada por bioensaios das cascas das frutas resultou no isolamento de dezessete composto [...] s incluindo principalmente triternóides e compostos fenólicos, dos quais um novo triterpeno do tipo oleanano (punicaone) juntamente com quatorze compostos conhecidos foram isolados pela primeira vez a partir desta planta. Sete dos componentes isolados foram avaliados para atividades inibitórias de FAS e dois deles apresentaram-se ativos. Em particular, o ácido flavogalônico que exibiu forte atividade inibitória de FAS com valor de IC50 de 10,3 µmol L-1. Abstract in english 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 co [...] mpounds, 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 µmol L-1.

  10. Endothelial and Neuronal Nitric Oxide Synthase Inhibitors Influences Angiotensin II Pressor Effect in Central Nervous System

    OpenAIRE

    Wilson Abrao Saad; Ismael Francisco Motta Siqueira Guarda; Luiz Antonio de Arruda. Camargo; William Abrao Saad; 6Renata Saad Guarda; Santos, Talmir A. F. B.; Sylvio Simoes

    2006-01-01

    The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP) in rats. Losartan and PD123349 AT1 and AT 2 (selective no peptides antagonists angiotensin receptors), as well as FK 409 (a nitric oxide donor), NW-nitro-L-arginine methyl ester (L-NAME) a constituve nitric oxide synthase inhibitor endothelial (eNOSI) and 7-nitroindazol (7NI) a specific neuronal nitric oxide synthase inhibitor (nNOSI) were used. Holtzman strain, (Rattus norvergi...

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

    OpenAIRE

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

    1994-01-01

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

  12. 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.

  13. Biochemistry: Acetohydroxyacid Synthase

    OpenAIRE

    Pham Ngoc Chien

    2010-01-01

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

  14. Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase

    International Nuclear Information System (INIS)

    Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-[3H]prop-CoA is incubated with the target enzymes, 3H2O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ? ?) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k2 = 86.7M-1min-1 at 300, pH 7.0)

  15. Fatty Acid Synthase Inhibitors from Plants and Their Potential Application in the Prevention of Metabolic Syndrome

    OpenAIRE

    Wei-xi Tian, Xiao-feng Ma

    2011-01-01

    Fatty acid synthase (FAS) attracts more and more attention recently as a potential target for metabolic syndrome, such as cancer, obesity, diabetes and cerebrovascular disease. FAS inhibitors are widely existed in plants, consisting of diversiform compounds. These inhibitors exist not only in herbs also in many plant foods, such as teas, allium vegetables and some fruits. These effective components include gallated catechins, theaflavins, flavonoids, condensed and hydrolysable tannins, thioet...

  16. 1,1-Bisphosphonate squalene synthase inhibitors: interplay between the isoprenoid subunit and the diphosphate surrogate.

    Science.gov (United States)

    Magnin, D R; Biller, S A; Dickson, J K; Logan, J V; Lawrence, R M; Chen, Y; Sulsky, R B; Ciosek, C P; Harrity, T W; Jolibois, K G

    1995-07-01

    Inhibitors of squalene synthase have the potential to be superior cholesterol-lowering agents. We previously disclosed that lipophilic 1,1-bisphosphonates I are potent squalene synthase inhibitors and orally active cholesterol-lowering agents in animal models (Ciosek, C. P., Jr.; et al. J. Biol. Chem. 1993, 268, 24832-24837). In this paper, we describe modifications to the bisphosphonate moiety, in an attempt to reduce the number of acidic functions contained in these inhibitors. Replacing one of the acidic groups with a methyl (II, R2 = CH3) results in potent inhibitors when paired with a close mimic of the naturally occurring farnesyl moiety (R1 = farnesylethyl) but not when paired with the shorter isoprene surrogates (R1 = geranylethyl or 4-biphenylpropyl). In contrast, all three corresponding bisphosphonates I are potent squalene synthase inhibitors. Inhibitory potency is recovered with the shorter isoprene surrogates when R2 is CH2OH or CH2OCH3. It is proposed that these R2 groups serve as hydrogen bond acceptors with the active site of the enzyme. The properties of these compounds as cholesterol biosynthesis inhibitors in rats are described, and synthetic routes to these and related compounds are detailed. PMID:7629799

  17. Germination and growth of Fimbristylis miliacea biotypes resistant and susceptible to acetolactate synthase-inhibiting herbicides / Germinação e crescimento de biótipos de Fimbristylis miliacea resistente e suscetível aos herbicidas inibidores da enzima acetolactato sintase

    Scientific Electronic Library Online (English)

    C.E., Schaedler; J.A., Noldin; D., Agostinetto; T., Dal Magro; L.C., Fontana.

    2013-09-01

    Full Text Available Biótipos de plantas daninhas suscetíveis e resistentes a herbicidas podem apresentar diferenças quanto ao seu valor adaptativo. Os objetivos deste trabalho foram comparar, em condição controlada e não competitiva, a análise de crescimento, características de germinação e peso de sementes de biótipos [...] de Fimbristylis miliacea resistente e suscetível a herbicidas inibidores da ALS. Experimentos foram conduzidos em casa de vegetação e em laboratório no período de outubro de 2008 a fevereiro de 2010. Para os estudos foram utilizados dois biótipos resistentes (FIMMI 10 e FIMMI 12) e um suscetível (FIMMI 13). No estudo de análise de crescimento, os tratamentos foram organizados em delineamento completamente casualizado com quatro repetições e oito épocas de coletas [21, 28, 35, 42, 49, 56, 69 dias após a emergência (DAE) e no florescimento]. Quanto aos estudos de velocidade de germinação, germinação e peso de sementes, foram determinados os índices de velocidade de germinação, porcentagem de germinação em diferentes temperaturas e peso de sementes dos biótipos. Os resultados demonstraram que o biótipo resistente FIMMI 12 apresentou diferença em todas as variáveis avaliadas em comparação ao biótipo resistente FIMMI 10 e, em comparação ao suscetível FIMMI 13, apenas no florescimento. O biótipo suscetível FIMMI 13 apresentou maior índice de velocidade de germinação e maior germinação em porcentagem quando comparado com os biótipos resistentes. Por outro lado, os biótipos resistentes FIMMI 10 e FIMMI 12 apresentaram maior massa de sementes. Abstract in english Weed biotypes resistant and susceptible to herbicides may have differences in their adaptive values. The aims of this study were to compare, under controlled and non-competitive condition, the growth analysis, germination features and seed weight of Fimbristylis miliacea (FIMMI) biotypes resistant a [...] nd susceptible to acetolactate synthase (ALS) inhibiting herbicides. Experiments were conducted in a greenhouse and in a laboratory from October 2008 to February 2010. Two resistant biotypes (FIMMI 10 and FIMMI 12) and one susceptible biotype (FIMMI 13) were used for the studies. For the study on growth analysis, the treatments were arranged in a completely randomized experimental design with four replications and sampled at 21, 28, 35, 42, 49, 56, 69 days after emergence (DAE) and at flowering stage. For the studies on germination speed, germination and seed weight, the indexes for germination speed, percentage of germination at different temperatures and seed weight of the biotypes were determined. The results showed that the resistant biotype FIMMI 12 shows differences in all variables compared to the resistant biotype FIMMI 10 and compared to the susceptible biotype FIMMI 13, only for the evaluation at flowering. The susceptible biotype FIMMI 13 showed a higher germination speed index and higher germination rate when compared with the resistant biotypes. On the other hand, the resistant biotypes FIMMI 10 and FIMMI 12 showed higher seed weight.

  18. Nitric oxide synthase inhibitors for the treatment of chronic tension-type headache.

    Science.gov (United States)

    Ashina, Messoud

    2002-04-01

    Chronic tension-type headache may be caused by prolonged painful input from pericranial myofacial tissues, for example tender points, resulting in central sensitisation (increased excitability of neurons in the central nervous system). Animal studies have shown that sensitisation of pain pathways may be caused by or associated with the activation of neuronal nitric oxide synthase and the generation of nitric oxide. Furthermore, it has been shown that nitric oxide synthase inhibitors reduce central sensitisation in animal models of persistent pain. On the basis of this information, the analgesic effect of the nitric oxide synthase inhibitor L-N(G) methyl arginine hydrochloride was investigated. This drug significantly reduced headache and myofacial factors in patients with chronic tension-type headache. These studies show that nitric oxide plays a crucial role in the pathophysiology of tension-type headache. The analgesic effect of nitric oxide synthase inhibition in patients with chronic tension-type headache is probably due to a reduction in central sensitisation at the level of the spinal dorsal horn, trigeminal nucleus or both. Furthermore, inhibition of nitric oxide synthase may become a novel principle in the future treatment of chronic headache. PMID:11934342

  19. New monomeric and dimeric uridinyl derivatives as inhibitors of chitin synthase.

    Science.gov (United States)

    Kral, Katarzyna; Bieg, Tadeusz; Nawrot, Urszula; W?odarczyk, Katarzyna; Lalik, Anna; Hahn, Przemys?aw; Wandzik, Ilona

    2015-08-01

    This study described the synthesis and in vitro evaluation of eight new derivatives of uridine as antifungal agents and inhibitors of chitin synthase. Dimeric uridinyl derivatives synthesized by us did not exhibit significant activity. One of the studied monomeric derivative, 5'-(N-succinyl)-5'-amino-5'-deoxyuridine methyl ester (compound 7) showed activities against several fungal strains (MIC range 0.06-1.00mg/mL) and inhibited chitin synthase from Saccharomyces cerevisiae (IC50=0.8mM). Moreover compound 7 exhibited synergistic interaction with caspofungin against Candida albicans (FIC index=0.28). PMID:26051755

  20. Acetohydroxyacid synthase: a target for antimicrobial drug discovery.

    Science.gov (United States)

    Pue, Nason; Guddat, Luke W

    2014-01-01

    Acetohydroxyacid synthase (AHAS) (EC 2.2.1.6) (also known as acetolactate synthase) is the first common enzyme in the branched chain amino acid (BCAA) biosynthesis pathway. This pathway is present in microorganisms and in plants but not in animals, making it an attractive target for both drug and herbicide discovery. The function of AHAS is to catalyze the conversion of two molecules of pyruvate to 2-acetolactate or to convert one molecule of pyruvate and a molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. Three cofactors are required for the activity of AHAS: thiamine diphosphate (ThDP), Mg²? and flavin-adenine dinucleotide (FAD). AHAS is the target for several classes of commercial herbicides that include the sulfonylurea and imidazolinone families. These herbicides are potent and selective inhibitors of AHAS with Ki values that can be in the low nM range. Such compounds also exhibit low application rates as herbicides (typically ~3 g ha?¹) and have low mammalian toxicity (LD?? values typically >4g/kg), thereby highlighting their utility and effectiveness as biocidal agents. However, somewhat surprisingly given the central importance of AHAS in the metabolism of microorganisms, no inhibitors of this enzyme have been commercialized into antimicrobial agents. Here we provide an overview of the biochemical characterization of AHASs from bacterial and fungal sources, analyse the structural features of these enzymes that are criticial to catalysis andprovide the current data on AHAS inhibitors that have potential to be developed into antimicrobial therapeutics. PMID:23688082

  1. Structural characterization of substrate and inhibitor binding to farnesyl pyrophosphate synthase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Schmidberger, Jason W; Schnell, Robert; Schneider, Gunter

    2015-03-01

    Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0?Å. The enzyme shows the typical ?-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme-ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes. PMID:25760619

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

    OpenAIRE

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

    2009-01-01

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

  3. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

    OpenAIRE

    Garcin, Elsa D.; Arvai, Andrew S.; Rosenfeld, Robin J.; Kroeger, Matt D.; Crane, Brian R.; Andersson, Gunilla; Andrews, Glen; Hamley, Peter J.; Mallinder, Philip R.; Nicholls, David J.; St-gallay, Stephen A.; Tinker, Alan C.; Gensmantel, Nigel P.; Mete, Antonio; Cheshire, David R.

    2008-01-01

    Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel...

  4. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    International Nuclear Information System (INIS)

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to Rcryst = 23.7% (Rfree = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis

  5. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    International Nuclear Information System (INIS)

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study

  6. Inhibitors of microsomal prostaglandin E2 synthase-1 enzyme as emerging anti-inflammatory candidates.

    Science.gov (United States)

    Bahia, Malkeet Singh; Katare, Yogesh Kumar; Silakari, Om; Vyas, Bhawna; Silakari, Pragati

    2014-07-01

    Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid (AA) into PGH2 that is further metabolized by terminal prostaglandin (PG) synthases into biologically active PGs, for example, prostaglandin E2 (PGE2), prostacyclin I2 (PGI2), thromboxane A2 (TXA2), prostaglandin D2 (PGD2), and prostaglandin F2 alpha (PGF2?). Among them, PGE2 is a widely distributed PG in the human body, and an important mediator of inflammatory processes. The successful modulation of this PG provides a beneficial strategy for the potential anti-inflammatory therapy. For instance, nonsteroidal anti-inflammatory agents (NSAIDs), both classical nonselective (cNSAIDs) and the selective COX-2 inhibitors (coxibs) attenuate the generation of PGH2 from AA that in turn reduces the synthesis of PGE2 and modifies the inflammatory conditions. However, the long-term use of these agents causes severe side effects due to the nonselective inhibition of other PGs, such as PGI2 and TXA2, etc. Microsomal prostaglandin E2 synthase-1 (mPGES-1), a downstream PG synthase, specifically catalyzes the biosynthesis of COX-2-derived PGE2 from PGH2, and describes itself as a valuable therapeutic target for the treatment of acute and chronic inflammatory disease conditions. Therefore, the small molecule inhibitors of mPGES-1 would serve as a beneficial anti-inflammatory therapy, with reduced side effects that are usually associated with the nonselective inhibition of PG biosynthesis. PMID:25019142

  7. Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.

    Science.gov (United States)

    Kumar, Vidya P; Frey, Kathleen M; Wang, Yiqiang; Jain, Hitesh K; Gangjee, Aleem; Anderson, Karen S

    2013-10-01

    Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2'-chlorophenyl with a sulfur bridge with a Ki of 8.83±0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors. PMID:23927969

  8. Identification of inhibitors of nitric oxide synthase that do not interact with the endothelial cell L-arginine transporter.

    OpenAIRE

    Bogle, R. G.; Moncada, S.; Pearson, J D; Mann, G. E.

    1992-01-01

    The effects of inhibitors of nitric oxide (NO) synthase and other cationic amino acids on unidirectional L-arginine transport were studied in porcine aortic endothelial cells cultured in microwell plates or perfused in microcarrier columns. L-Homoarginine, L-lysine and L-ornithine inhibited transport of L-arginine. The NO synthase inhibitors NG-monomethyl-L-arginine and NG-iminoethyl-L-ornithine also reduced L-arginine uptake, whereas NG-nitro-L-arginine and its methyl-ester had no inhibitory...

  9. Fatty Acid Synthase Inhibitors from Plants and Their Potential Application in the Prevention of Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Wei-xi Tian, Xiao-feng Ma, Shu-yan Zhang, Ying-hui Sun, Bing-hui Li

    2011-03-01

    Full Text Available Fatty acid synthase (FAS attracts more and more attention recently as a potential target for metabolic syndrome, such as cancer, obesity, diabetes and cerebrovascular disease. FAS inhibitors are widely existed in plants, consisting of diversiform compounds. These inhibitors exist not only in herbs also in many plant foods, such as teas, allium vegetables and some fruits. These effective components include gallated catechins, theaflavins, flavonoids, condensed and hydrolysable tannins, thioethers, pentacyclic triterpenes, stilbene derivatives, etc, and they target at the different domains of FAS, showing different inhibitory mechanisms. Interestingly, these FAS inhibitor-contained herbs and plant foods and their effective components are commonly related to the prevention of metabolic syndromes including fat-reducing and depression of cancer. From biochemical angle, FAS can control the balance between energy provision and fat production. Some studies have shown that the effects of those effective components in plants on metabolic syndromes are mediated by inhibiting FAS. This suggests that FAS plays a critical role in the regulation of energy metabolism, and the FAS inhibitors from plants have signi? cant potential application value in the treatment and prevention of metabolic syndromes.

  10. Manejo de Bidens subalternans resistente aos herbicidas inibidores da acetolactato sintase Management of Bidens subalternans resistant to acetolactate synthase inhibitor herbicides

    OpenAIRE

    Gazziero, D. L. P.; Prete, C. E. C.; Sumiya, M.

    2003-01-01

    A extensão das áreas com seleção de populações de plantas daninhas resistentes a herbicidas tem aumentado rapidamente no Brasil nos últimos anos, sendo citado como causa principal desta seleção a recomendação inadequada de produtos. Com o objetivo de avaliar a eficácia de controle de plantas daninhas através de herbicidas, com diferentes mecanismos de ação, sobre plantas de Bidens subalternans, foi conduzido o presente trabalho, que envolveu um experimento de casa de vegetaçã...

  11. Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors

    OpenAIRE

    Duhoux, Arnaud; De?lye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used...

  12. Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

    Directory of Open Access Journals (Sweden)

    Carla S. Jung

    2014-03-01

    Full Text Available Previous results on nitric oxide (NO metabolism after traumatic brain injury (TBI show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA were reported in cerebro-spinal fluid (CSF after traumatic subarachnoid hemorrhage (SAH. Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH and protein-arginine methyltransferase 1 (PRMT1 was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment.

  13. Endothelial and Neuronal Nitric Oxide Synthase Inhibitors Influences Angiotensin II Pressor Effect in Central Nervous System

    Directory of Open Access Journals (Sweden)

    Wilson Abrao Saad

    2006-01-01

    Full Text Available The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP in rats. Losartan and PD123349 AT1 and AT 2 (selective no peptides antagonists angiotensin receptors, as well as FK 409 (a nitric oxide donor, NW-nitro-L-arginine methyl ester (L-NAME a constituve nitric oxide synthase inhibitor endothelial (eNOSI and 7-nitroindazol (7NI a specific neuronal nitric oxide synthase inhibitor (nNOSI were used. Holtzman strain, (Rattus norvergicus weighting 200-250 g were anesthetized with zoletil 50 mg kg-1 (tiletamine chloridrate 125 mg and zolazepan chloridrate 125 mg into quadriceps muscle and a stainless steel cannula was stereotaxically implanted into their Lateral Ventricle (LV. Controls were injected with a 0.5 ?l volume of 0.15 M NaCl. Angiotensin II injected into LV increased MAP (19±3 vs. control 3±1 mm Hg, which is potentiated by prior injection of L-NAME in the same site 26±2 mm Hg. 7NI injected prior to ANG II into LV also potentiated the pressor effect of ANG II but with a higher intensity than L-NAME 32±3 mm Hg. FK 409 inhibited the pressor effect of ANG II (6±1 mm Hg. Losartan injected into LV before ANG II influences the pressor effect of ANG II (8±1 mm Hg. The PD 123319 decreased the pressor effects of ANG II (16±1 mm Hg. Losartan injected simultaneously with FK 409 blocked the pressor effect of ANG II (3±1 mm Hg. L-NAME produced an increase in the pressor effect of ANG II, may be due to local vasoconstriction and all at once by neuronal NOS inhibition but the main effect is of the 7-NIT an specific nNOS inhibitor. The AT1 antagonist receptors improve basal nitric oxide (NO production and release. These data suggest the involvement of constitutive and neuronal NOS in the control of arterial blood pressure induced by ANG II centrally, evolving AT1 receptor-mediated vasoconstriction and AT2 receptor-mediated vasodilatation. These results were confirmed by the experiment using FK 409.

  14. Nitric oxide synthase inhibitors exert differential time-dependent effects on LPS-induced uveitis.

    Science.gov (United States)

    Allen, J B; McGahan, M C; Ferrell, J B; Adler, K B; Fleisher, L N

    1996-01-01

    Nitric oxide (NO) is a highly reactive radical which plays an integral role in physiological and pathophysiological processes. NO is produced endogenously in small amounts by a constitutive NO synthase (cNOS) as a regulator of vascular tone and neurotransmission. NO can also be produced in large amounts by an inducible NOS (iNOS) in response to endotoxin and cytokines, and has been reported to be a mediator of lipopolysaccharide (LPS)-induced uveitis in rats. The purpose of the present study was to investigate the effects of NOS inhibitors with different NOS isoform specificities in the rabbit model of endotoxin-induced ocular inflammation. LPS and/or inhibitors of NOS. NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG), were injected intravitreally and the eyes observed by slit lamp for 24 hr. Coinjection of LPS with L-NAME inhibited anterior inflammation in rabbits. Iridal hyperemia (IH) and aqueous flare (AF) were completely abolished in eight out of nine rabbits in a dose-dependent manner. In addition, total cell counts were significantly suppressed (7393 +/- 697 vs. 325 +/- 188, P aqueous protein levels were reduced to near control levels (25 +/- 0.75 vs. 1.72 +/- 0.36, P aqueous humor. In fact, our results suggest that cNOS may play a greater role in the earlier stages of this developing inflammatory response. These results extend others' observations that NO is a key mediator in uveitis, that induction of iNOS plays a critical role in experimental uveitis, and suggest that NO has a complex role in the ocular inflammatory process. Inhibitors of NOS can abort the LPS-induced inflammatory response if administered early enough, but could potentially exacerbate an established inflammatory episode. PMID:8674509

  15. Características de ??acetolactato sintetasa y producción de diacetilo por Enterococcus faecium ETw7 y Enterococcus faecalis ETw23 / Characteristics of ??acetolactate synthase and diacetyl production by Enterococcus faecium ETw7 and Enterococcus faecalis ETw23

    Scientific Electronic Library Online (English)

    Marisol, Vallejo; Emilio, Marguet; Valeria, Etchechoury.

    Full Text Available El diacetilo es un compuesto aromático esencial en productos lácteos fermentados como el queso. En este trabajo se estudiaron características cinéticas y bioquímicas de la ?-acetolactato sintetasa (?-ALS) y su influencia en la producci?n de diacetilo en Enterococcus faecium ETw7 y Enteroccoccus faec [...] alis ETw23. En ambos casos, los par?metros cinéticos revelaron una baja afinidad por el piruvato, como ha sido descrito en otras bacterias ácido lácticas. E. faecium ETw7 desarrolló la máxima actividad enzimática a pH 5,8-6,2 y 40 ºC, sin embargo bajo las condiciones de maduración de quesos (pH 5,0 y 15 oC) la actividad remanente fue baja. La ?-ALS de E. faecalis ETw23 mostró la máxima actividad al pH de maduración, la temperatura óptima fue determinada a 40 ºC y la actividad remanente a 15 ºC fue aproximadamente el 30% de la máxima. El crecimiento y la producción de diacetilo fue estudiada en el medio De Man-Rogosa-Sharpe (MRS) y MRS suplementado con citrato (MRScit). La tasa de crecimiento de E. faecium ETw7 fue comparable en ambos medios, pero se observó un aumento de la biomasa en MRScit. En el caso de E. faecalis ETw23 se logró una mayor tasa de crecimiento entre las 6 y 10 h, y una mayor biomasa en MRScit. Después de 24 h de crecimiento E. faecium ETw7 alcanzó un nivel de 20,4 ?M de diacetilo en MRS y 26,1 ?M en MRScit, mientras que E. faecalis ETw23 logr? niveles de 41,8 ?M y 61,7 ?M, respectivamente. Los resultados de este estudio sugieren que E. faecalis ETw23 puede contribuir en el desarrollo de aromas en quesos a trav?s de su rol en la producci?n de diacetilo. Abstract in english Diacetyl is an essential flavor compound in fermented dairy products such as cheese. In this work kinetic and biochemical characteristics of ??acetolactate sinthase (?-ALS) and its influence on the formation of diacetyl were studied in Enterococcus faecium ETw7 and Enteroccoccus faecalis ETw23. In b [...] oth cases, the kinetic parameters revealed a low affinity for piruvate, as has been described in other lactic acid bacteria. E. faecium ETw7 displayed its maximal enzimatic activity at pH 5.8-6.2 and 40 ºC, however under cheese ripening condition (pH 5.0 and 15 oC) the remaining activity was low. ??ALS from E. faecalis ETw23 showed its maximal activity at ripening pH, the optimun temperature was determined at 40 ºC and the remaining activity at 15 ºC was about 30% of its maximal one. The growth and diacetyl formation by both strains were studied in De Man-Rogosa-Sharpe medium (MRS) and MRS supplemented with citrate (MRScit). In both medium the growth rate of E. faecium ETw7 was comparable but an enhancement in biomass was observed in MRScit. In the case of E. faecalis ETw23 a higher growth rate, between 6 h and 10 h, and a higher biomass were achieved in MRScit. After 24 h of growth, E. faecium ETw7 reached a level of 20.4 ?M of diacetyl in MRS and 26.1 ?M in MRScit, while E. faecalis ETw23 achieved levels of 41.8 ?M and 61.7 ?M, respectively. The results of the study suggest that E. faecalis ETw23 may contribute to flavor development in cheese through its role in diacetyl production.

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

    Science.gov (United States)

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

    1993-11-25

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

  17. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase ? subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-? suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on ?-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  18. Effect of centrally administered C75, a fatty acid synthase inhibitor, on ghrelin secretion and its downstream effects

    OpenAIRE

    Hu, Zhiyuan; Cha, Seung Hun; Haasteren, Goedelle; Wang, Jing; Lane, M. Daniel

    2005-01-01

    The central administration of the fatty acid synthase (FAS) inhibitor, C75, rapidly suppresses the expression of orexigenic neuropeptides [neuropeptide Y (NPY) and agouti-related protein (AgRP)] and activates expression of anorexigenic neuropeptides [proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART)] in the hypothalamus. The combined actions of these changes inhibit food intake and decrease body weight. Intracerebroventricular injection of C75 appears to rapi...

  19. Comparison of plasma and tissue levels of ZD1694 (Tomudex), a highly polyglutamatable quinazoline thymidylate synthase inhibitor, in preclinical models.

    OpenAIRE

    Aherne, G. W.; Ward, E.; Lawrence, N; Dobinson, D.; Clarke, S. J.; Musgrove, H.; Sutcliffe, F; Stephens, T; Jackman, A.L.

    1998-01-01

    ZD1694 (Tomudex, raltitrexed) is a specific quinazoline antifolate thymidylate synthase inhibitor that relies on polyglutamation for high potency. Antibodies to ZD1694 have been used to establish a sensitive radioimmunoassay as an alternative to high-performance liquid chromatography (HPLC). The radioimmunoassay is reproducible, accurate and provides a means of determining low levels of ZD1694 in plasma (< 1 nM). By virtue of the high cross-reactivity of the antibodies with polyglutamated for...

  20. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis.

    Science.gov (United States)

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C

    2015-07-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

  1. The effects of tempol, 3-aminobenzamide and nitric oxide synthase inhibitors on acoustic injury of the mouse cochlea.

    Science.gov (United States)

    Murashita, Hidekazu; Tabuchi, Keiji; Hoshino, Tomofumi; Tsuji, Shigeki; Hara, Akira

    2006-04-01

    Oxygen free radicals have been implicated in the pathogenesis of acoustic injury of the cochlea. The purpose of this study was to evaluate the effects of tempol (a superoxide anion scavenger), 3-aminobenzamide (a poly (ADP-ribose) synthetase (PARS) inhibitor), N-nitro-l-arginine (a non-selective nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (a selective neuronal NOS inhibitor) and aminoguanidine (a selective inducible NOS inhibitor) on acoustic injury. Mice were exposed to a 4 kHz pure tone of 110-128 dB SPL for 4h. Tempol, 3-aminobenzamide or N-nitro-l-arginine was intraperitoneally administered immediately before the onset of acoustic overexposure, while 7-nitroindazole or aminoguanidine was intraperitoneally administered every 12h starting immediately before the onset of acoustic overexposure. The threshold shift of the auditory brainstem response (ABR) and hair cell loss were then evaluated one and two weeks after acoustic overexposure. Tempol and 3-aminobenzamide significantly protected the cochlea against acoustic injury, whereas the NOS inhibitors did not exert any protective effect. These findings suggest that reactive oxygen species and PARS are involved in acoustic injury of the cochlea. However, further study is necessary to elucidate the roles of nitric oxide and nitric oxide synthase in acoustic injury. PMID:16516419

  2. Stimulation by nitric oxide synthase inhibitors of gastric and duodenal HCO3- secretion in rats.

    Science.gov (United States)

    Takeuchi, K; Ohuchi, T; Miyake, H; Okabe, S

    1993-09-01

    The role of nitric oxide (NO) in the regulation of gastroduodenal HCO3- secretion was investigated in anesthetized rats using the NO biosynthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME). HCO3- secretion was measured at pH 7.0 using a pH-stat method in the chambered stomach in the presence of omeprazole or in the proximal duodenum. Intravenous administration of L-NAME (1-5 mg/kg) increased HCO3- secretion in a dose-dependent manner in both the stomach and duodenum, with a concomitant elevation of arterial blood pressure. The stimulatory effect of L-NAME on HCO3- secretion was mimicked by another NO synthase inhibitor, NG-monomethyl-L-arginine (50 mg/kg), but not by the enantiomer NG-nitro-D-arginine methyl ester, and was significantly antagonized by concurrent administration of L-arginine, but not D-arginine, at 200 mg/kg. The exogenous NO donor nitroprusside (4 mg/kg) by itself decreased the rate of HCO3- secretion and significantly antagonized the HCO3- stimulatory action of L-NAME. Furthermore, the increased HCO3- secretion caused by L-NAME was significantly attenuated by prior administration of atropine (1 mg/kg, s.c.) or indomethacin (5 mg/kg, s.c.) and by bilateral vagotomy but was not influenced by sensory deafferentation after capsaicin pretreatment, though none of the treatments had any effect on the changes in blood pressure induced by L-NAME. These results suggest that L-NAME stimulates HCO3- secretion in the gastroduodenal mucosa. This action is associated with the inhibition of NO biosynthesis and may be partly dependent on vagal-cholinergic innervation and mediated by endogenous prostaglandins. PMID:7690403

  3. Deltamethrin-induced testicular apoptosis in rats: the protective effect of nitric oxide synthase inhibitor.

    Science.gov (United States)

    El-Gohary, M; Awara, W M; Nassar, S; Hawas, S

    1999-01-01

    This study is the first to examine and characterize the testicular apoptosis which might be induced due to exposure of male rats to deltamethrin. Furthermore, the role which might be played by nitric oxide (NO), as well as the other reactive oxygen species (ROS) in controlling this testicular apoptosis was assessed. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis and cellular morphology on testicular tissue sections. It was found that administration of deltamethrin (1 mg/kg daily for 21 days) to animals resulted in characteristic DNA migration patterns (laddering), thereby providing evidence that apoptosis is the major mechanism of cell death in the testicular tissues. In addition, histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells, primary and secondary spermatocytes. These changes, in addition to the appearance of Sertoli cell vacuoles in deltamethrin-intoxicated animals, indicates the suppression of spermatogenesis. At the same time, the plasma levels of both NO and lipid peroxides measured as malondialdehyde (MDA) were found to be significantly increased in deltamethrin-treated animals. Administration of NO synthase (NOS) inhibitors such as N(G)-nitro monomethyl L-arginine hydrochloride (L-NMMA, 1 mg/kg) to rats 2 h before exposure to deltamethrin was effective in the reduction of the typically testicular apoptotic DNA fragmentation pattern and the associated histopathological changes. These findings may suggest that deltamethrin-induced testicular apoptosis is mediated by NO. Therefore, the pharmacological manipulation of apoptosis by selective NOS inhibitors such as L-NMMA may offer new possibilities for the control of deltamethrin-induced testicular dysfunction and infertility in the future. PMID:10199576

  4. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    Energy Technology Data Exchange (ETDEWEB)

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  5. Allosteric inhibitor specificity of Thermotoga maritima 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

    Science.gov (United States)

    Cross, Penelope J; Parker, Emily J

    2013-09-17

    3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Allosteric regulation of Thermotoga maritima DAH7PS is mediated by L-Tyr binding to a discrete ACT regulatory domain appended to a core catalytic (?/?)8 barrel. Variants of T. maritima DAH7PS (TmaDAH7PS) were created to probe the role of key residues in inhibitor selection. Substitution Ser31Gly severely reduced inhibition by L-Tyr. In contrast both L-Tyr and L-Phe inhibited the TmaHis29Ala variant, while the variant where Ser31 and His29 were interchanged (His29Ser/Ser31His), was inhibited to a greater extent by L-Phe than L-Tyr. These studies highlight the role and importance of His29 and Ser31 for determining both inhibitory ligand selectivity and the potency of allosteric response by TmaDAH7PS. PMID:23916814

  6. Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

    Scientific Electronic Library Online (English)

    C.A.A., Perotti; M.S., Nogueira; J., Antunes-Rodrigues; E.C., Cárnio.

    1999-11-01

    Full Text Available It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg [...] /kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P

  7. Pharmacokinetics and biochemical efficacy of pirmagrel, a thromboxane synthase inhibitor, in renal allograft recipients.

    Science.gov (United States)

    Chouinard, M L; Martin, L L; Coffman, T; Hamilton, B H; Linberg, L F; Pamidi, A; Simke, J P; Rakhit, A

    1992-12-01

    The effects of a 48-hour 0.5 mg/kg/hr infusion of the thromboxane synthase inhibitor pirmagrel were studied in 10 renal allograft recipients with cyclosporine nephrotoxicity. Plasma concentrations reached a mean steady-state plasma level of 1798 +/- 481 ng/ml. Biphasic, rapid elimination of pirmagrel was observed with a distribution half-life of 6.7 minutes and a terminal half-life of 73 minutes. Plasma clearance and the volume of distribution of the drug were 300 +/- 87 ml/hr/kg and 497 +/- 232 ml/kg, respectively. The pharmacodynamic effects of pirmagrel were marked by a mean 96% suppression of serum thromboxane B2 (TXB2), which coincided with a suppression of urinary excretion of TXB2, 2,3-dinor-TXB2, and 11-dehydro-TXB2 of 85% +/- 8%, 91% +/- 5%, and 89% +/- 9%, respectively. Urinary excretion of all thromboxane metabolites measured at the end of 1 week after termination of infusion was returned to the baseline. In conclusion, pirmagrel caused effective and sustained suppression of all thromboxane derived metabolites in plasma and urine during continuous infusion in kidney transplant patients receiving cyclosporine. PMID:1458769

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

    Science.gov (United States)

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

    2009-01-01

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

  9. A glucosylceramide synthase inhibitor protects rats against the cytotoxic effects of shiga toxin 2.

    Science.gov (United States)

    Silberstein, Claudia; Lucero, María S; Zotta, Elsa; Copeland, Diane P; Lingyun, Li; Repetto, Horacio A; Ibarra, Cristina

    2011-05-01

    Postdiarrhea hemolytic uremic syndrome is the most common cause of acute renal failure in children in Argentina. Renal damage has been strongly associated with Shiga toxin (Stx), which binds to the globotriaosylceramide (Gb3) receptor on the plasma membrane of target cells. The purpose of the study was to evaluate the in vivo effects of C-9, a potent inhibitor of glucosylceramide synthase and Gb3 synthesis, on kidney and colon in an experimental model of hemolytic uremic syndrome in rats. Rats were i.p. injected with supernatant from recombinant Escherichia coli expressing Stx2 (sStx2). A group of these rats were orally treated with C-9 during 6 d, from 2 d prior until 4 d after sStx2 injection. The injection of sStx2 caused renal damage as well as a loss of goblet cells in colonic mucosa. Oral treatment with C-9 significantly decreased rat mortality to 50% and reduced the extension of renal and intestinal injuries in the surviving rats. The C-9 also decreased Gb3 and glucosylceramide expression levels in rat kidneys. It is particularly interesting that an improvement was seen when C-9 was administered 2 d before challenge, which makes it potentially useful for prophylaxis. PMID:21270676

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

    Science.gov (United States)

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

    2012-12-14

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

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

    Science.gov (United States)

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

    2012-06-19

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

  12. The Effects of nitric oxide synthase inhibitor (L-NAME on epididymal sperm count, motility, and morphology in varicocelized rat

    Directory of Open Access Journals (Sweden)

    Bahmanzadeh M.

    2008-03-01

    Full Text Available Introduction: Increase in the nitric oxide in the spermatic veins of men by varicocele has been reported. Although Several studies have considered the relationship between varicocele and semen NO concentrations, no study on the effects of nitric oxide synthase inhibitor (L-NAME on epididymal sperm count, motility and morphology which are important in fertility of the individual has been reported. The aim of study was to evaluate the effects of nitric oxide synthase inhibitor (L-NAME on epididymal sperm count, motility, and morphology in varicocelized rat.Methods: Twenty four Wistar male rats divided into four groups. The group A and B underwent a left experimental varicocele (by 20-gauge needle. Group C, underwent a procedure similar to groups A and B without any change on spermatic vein (as sham group. Group D referred to as control. Animals in group A were killed 10 weeks after the operation and both left and right epididymal sperm were counted and their morphology and motility were analyzed. Animals in group B received 10mg/kg L-NAME intraperitoneally daily for ten weeks.Results: In group A, Sperm count decreased and the morphology changed significantly in comparison with the groups C and D. The sperm morphology in groups A and B showed statistically significant differences (P<0.0001. Sperm motility decreased significantly in the group A in comparison with the groups C and D. Although motility in group A of animals were different in comparison with group B , it was not statistically significant.Conclusion: These findings suggest that nitric oxide synthase inhibitor (L-NAME improved sperm count and morphology.

  13. Effect of Potential Amine Prodrugs of Selective Neuronal Nitric Oxide Synthase Inhibitors on Blood-Brain Barrier Penetration

    OpenAIRE

    Silverman, Richard B.; Lawton, Graham R.; Ranaivo, Hantamalala Ralay; Seo, Jiwon; Watterson, D. Martin

    2009-01-01

    Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood-brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an...

  14. Selective inhibitors of neuronal nitric oxide synthase--is no NOS really good NOS for the nervous system?

    Science.gov (United States)

    Moore, P K; Handy, R L

    1997-06-01

    It is now ten years since NO was shown to account for the biological activity of endothelium-derived relaxing factor (EDRF). It is also the tenth anniversary of the identification of L-NG monomethyl arginine (L-NMMA) as the very first inhibitor of NO biosynthesis. That EDRF and NO were one and the same sparked an explosion of interest in the biochemistry and pharmacology of NO which has yet to subside. In contrast, the first ever nitric oxide synthase (NOS) inhibitor slipped seamlessly into the literature virtually without comment at the time. Over the following decade, L-NMMA (and like NOS inhibitors) have proved invaluable as tools for probing the biological roles of NO in health and disease and, in particular, have increased our understanding of the function of NO in the nervous system. Further advances in this important area now require the development of inhibitors selective for the neuronal isoform of NOS (nNOS). Here, Philip Moore and Rachel Handy provide an up-to-date account of the literature regarding the biochemical and pharmacological characterization of NOS inhibitors with particular reference to compounds with greater selectivity for the nNOS isoform. PMID:9226999

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

    OpenAIRE

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

    2009-01-01

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

  16. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound

    OpenAIRE

    ZHAO, GENGXIANG; Haskins, Nantaporn; Jin, Zhongmin; ALLEWELL, NORMA M.; Tuchman, Mendel; SHI, DASHUANG

    2013-01-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-argini...

  17. Brain-derived neurotrophic factor and nitric oxide synthase inhibitor protect the vestibular organ against gentamicin ototoxicity.

    Science.gov (United States)

    Takumida, Masaya; Anniko, Matti

    2002-01-01

    In order to find a way to develop a new treatment for inner ear disorders, the effects of a nitric oxide synthase (NOS) inhibitor [N-nitro-L-arginine methylester (L-NAME)] and a neurotrophin [brain-derived neurotrophic factor (BDNF)] were investigated. The effect of L-NAME and BDNF on gentamicin-induced vestibular hair cell damage was investigated by using the in vitro LIVE/DEAD system. Both L-NAME and BDNF individually reduced the vestibular hair cell damage caused by gentamicin but the combination of L-NAME and BDNF was more successful in preventing damage. It is therefore suggested that treatment with a combination of an NOS inhibitor and a neurotrophin will help us to treat inner ear disorders. PMID:11876587

  18. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    International Nuclear Information System (INIS)

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in As induced VaD

  19. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Bhupesh, E-mail: drbhupeshresearch@gmail.com; Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in As induced VaD.

  20. L-NAME, a nitric oxide synthase inhibitor, as a potential countermeasure to post-suspension hypotension in rats

    Science.gov (United States)

    Bayorh, M. A.; Socci, R. R.; Watts, S.; Wang, M.; Eatman, D.; Emmett, N.; Thierry-Palmer, M.

    2001-01-01

    A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30 degrees tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

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

    International Nuclear Information System (INIS)

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

  2. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs

    International Nuclear Information System (INIS)

    High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (10ity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy

  3. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    Energy Technology Data Exchange (ETDEWEB)

    Sprenger, Janina [Lund University, SE-221 00 Lund (Sweden); Lund University, SE-221 84 Lund (Sweden); Svensson, Bo [Lund University, SE-221 00 Lund (Sweden); SARomics Biostructures AB, Box 724, SE-220 07 Lund (Sweden); Hålander, Jenny [Lund University, SE-221 00 Lund (Sweden); Carey, Jannette [Princeton University, Princeton, New Jersey (United States); Persson, Lo [Lund University, SE-221 84 Lund (Sweden); Al-Karadaghi, Salam, E-mail: salam.al-karadaghi@biochemistry.lu.se [Lund University, SE-221 00 Lund (Sweden)

    2015-03-01

    In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5?-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

  4. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    International Nuclear Information System (INIS)

    In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5?-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS

  5. Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.

    Science.gov (United States)

    Ashe, Karen M; Bangari, Dinesh; Li, Lingyun; Cabrera-Salazar, Mario A; Bercury, Scott D; Nietupski, Jennifer B; Cooper, Christopher G F; Aerts, Johannes M F G; Lee, Edward R; Copeland, Diane P; Cheng, Seng H; Scheule, Ronald K; Marshall, John

    2011-01-01

    The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects. PMID:21738789

  6. Thiolactomycin-based ?-ketoacyl-AcpM synthase A (KasA) inhibitors: fragment-based inhibitor discovery using transient one-dimensional nuclear overhauser effect NMR spectroscopy.

    Science.gov (United States)

    Kapilashrami, Kanishk; Bommineni, Gopal R; Machutta, Carl A; Kim, Pilho; Lai, Cheng-Tsung; Simmerling, Carlos; Picart, Francis; Tonge, Peter J

    2013-03-01

    Thiolactomycin (TLM) is a natural product inhibitor of KasA, the ?-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy. PMID:23306195

  7. Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening.

    Science.gov (United States)

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J; Teitelbaum, Aaron M; Remmel, Rory P; Aldrich, Courtney C

    2010-12-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M.?tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class?I), diarylsulfones (class?II), and benzimidazole-2-thiones (class?III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

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

    Science.gov (United States)

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

    2005-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Sheela Sharma

    2002-01-01

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

  10. The mTOR Inhibitor Rapamycin Synergizes with a Fatty Acid Synthase Inhibitor to Induce Cytotoxicity in ER/HER2-Positive Breast Cancer Cells

    Science.gov (United States)

    Yan, Chen; Yan, Xue; Jingyue, Yang; Wenchao, Liu; Sheng, Han

    2014-01-01

    Patients with ER/HER2-positive breast cancer have a poor prognosis and are less responsive to selective estrogen receptor modulators; this is presumably due to the crosstalk between ER and HER2. Fatty acid synthase (FASN) is essential for the survival and maintenance of the malignant phenotype of breast cancer cells. An intimate relationship exists between FASN, ER and HER2. We hypothesized that FASN may be the downstream effector underlying ER/HER2 crosstalk through the PI3K/AKT/mTOR pathway in ER/HER2-positive breast cancer. The present study implicated the PI3K/AKT/mTOR pathway in the regulation of FASN expression in ER/HER2-positive breast cancer cells and demonstrated that rapamycin, an mTOR inhibitor, inhibited FASN expression. Cerulenin, a FASN inhibitor, synergized with rapamycin to induce apoptosis and inhibit cell migration and tumorigenesis in ER/HER2-positive breast cancer cells. Our findings suggest that inhibiting the mTOR-FASN axis is a promising new strategy for treating ER/HER2-positive breast cancer. PMID:24866893

  11. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    OpenAIRE

    Martin C. Sadowski; Pouwer, Rebecca H.; Gunter, Jennifer H.; Lubik, Amy A.; Quinn, Ronald J.; Nelson, Colleen C.

    2014-01-01

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular e...

  12. A Small Molecule Deubiquitinase Inhibitor Increases Localization of Inducible Nitric Oxide Synthase to the Macrophage Phagosome and Enhances Bacterial Killing?†

    Science.gov (United States)

    Burkholder, Kristin M.; Perry, Jeffrey W.; Wobus, Christiane E.; Donato, Nicholas J.; Showalter, Hollis D.; Kapuria, Vaibhav; O'Riordan, Mary X. D.

    2011-01-01

    Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking. PMID:21911458

  13. A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

    Science.gov (United States)

    Burkholder, Kristin M; Perry, Jeffrey W; Wobus, Christiane E; Donato, Nicholas J; Showalter, Hollis D; Kapuria, Vaibhav; O'Riordan, Mary X D

    2011-12-01

    Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking. PMID:21911458

  14. Substrate channeling: alpha-ketobutyrate inhibition of acetohydroxy acid synthase in Salmonella typhimurium.

    OpenAIRE

    Shaw, K. J.; Berg, C. M.

    1980-01-01

    Excess alpha-ketobutyrate inhibited the growth of Salmonella typhimurium LT2 by inhibiting the acetohydroxy acid synthase-catalyzed synthesis of alpha-acetolactate (a valine precursor). As a result, cells were starved for valine, and both ilvB (encoding acetohydroxy acid synthase I) and ilvGEDA (ilvG encodes acetohydroxy acid synthase II) were derepressed. The addition of valine reversed the effects of alpha-ketobutyrate.

  15. Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges.

    Czech Academy of Sciences Publication Activity Database

    Víte?ek, J.; Lojek, Antonín; Valacchi, G.; Kubala, Lukáš

    2012-01-01

    Ro?. 2012, ?. 2012 (2012), ID 318087. ISSN 0962-9351 R&D Projects: GA ?R(CZ) GA524/08/1753 Institutional support: RVO:68081707 Keywords : METHYL-L-ARGININE * HIGHLY SELECTIVE INHIBITORS * CARDIOVASCULAR RISK-FACTOR Subject RIV: BO - Biophysics Impact factor: 3.882, year: 2012

  16. In silico deconstruction of ATP-competitive inhibitors of glycogen synthase kinase-3?.

    Science.gov (United States)

    Bisignano, Paola; Lambruschini, Chiara; Bicego, Manuele; Murino, Vittorio; Favia, Angelo D; Cavalli, Andrea

    2012-12-21

    Fragment-based methods have emerged in the last two decades as alternatives to traditional high throughput screenings for the identification of chemical starting points in drug discovery. One arguable yet popular assumption about fragment-based design is that the fragment binding mode remains conserved upon chemical expansion. For instance, the question of the binding conservation upon fragmentation of a molecule is still unclear. A number of papers have challenged this hypothesis by means of experimental techniques, with controversial results, "underlining" the idea that a simple generalization, maybe, is not possible. From a computational standpoint, the issue has been rarely addressed and mostly to test novel protocols on limited data sets. To fill this gap, we here report on a computational retrospective study concerned with the in silico deconstruction of leadlike compounds, active on the pharmaceutically relevant enzyme glycogen synthase kinase-3?. PMID:23198830

  17. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    Directory of Open Access Journals (Sweden)

    Tomishima Yoshiro

    2013-01-01

    Full Text Available Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2 synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg. The effects of ozagrel (200 mg/kg treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL on cytochrome P450 2E1 (CYP2E1 activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos and C/EBP homologous protein (chop, but did not suppress B-cell lymphoma 2-like protein11 (bim expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury.

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

    OpenAIRE

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

    2013-01-01

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

  19. Characterization of Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitors as Stimulators of Steroidogenesis

    OpenAIRE

    Gunosewoyo, Hendra; Midzak, Andrew; Gaisina, Irina N.; Sabath, Emily V.; Fedolak, Allison; Hanania, Taleen; Brunner, Dani; Papadopoulos, Vassilios; Kozikowski, Alan P.

    2013-01-01

    Inhibition of GSK-3? has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3? resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3? inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these...

  20. Amaranthus palmeri resistance and differential tolerance of Amaranthus palmeri and Amaranthus hybridus to ALS-inhibitor herbicides.

    Science.gov (United States)

    Burgos, N R; Kuk, Y I; Talbert, R E

    2001-05-01

    Suspected imazaquin-resistant accessions of Amaranthus palmeri were studied to determine the magnitude of resistance and cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides and compare differential tolerance of A palmeri and Amaranthus hybridus to ALS inhibitors. Five of seven A palmeri accessions were resistant to imazaquin. The most imazaquin-resistant accession, accession 7, also showed 74, 39 and 117 times higher resistance than the susceptible biotype to chlorimuron, diclosulam and pyrithiobac, respectively. Resistance to imazaquin and cross-resistance to other ALS inhibitors in A palmeri was due to a less-sensitive ALS enzyme. A palmeri was 70 times more tolerant to imazaquin than A hybridus. A palmeri was also seven times more tolerant to pyrithiobac than A hybridus. Differences in ALS enzyme sensitivity could not fully account for the high tolerance of A palmeri to imazaquin compared to A hybridus. Both species were equally affected by chlorimuron and diclosulam. PMID:11374163

  1. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.

    Science.gov (United States)

    Jensen, Samuel A; Calvert, Andrea E; Volpert, Giora; Kouri, Fotini M; Hurley, Lisa A; Luciano, Janina P; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H; Stegh, Alexander H

    2014-04-15

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

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

    Science.gov (United States)

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

    2005-02-10

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

  3. N-nitro-L-arginine, a nitric oxide synthase inhibitor, aggravates iminodipropionitrile-induced neurobehavioral and vestibular toxicities in rats.

    Science.gov (United States)

    Khan, Haseeb Ahmad

    2012-11-01

    Exposure of iminodipropionitrile (IDPN) to rodents produces permanent behavioral syndrome characterized by repetitive head movements, circling and back walking. Other synthetic nitriles of industrial importance such as crotonitrile and allylnitrile are also able to produce similar motor deficits in experimental animals. However, due to the well-defined behavioral deficits and their easy quantification, IDPN-induced behavioral syndrome is a preferential animal model to test the interaction of various agents with synthetic nitriles. This study reports the effect of non-specific nitric oxide synthase inhibitor, N-nitro-L-arginine (NARG) on IDPN-induced neurobehavioral toxicity in adult male Wistar rats. Four groups of animals were given i.p. injections of IDPN (100 mg/kg) for 6 days. These rats were treated with oral administration of NARG in the doses of 0 (IDPN alone group), 50, 150 and 300 mg/kg, 60 min before IDPN, respectively. Control rats received vehicle only, whereas another group was treated with 300 mg/kg of NARG alone (without IDPN). The results showed that NARG significantly exacerbated the incidence and intensity of IDPN-induced dyskinetic head movements, circling and back walking. The histology of inner ear showed massive degeneration of the sensory hair cells in the crista ampullaris of rats receiving the combined treatment with IDPN and NARG, suggesting a possible role of nitric oxide in IDPN-induced neurobehavioral syndrome in rats. PMID:21388795

  4. Effect of nitric oxide synthase inhibitor L-NAME on fear extinction in rats: a task-dependent effect.

    Science.gov (United States)

    Luo, Huaiqing; Han, Li; Tian, Shaowen

    2014-06-20

    There is increasing evidence that nitric oxide may be involved in learning and memory. However, there remain comparatively few studies that have explored the relationship between nitric oxide signaling and fear extinction, an inhibitory learning model. In the present study, we tested the effects of nitric oxide synthase inhibitor l-NAME on three tone fear extinction tasks in rats. In task 1, rats received fear conditioning, extinction training and extinction test in the same context (AAA design). In task 2, rats received fear conditioning in context A, extinction training in context B and extinction test in context A (ABA design). In task 3, rats received fear conditioning in context A, extinction training and extinction test in context B (ABB design). l-NAME (10, 20 and 40 mg/kg) was injected intraperitoneally 30 min prior to extinction training in each task. Percent of time spent freezing was used to measure conditioned fear response. We found that l-NAME administrations had no effect on freezing in task 1 and 2 but produced a dose-dependent increase in task 3. Further results indicated that the increased freezing in task 3 was not attributed to state-dependency effects or nonspecific changes of locomotor activity that followed l-NAME injection. These results showed that l-NAME produced a task-dependent impairment of fear extinction, and implied that nitric oxide signaling was involved in memory process of certain extinction tasks. PMID:24792396

  5. Inhibition of melatonin-induced ascorbic acid and LHRH release by a nitric oxide synthase and cyclic GMP inhibitor.

    Science.gov (United States)

    Karanth, Sharada; Yu, Wen H; Mastronardi, Claudio A; McCann, Samuel M

    2004-07-01

    Melatonin (MEL), the principle secretory product of the pineal gland, has been shown to function as an antioxidant and free-radical scavenger. We previously showed that the release of ascorbic acid (AA) and luteinizing hormone releasing hormone (LHRH) from medial basal hypothalamus (MBH) was mediated by nitric oxide (NO) that released cyclic guanosine 3'5'-mono-phosphate (cGMP). Therefore, it was of interest to evaluate the effect of MEL on AA and LHRH release and study the effect of a nitric oxide synthase (NOS) inhibitor, 6-anilino-5,8-quinoline-dione (LY 83583), and a guanylyl cyclase (GC) inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (O.D.Q.), on the release process. Because NO has been shown to activate soluble guanylyl cyclase that elicited an elevation of cGMP in target cells, in the current investigation LY 83583, O.D.Q., or N(G)-monomethyl-l-arginine (NMMA), a competitive inhibitor of NOS, were used to evaluate their effects on MEL-induced AA and LHRH release. Medial basal hypothalami were incubated in 0.5 ml of Krebs-Ringer bicarbonate (KRB) buffer for 1 hr. Subsequently, the tissues were incubated with graded concentrations of MEL (10(-8) to 10(-4) M), MEL + NMMA (3 x 10(-4) M), MEL + LY 83583 (10(-6) M), or MEL + O.D.Q. (10(-5) M) for 1 hr. Ascorbic acid and LHRH released into the medium were measured by high-performance liquid chromatography (HPLC) and radio-immunoassay (RIA), respectively. Melatonin (10(-6) and 10(-5) M) significantly stimulated both AA and LHRH release, but the lower and the highest concentrations were ineffective. A combination of MEL + NMMA completely blocked both AA and LHRH release, supporting a role for NO in the releasing action. Both LY 83583 and O.D.Q. significantly suppressed MEL-induced AA and LHRH release, emphasizing the role of NOS, GC, and cGMP in mediating the action of MEL. The data of these in vitro experiments support a role for MEL in the hypothalamic control of AA and LHRH release. PMID:15229359

  6. Effects of selective inhibitors of neuronal and inducible NO-synthase on ATP content and survival of cultured rat cerebellar neurons during hyperstimulation of glutamate receptors.

    Science.gov (United States)

    Salykina, M A; Sorokina, E G; Krasilnikova, I A; Reutov, V P; Pinelis, V G

    2013-05-01

    We studied the effects of selective inhibitors of neuronal and inducible NO-synthase (7-nitroindazole and aminoguanidine) and non-selective NO-synthase inhibitor L-NAME on ATP content and survival of cultured rat cerebellar neurons during hyperstimulation of glutamate receptors with toxic doses of glutamate. Application of 100 ?M glutamate reduced ATP content in the primary culture of 7-8- and 14-15-day-old cerebellar granule cells by 66 and 49%, respectively, in comparison with the control. Inhibition of nitric oxide synthesis with 7-nitroindazole during glutamate exposure in the culture of 7-8-day-old neurons and with 7-nitroindazole and aminoguanidine in the culture of 14-15-day-old neurons ensured better protection of cells from ATP level decrease than non-specific inhibition with L-NAME. In addition, inhibition of neuronal and inducible NO-synthase during glutamate exposure decreased death of "young" neurons, whereas death of "old" neurons remained high under these conditions. PMID:23667868

  7. Resistência de Bidens subalternans aos herbicidas inibidores da enzima acetolactato sintase utilizados na cultura da soja Resistance of Bidens subalternans to the acetolactate synthase inhibitor herbicides used in soybean crop

    OpenAIRE

    Gelmini, G. A.; Victo?ria Filho, R.; Novo, M. C. S. S.; Adoryan, M. L.

    2002-01-01

    O uso contínuo e prolongado de produtos com o mesmo mecanismo de ação pode provocar a manifestação de biótipos resistentes. Para verificar possíveis novos casos de resistência, bem como alternativas para prevenção e manejo, foram coletadas sementes de Bidens subalternans na região de São Gabriel D' Oeste-MS, em plantas que sobreviveram a tratamentos em que inibidores da ALS foram sistematicamente utilizados. Em experimento conduzido em vasos em casa de vegetação, o biótipo com ...

  8. CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

    Science.gov (United States)

    Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-02-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  9. CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

    Science.gov (United States)

    Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-01-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  10. NO-synthase inhibitors provide influence on protective effect of modified endotoxine diphosphoryl lipid A in a rat heart model of ischemic-reperfusion injury.

    Science.gov (United States)

    Kuzelová, M; Mladonická, M; Bukovský, M; Dubnicková, M; Adameová, A; Svec, P

    2006-06-01

    The present study was designed to assess whether a protective effect of the modified diphosphoryl lipid A (modLA) against myocardial ischemia-reperfusion injury (IRI) in rats can be related to the mechanism involving inducible nitric oxide synthase (iNOS). Pre-treatment with modLA significantly reduced the duration of both ventricular tachycardia (p modLA was markedly attenuated by the prior administration of selective iNOS inhibitor S-methylisothiourea (SMT). In this animal group, mortality was significantly increased (p modLA. PMID:16826982

  11. Glycogen synthase kinase-3? (GSK-3?) inhibitors AR-A014418 and B6B3O prevent human immunodeficiency virus–mediated neurotoxicity in primary human neurons

    OpenAIRE

    Nguyen, Timothy B.; Lucero, Ginger R.; Chana, Gursharan; Hult, Britta J.; Tatro, Erick T.; Masliah, Eliezer; Grant, Igor; Achim, Cristian L.; Everall, Ian P.

    2009-01-01

    Glycogen synthase kinase-3? (GSK3?) role in human immunodeficiency virus (HIV)-associated neurodegeneration has been evidenced by previous investigations. In this study, we investigated the specificity of two GSK3?-specific inhibitors, AR-A014418 (A) and B6B30 (B) to prevent direct neurotoxicity in primary human neurons exposed to HIV (BaL). Neurons were exposed to HIV (500 pg/ml) for 12-h and 6-day periods in the presence and absence of A (1 µM, 100 nM, 10 nM) and B (50 nM, 5 nM, 500 pM)...

  12. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.

    OpenAIRE

    Jackman, A.L.; Kelland, L R; Kimbell, R; BROWN, M.; GIBSON, W; Aherne, G. W.; Hardcastle, A; Boyle, F.T.

    1995-01-01

    Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to an...

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

    Science.gov (United States)

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

    2009-03-20

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

  14. Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2014-07-01

    Full Text Available A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS. Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS. The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration.

  15. Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase.

    Czech Academy of Sciences Publication Activity Database

    von Leitner, E.C.; Klinke, A.; Atzler, D.; Slocum, J.L.; Lund, N.; Kielstein, J.T.; Maas, R.; Schmidt-Haupt, R.; Pekarová, Michaela; Hellwinkel, O.; Tsikas, D.; D'Alecy, L.G.; Lau, D.; Willems, S.; Kubala, Lukáš; Ehmke, H.; Meinertz, T.; Blankenberg, S.; Schwedhelm, E.; Gadegbeku, C.A.; Boger, R.H.; Baldus, S.; Sydow, K.

    2011-01-01

    Ro?. 124, ?. 4 (2011), s. 2735-U342. ISSN 0009-7322 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : arteriosclerosis * leukocytes * nitric oxide synthase Subject RIV: BO - Biophysics Impact factor: 14.739, year: 2011

  16. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae.

    Science.gov (United States)

    Demaeght, Peter; Osborne, Edward J; Odman-Naresh, Jothini; Grbi?, Miodrag; Nauen, Ralf; Merzendorfer, Hans; Clark, Richard M; Van Leeuwen, Thomas

    2014-08-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as 'mite growth inhibitors', and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  17. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

    2013-11-15

    Highlights: •EV-077 reduced TNF-? induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNF? incubation, whereas concentrations of 6-keto PGF1? in supernatants of endothelial cells incubated with TNF? were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNF?-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  18. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Highlights: •EV-077 reduced TNF-? induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNF? incubation, whereas concentrations of 6-keto PGF1? in supernatants of endothelial cells incubated with TNF? were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNF?-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy

  19. Protection of outer hair cells from reperfusion injury by an iron chelator and a nitric oxide synthase inhibitor in the guinea pig cochlea.

    Science.gov (United States)

    Tabuchi, K; Okubo, H; Fujihira, K; Tsuji, S; Hara, A; Kusakari, J

    2001-07-01

    To examine whether an active process of the cochlea was injured by ischemia-reperfusion, time courses of distortion-product otoacoustic emissions (DPOAEs) were examined before, during and after 30 min cochlear ischemia using albino guinea pigs. DPOAEs decreased to the minimum level when the animals were subjected to ischemia. When the cochlea was recirculated, DPOAEs initially recovered with time until 20 min after the onset of reperfusion. However, thereafter the amplitude of DPOAEs gradually decreased toward the noise level. Administration of deferoxamine (an iron chelator) or N-nitro-L-arginine (a nitric oxide synthase inhibitor) ameliorated this decrease of DPOAEs during reperfusion and significantly increased the DPOAE amplitudes 60 min after the onset of reperfusion as compared with those in non-treated animals. These results suggest that cochlear reperfusion as well as ischemia injured the active process of the cochlea and that free radicals and nitric oxide play important roles in this injury. PMID:11516567

  20. Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

    International Nuclear Information System (INIS)

    The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

  1. Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening

    OpenAIRE

    Vasan, Mahalakshmi; Neres, Joa?o; Williams, Jessica; Wilson, Daniel J.; Teitelbaum, Aaron M.; Remmel, Rory P.; Aldrich, Courtney C.

    2010-01-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at t...

  2. BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: A promising neuroprotective strategy

    OpenAIRE

    Chabrier, Pierre-etienne; Auguet, Michel; Spinnewyn, Brigitte; Auvin, Serge; Cornet, Sylvie; Demerle?-pallardy, Caroline; Guilmard-favre, Christine; Marin, Jean-gre?goire; Pignol, Bernadette; Gillard-roubert, Ve?ronique; Roussillot-charnet, Christelle; Schulz, Jocelyne; Viossat, Isabelle; Bigg, Dennis; Moncada, Salvador

    1999-01-01

    Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-{4-...

  3. Branched-chain amino acid biosynthesis inhibitors: herbicide efficacy is associated with an induced carbon-nitrogen imbalance.

    Science.gov (United States)

    Zabalza, Ana; Zulet, Amaia; Gil-Monreal, Miriam; Igal, Maria; Royuela, Mercedes

    2013-06-15

    Acetolactate synthase (ALS; EC 4.1.3.18) and ketol-acid reductoisomerase (KARI; EC 1.1.1.86) are two consecutive enzymes in the biosynthesis of branched-chain amino acids. Several commercial herbicides inhibit ALS as their primary site of action. KARI has also attracted attention as a potential target for herbicides. Although potent and selective inhibitors of KARI have been discovered, these inhibitors display less herbicidal activity than ALS-inhibiting herbicides. To obtain a better understanding of these findings, we have compared the physiological effects induced in pea plants after KARI or ALS inhibition. Although, both types of inhibitors induce growth arrest and photosynthesis inhibition, plant death occurs more rapidly under ALS inhibition than KARI inhibition. Carbohydrates accumulated in the leaves and roots following treatments with both inhibitors. The carbohydrate accumulation in the leaves occurred as a consequence of a decrease in sink strength. In contrast, the free amino acid content was only affected through ALS inhibition. These results indicate that although KARI and ALS inhibition block the same biosynthetic pathway and exert common effects on carbon metabolism, nitrogen metabolism is more affected via ALS than KARI inhibition. Thus, metabolic alterations in nitrogen metabolism induced through ALS inhibitors might contribute to the increased efficacy of these chemicals as herbicides. PMID:23394788

  4. Inhibitors

    Science.gov (United States)

    ... this page: About CDC.gov . Hemophilia Share Compartir Inhibitors People with hemophilia have a higher quality of ... most serious and costly complications of hemophilia. About Inhibitors People with hemophilia use treatment products called factor ...

  5. Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

    OpenAIRE

    Weichsel, A; Montfort, W. R.; Cie?la, J; Maley, F

    1995-01-01

    A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP rela...

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

    Science.gov (United States)

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

    2002-01-01

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

  7. Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Ying; Hammoudeh, Dalia; Yun, Mi-Kyung; Qi, Jianjun; White, Stephen W.; Lee, Richard E. (Tennessee-HSC); (SJCH)

    2012-05-29

    Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

  8. Anti-diabetic efficacy of KICG1338, a novel glycogen synthase kinase-3? inhibitor, and its molecular characterization in animal models of type 2 diabetes and insulin resistance.

    Science.gov (United States)

    Kim, Kyoung Min; Lee, Kuy-Sook; Lee, Gha Young; Jin, Hyunjin; Durrance, Eunice Sung; Park, Ho Seon; Choi, Sung Hee; Park, Kyong Soo; Kim, Young-Bum; Jang, Hak Chul; Lim, Soo

    2015-07-01

    Selective inhibition of glycogen synthase kinase-3 (GSK3) has been targeted as a novel therapeutic strategy for diabetes mellitus. We investigated the anti-diabetic efficacy and molecular mechanisms of KICG1338 (2-(4-fluoro-phenyl)-3H-imidazo[4,5-b]pyridine-7-carboxylic acid(4-methyl-pyridin-3-yl)-amide), a GSK3? inhibitor, in three animal models: Otsuka Long-Evans Tokushima Fatty (OLETF) rats, leptin receptors-deficient db/db mice, and diet-induced obese (DIO) mice. Biochemical parameters including glucose tolerance tests and gene expressions associated with glucose metabolism were investigated. Glucose excursion decreased significantly by KICG1338-treated OLETF rats, accompanied by increase in insulin receptor substrate-1 and glucose transporter (GLUT)-4 expressions in muscle and decreased GLUT-2 expression in liver. Glucose-lowering effects were similarly observed in KICG1338-treated db/db and DIO mice. KICG1338 treatment increased adiponectin levels and decreased TNF-? levels. KICG1338 therapy also led to greater ?-cell preservation and less hepatic fat infiltration with decreased expressions of genes involved in inflammation and endoplasmic reticulum stress. These data demonstrate anti-diabetic efficacy of KICG1338, a novel GSK3? inhibitor. PMID:25802191

  9. The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

    Directory of Open Access Journals (Sweden)

    Weiss Ingrid M

    2007-11-01

    Full Text Available Abstract Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc. The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 ?M – 10 ?M dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

  10. Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice.

    Science.gov (United States)

    Nietupski, Jennifer B; Pacheco, Joshua J; Chuang, Wei-Lien; Maratea, Kimberly; Li, Lingyun; Foley, Joseph; Ashe, Karen M; Cooper, Christopher G F; Aerts, Johannes M F G; Copeland, Diane P; Scheule, Ronald K; Cheng, Seng H; Marshall, John

    2012-04-01

    Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction. PMID:22366055

  11. 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.

  12. Synthesis and Biological Evaluation of 3-Benzisoxazolyl-4-indolylmaleimides as Potent, Selective Inhibitors of Glycogen Synthase Kinase-3?

    OpenAIRE

    Jianrong Gao; Weisi Wang; Yujin Li; Liang Han; Lei Xu; Jiayi Cao; Tao Pang; Yubo Zhou; Meng Li; Qing Ye; Jia Li

    2013-01-01

    A series of novel 3-benzisoxazolyl-4-indolyl-maleimides were synthesized and evaluated for their GSK-3? inhibitory activity. Most compounds exhibited high inhibitory potency towards GSK-3?. Among them, compound 7j with an IC50 value of 0.73 nM was the most promising GSK-3? inhibitor. Preliminary structure-activity relationships were examined and showed that different substituents on the indole ring and N1-position of the indole ring had varying degrees of influence on the GSK-3? inhibitor...

  13. Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms.

    OpenAIRE

    Gollop, N.; Damri, B.; Chipman, D. M.; Barak, Z.

    1990-01-01

    Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as f...

  14. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    International Nuclear Information System (INIS)

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/?CT imaging. GSK-3 inhibitors caused ?-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/?CT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and mineralisation produced by GSK-3 inhibition. • In rats, 3 GSK-3 inhibitors produced a unique serum bone turnover biomarker profile. • Enhanced bone formation was seen within 7 to 14 days of compound treatment in rats

  15. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/?CT imaging. GSK-3 inhibitors caused ?-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/?CT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and mineralisation produced by GSK-3 inhibition. • In rats, 3 GSK-3 inhibitors produced a unique serum bone turnover biomarker profile. • Enhanced bone formation was seen within 7 to 14 days of compound treatment in rats.

  16. Experiments with nitric oxide synthase inhibitors in spinal nerve ligated rats provide no evidence of a role for nitric oxide in neuropathic mechanical allodynia.

    Science.gov (United States)

    Lee, Doo H; Singh, Jai Pal; Lodge, David

    2005-09-16

    We have investigated the effect of treatment with N(omega)-nitro-l-arginine methylester (l-NAME), a non-selective nitric oxide synthase inhibitor (NOS), both before and after the induction of mechanical allodynia by tight ligation of the left L5 and L6 spinal nerves in rats (SNL rats). The degree of mechanical allodynia was measured by tactile threshold for paw flinching with von Frey filaments. Intraperitoneal (i.p.) administration of l-NAME (3-30 mg/kg) 1 week after the spinal nerve ligation produced a dose-dependent reduction of the behavioral signs of mechanical allodynia, but the effect was not reversed by pretreatment with l-arginine (300 mg/kg). N(omega)-Nitro-l-arginine (l-NNA, i.p., 30 mg/kg), aminoguanidine (AG, i.p., 30 mg/kg) and a potent neuronal NOS inhibitor (LY457963, i.p., 30 mg/kg) did not reduce mechanical sensitivity in the SNL rats. Furthermore, using an ex vivo NOS activity assay, l-NAME partially inhibited the spinal NOS activity, whereas LY457963 almost completely inhibited the spinal NOS activity. Prior administration of l-NAME (i.p., 30 mg/kg) or of MK-801 (0.5 mg/kg), an NMDA antagonist, 30 min before the spinal nerve ligation significantly prevented the development of mechanical allodynia after spinal nerve ligation for an extended period of time. High doses of l-arginine (100 mg/kg or 300 mg/kg, i.p.), however, did not reverse the preemptive effect of l-NAME. These results suggest that neither the anti-allodynic nor the preemptive effects of l-NAME are mediated by NOS inhibition. PMID:15964141

  17. Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines.

    Science.gov (United States)

    van Triest, B; Pinedo, H M; van Hensbergen, Y; Smid, K; Telleman, F; Schoenmakers, P S; van der Wilt, C L; van Laar, J A; Noordhuis, P; Jansen, G; Peters, G J

    1999-03-01

    Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics. PMID:10100718

  18. Effect of the ATPase inhibitor protein IF1 on H+ translocation in the mitochondrial ATP synthase complex

    International Nuclear Information System (INIS)

    The H+ FoF1-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F1 moiety of the complex protrudes at the inner side of the membrane, the Fo sector spans the membrane reaching the outer side. The IF1 component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the FoF1-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF1 (residue 42-58) to the F1-?/? subunits. We have analyzed the effect of native purified IF1 the IF1-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K+] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted FoF1 complex. The results show that IF1, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F1 to the Fo side and in the opposite direction. Cross-linking of IF1 to F1-?/? subunits inhibits the ATP-driven H+ translocation but enhances H+ conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the FoF1 complex.

  19. Effects of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester on phencyclidine-induced effects in rats.

    Science.gov (United States)

    Bujas-Bobanovic, M; Robertson, H A; Dursun, S M

    2000-12-01

    Phencyclidine (PCP) is widely used as an animal model of schizophrenia. In rats, acute PCP treatment increased locomotor activity and induced stereotyped behaviours consisting of head weaving, turning and backpedalling. PCP had differential regional effects on c-fos expression in rat brain, suggesting different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. To elucidate the role of nitric oxide, an important intracellular messenger, in the mechanism of action of PCP the effects of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were studied in PCP-treated animals. L-NAME potentiated PCP-induced behaviours and c-fos expression in many brain regions. The greatest increases were observed in the frontal, retrosplenial granular cortex, cerebellum, thalamic and subthalamic nuclei. While PCP alone induced low c-fos expression in the entorhinal cortex, with almost no expression in the rostral part of caudate putamen, animals pretreated with L-NAME showed marked activation in these brain areas. These results strongly indicate the involvement of the nitric oxide system in the mechanism of action of PCP. PMID:11099700

  20. Chronic treatment with the nitric oxide synthase inhibitor, L-NAME, attenuates estradiol-mediated improvement of learning and memory in ovariectomized rats

    Scientific Electronic Library Online (English)

    Hamid, Azizi-Malekabadi; Mahmoud, Hosseini; Fatima, Saffarzadeh; Reza, Karami; Fatimeh, Khodabandehloo.

    Full Text Available INTRODUCTION: The role of ovarian hormones and nitric oxide in learning and memory has been widely investigated. OBJECTIVE: The present study was carried out to evaluate the effect of the nitric oxide synthase (NOS) inhibitor, N (G)-nitro-L-arginine methyl ester (L-NAME), on the ability of estradiol [...] to improve learning in OVX rats using the Morris water maze. METHODS: Forty rats were divided into five groups: (1) ovariectomized (OVX), (2) ovariectomized-estradiol (OVX-Est), (3) ovariectomized-L-NAME 10 (OVX-LN 10), (4) ovariectomized-L-NAME 50 (OVX-LN 50) and (5) ovariectomized-estradiol-L-NAME 50 (OVX-Est-LN 50). The animals in the OVX-Est group were treated with a weekly injection of estradiol valerate (2 mg/kg; i.m.). The OVX-LN 10 and OVX-LN 50 groups were treated with daily injections of 10 and 50 mg/kg L-NAME (i.p.), respectively. The animals in the OVX-Est-LN 50 group received a weekly injection of estradiol valerate and a daily injection of 50 mg/kg L-NAME. After 8 weeks, all animals were tested in the Morris water maze. RESULTS: The animals in the OVX-Est group had a significantly lower latency in the maze than the OVX group (p

  1. Effects of soluble epoxide hydrolase inhibitor on the expression of fatty acid synthase in peripheral blood mononuclear cell in patients with acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    Zhao Xuan

    2013-01-01

    Full Text Available Abstract Background Researches have shown that soluble epoxide hydrolase inhibitors (sEHi can protect against the development of atherosclerosis. Simultaneously, emerging evidences have implicated the association between fatty acid synthase (FAS and acute coronary syndrome (ACS. We tested the hypothesis that sEHi could reduce the occurrence of ACS by regulating FAS. Methods Hospitalized ACS patients were selected as the ACS group (n = 65 while healthy normal subjects as the control group (n = 65. The blood levels of lipoproteins, fasting glucose, myocardial enzyme and high-sensitivity C-reactive protein (hs-CRP were measured within 24 hours after admission. The peripheral blood mononuclear cells (PBMCs were isolated and cultured. Trans-4-[4-(3-Adamantan-1-ylureidocyclohexyloxy] benzoic acid (t-AUCB, a kind of sEHi, was then added to cells in various concentrations (0, 10, 50, 100 ?mol/L. The expression of FAS, interleukin-6 (IL-6 mRNA and protein was detected by real-time PCR or Western blot, respectively. Results (1 Compared with the control group, the serum concentration of hs-CRP in the ACS group was increased (PPPPP Conclusions sEH inhibition regulated FAS and inhibited inflammation in cultured PBMCs from ACS patients, a mechanism that might prevent rupture of atherosclerotic lesions and protect against development of ACS.

  2. The potentiation of radiation response in human colon carcinoma cells in vitro and murine lymphoma in vivo by AG337 (thymitaqTM), a novel thymidylate synthase inhibitor

    International Nuclear Information System (INIS)

    Purpose: To determine whether the administration of ThymitaqTM (AG337), a selective inhibitor of thymidylate synthase (TS), enhances radiation-induced cytotoxicity in vitro and increases tumor control rate in vivo. Methods and Materials: In vitro studies were carried out with HT-29 human colon carcinoma cells. In vivo studies were carried out using L5178Y(TK-) murine lymphoma implanted in DBA/2 mice. Results: Pretreatment of HT-29 cells to nontoxic concentration of AG337 (-) tumors, the drug alone (50 mg/kg, i.p. x 5) had a minimal tumor growth delay, while a single dose of radiation (17 Gy) resulted in 50), the radiation dose modification factor after a single dose of radiation was 2.6. Conclusion: The concentration of drug shown to be of radiosensitizing value in the in vivo studies is achievable in humans. The results of the present study further supports the potential utility of AG337 in the treatment of human tumors by radiotherapyumors by radiotherapy

  3. Chromosomal Integration and Expression of Two Bacterial ?-Acetolactate Decarboxylase Genes in Brewer's Yeast

    OpenAIRE

    Blomqvist, K.; Suihko, M. -l; Knowles, J.; Penttila?, M.

    1991-01-01

    A bacterial gene encoding ?-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the ?-acetolactate decarboxylase gene of the PGK1 integrant strains w...

  4. Lack of tolerance for the anti-dyskinetic effects of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in rats

    Scientific Electronic Library Online (English)

    N., Novaretti; F.E., Padovan-Neto; V., Tumas; C.A., da-Silva; E.A., Del Bel.

    1047-10-01

    Full Text Available 7-Nitroindazole (7-NI) inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could i [...] nterfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P

  5. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    International Nuclear Information System (INIS)

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS+ and cyclooxygenase-2+) and alternatively activated profibrotic (YM-1+ and galectin-3+) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ? Nitrogen mustard (NM) induces acute lung injury and fibrosis. ? Pulmonary toxicity is associated with increased expression of iNOS. ? Transient inhibition of iNOS attenuates acute lung injury induced by NM.

  6. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama; Venosa, Alessandro [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Hall, LeRoy [Drug Safety Sciences, Johnson and Johnson, Raritan, NJ 08869 (United States); Gow, Andrew J. [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Sinko, Patrick J. [Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ? Nitrogen mustard (NM) induces acute lung injury and fibrosis. ? Pulmonary toxicity is associated with increased expression of iNOS. ? Transient inhibition of iNOS attenuates acute lung injury induced by NM.

  7. Synthesis and Biological Evaluation of 3-Benzisoxazolyl-4-indolylmaleimides as Potent, Selective Inhibitors of Glycogen Synthase Kinase-3?

    Directory of Open Access Journals (Sweden)

    Jianrong Gao

    2013-05-01

    Full Text Available A series of novel 3-benzisoxazolyl-4-indolyl-maleimides were synthesized and evaluated for their GSK-3? inhibitory activity. Most compounds exhibited high inhibitory potency towards GSK-3?. Among them, compound 7j with an IC50 value of 0.73 nM was the most promising GSK-3? inhibitor. Preliminary structure-activity relationships were examined and showed that different substituents on the indole ring and N1-position of the indole ring had varying degrees of influence on the GSK-3? inhibitory potency. Compounds 7c, 7f, 7j–l and 7o–q could obviously reduce A?-induced Tau hyperphosphorylation by inhibiting GSK-3? in a cell-based functional assay.

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

    OpenAIRE

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

    1990-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Suborov Evgeny V

    2012-06-01

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

  10. Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

    OpenAIRE

    Edivaldo Domingues Velini; Maria Lúcia Bueno Trindade; Elza Alves; Ana Catarina Catâneo; Celso Luis Marino; Ivan Godoy Maia; Edson Seizo Mori; Edson Luiz Furtado; Iraê Amaral Guerrini; Carlos Frederico Wilcken

    2005-01-01

    Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glypho...

  11. Vascular hyporeactivity to angiotensin II induced by Escherichia coli endotoxin is reversed by N?-Nitro-L-Arginine, an inhibitor of nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    L. A. RODRIGUES

    2009-01-01

    Full Text Available

    Septic shock or sepsis is reported to be one of the major causes of death when followed by systemic infectious trauma in humans and other mammals. Its development leads to a large drop in blood pressure and a reduction in vascular responsiveness to physiological vasoconstrictors which, if not contained, can lead to death. It is proposed that this vascular response is due to the action of bacterial cell wall products released into the bloodstream by the vascular endothelium and is considered a normal response of the body`s defenses against infection. A reduction in vascular reactivity to epinephrine and norepinephrine is observed under these conditions. In the present study in rats, the aim was to assess whether those effects of hypotension and hyporeactivity are also related to another endogenous vasoconstrictor, angiotensin II (AII. We evaluated the variation in the power of this vasoconstrictor over the mean arterial pressure in anesthetized rats, before and after the establishment of hypotension by Escherichia coli endotoxin (Etx. Our results show that in this model of septic shock, there is a reduction in vascular reactivity to AII and this reduction can be reversed by the inhibitor of nitric oxide synthase, N?-Nitro-L-Arginine (N?NLA. Our results also suggest that other endogenous factors (not yet fully known are involved in the protection of rats against septic shock, in addition to the L-arginine NO pathway. Keywords: vascular hyporeactivity; NO; rat; angiotensin II; N?NLA Escherichia coli endotoxin.

  12. Role of L-NAME, a nitric oxide synthase inhibitor, in the improvement of morphine-induced amnesia induced by nicotine

    Directory of Open Access Journals (Sweden)

    Morteza Piri

    2011-01-01

    Full Text Available Introduction: Drugs of abuse such as nicotine and morphine used systemically by addicts produce their effects via the mesolimbic dopaminergic pathway. Furthermore, evidence indicates that some behavioral effects of nicotine and morphine are mediated by nitric oxide (NO. Based on these observations, the aim of the present study was to investigate the effects of intra-nucleus accumbens (NAc injection of a nitric oxide synthase (NOS inhibitor, L-NAME, on the nicotine’s effect on the morphine-induced amnesia. Methods: As a model of memory assessment, a step-through type passive avoidance task was used. All animals were bilaterally implanted with a chronic cannulae in the NAc shell and trained by using a 1 mA foot shock. Animals were tested 24 h after training to measure step-through latency. Results: Post-training injection of morphine impaired memory performance on the test day. Pre-test administration of the same doses of morphine reversed amnesia induced by post-training administration of morphine. Moreover, administration of nicotine before the test prevented morphine amnesia. Impairment of memory because of post-training injection of morphine was also prevented by pretest administration of L-NAME. Co-administration of an ineffective dose of nicotine with ineffective doses of L-NAME synergistically improved memory that was impaired by morphine. On the other hand, pre-test intra-NAc injection of L-NAME impaired passive avoidance memory by itself. Conclusion: Considering the effects of pre-test intra-NAc injection of L-NAME alone or in combination with ineffective dose of nicotine on morphine amnesia, it may be concluded that nitric oxide system of nucleus accumbens has an important role in the improvement of morphine-induced amnesia and morphine state-dependent memory caused by nicotine.

  13. ?-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions.

    Science.gov (United States)

    Zuljan, Federico A; Repizo, Guillermo D; Alarcon, Sergio H; Magni, Christian

    2014-10-01

    Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain. PMID:25100661

  14. Mutations in the small subunit of acetolactate synthase from Streptomyces cinnamonensis.

    Czech Academy of Sciences Publication Activity Database

    Kopecký, Jan; Pospíšil, Stanislav; Janata, Ji?í

    SissiHeraklion : Hellenic Society of Biological Sciences, 1999. s. 30. [International Symposium on the Biology of Actinomycetes /11./. 24.10.1999-28.10.1999, Sissi-Heraklion] Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology

  15. Diverse mechanisms of growth inhibition by luteolin, resveratrol, and quercetin in MIA PaCa-2 cells: a comparative glucose tracer study with the fatty acid synthase inhibitor C75

    OpenAIRE

    Harris, Diane M.; Li, Luyi; Chen, Monica; Lagunero, F. Tracy; Go, Vay Liang W.; Boros, Laszlo G.

    2011-01-01

    The rationale of this dose matching/dose escalating study was to compare a panel of flavonoids—luteolin, resveratrol, and quercetin—against the metabolite flux-controlling properties of a synthetic targeted fatty acid synthase inhibitor drug C75 on multiple macromolecule synthesis pathways in pancreatic tumor cells using [1,2-13C2]-d-glucose as the single precursor metabolic tracer. MIA PaCa-2 pancreatic adenocarcinoma cells were cultured for 48 h in the presence of 0.1% DMSO (control), o...

  16. Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

    OpenAIRE

    Goupil-feuillerat, N.; Cocaign-bousquet, M.; Godon, J. J.; Ehrlich, S. D.; Renault, P.

    1997-01-01

    The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid s...

  17. Variation in ?-acetolactate production within the hybrid lager yeast group Saccharomyces pastorianus and affirmation of the central role of the ILV6 gene.

    Science.gov (United States)

    Gibson, Brian; Krogerus, Kristoffer; Ekberg, Jukka; Monroux, Adrien; Mattinen, Laura; Rautio, Jari; Vidgren, Virve

    2015-01-01

    A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling ?-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. PMID:24965182

  18. Synthesis and in vivo distribution of no-carrier-added N(?)-nitro-L-arginine [11C]methyl ester, a nitric oxide synthase inhibitor

    International Nuclear Information System (INIS)

    N(?)-nitro-L-arginine methyl ester (L-NAME) was labelled with carbon-11 as a potential PET tracer for NO synthase. N(?)-t-butoxycarbonyl-N(?)-nitro-L-arginine was reacted with [11C]diazomethane. After deprotection with trifluoroacetic acid the formed [11C]L-NAME was purified using HPLC. Biodistribution studies in rats and PET studies in monkeys and dogs showed no correlation between radioactivity distribution and NO synthase localization in brain and heart. Substantial amounts of [11C]methanol were detected in dog plasma shortly after injection. These findings preclude the use of [11C]L-NAME as a PET tracer

  19. Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source.

    OpenAIRE

    Dailey, F. E.; Cronan, J. E.

    1986-01-01

    Escherichia coli K-12 has two acetohydroxy acid synthase (AHAS) isozymes (AHAS I and AHAS III). Both of these isozymes catalyze the synthesis of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are key intermediates of the isoleucine-valine biosynthetic pathway. Strains lacking either isozyme but not both activities have been previously shown to grow well in minimal media in the absence of isoleucine and valine on any of several commonly used carbon sources (e.g., glucose or su...

  20. Production of a monocot-specific monoclonal antibody against acetohydroxyacid synthase and its use in the purification and characterization of the enzyme.

    OpenAIRE

    Singh, B. K.; Lumanglas, A.; Wang, B. S.

    1991-01-01

    Acetohydroxyacid synthase [AHAS; acetolactate pyruvate-lyase (carboxylating), EC 4.1.3.18], the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine, is a known target for several different chemical classes of herbicides. Antibodies required for immunological characterization of the enzyme have not been generated by the conventional method of antibody production using purified protein. Monoclonal antibodies were raised against AHAS from corn by using as immunogen a synth...

  1. Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

    Science.gov (United States)

    Heyes, Logan C; Reichau, Sebastian; Cross, Penelope J; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4?M. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated. PMID:25245459

  2. Effect of an inhibitor of neuronal nitric oxide synthase 7-nitroindazole on cerebral hemodynamic response and brain excitability in urethane-anesthetized rats.

    Czech Academy of Sciences Publication Activity Database

    Broží?ková, Carole; Otáhal, Jakub

    2013-01-01

    Ro?. 62, Suppl.1 (2013), S57-S66. ISSN 0862-8408 R&D Projects: GA ?R(CZ) GAP303/10/0999; GA ?R(CZ) GPP304/11/P386; GA ?R(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : cerebral hemodynamic response * brain excitability * neuronal nitric oxide synthase * 7-nitroindazole * rat Subject RIV: FH - Neurology Impact factor: 1.487, year: 2013

  3. Patterns of resistance to ALS herbicides in inhibitors in Smallflower Umbrella Sedge (Cyperus difformis) and Ricefield Bulrush (Schoenoplectus mucronatus)

    OpenAIRE

    Ferrero, Aldo; Vidotto, Francesco; Busi, Roberto

    2006-01-01

    Biotypes of smallflower umbrella sedge and ricefield bulrush resistant to acetolactate synthase (ALS)-inhibiting herbicides have been reported in several rice areas of the world. Here, we present results of a study conducted on whole plants of seven smallflower umbrella sedge and four ricefield bulrush biotypes collected in Italian, Spanish, and Californian rice fields to evaluate cross-resistance to ALS herbicides in these important weeds of temperate rice. The following herbicides were test...

  4. ATP synthase.

    Science.gov (United States)

    Junge, Wolfgang; Nelson, Nathan

    2015-06-01

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

  5. 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.

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

    OpenAIRE

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

    1988-01-01

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

  7. Genome-wide transcriptional responses of Escherichia coli to glyphosate, a potent inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

    Science.gov (United States)

    Lu, Wei; Li, Liang; Chen, Ming; Zhou, Zhengfu; Zhang, Wei; Ping, Shuzhen; Yan, Yongliang; Wang, Jin; Lin, Min

    2013-03-01

    The shikimate pathway enzymes offer attractive targets for the development of antimetabolites. Glyphosate is an effective antimetabolite that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway, thereby resulting in a shortage of the chorismate-derived essential aromatic amino acids. However, little is known about the genome-wide transcriptional responses of bacteria to glyphosate shock. In the current study, a transcriptome analysis of Escherichia coli (E. coli) exposed to glyphosate identified the differential expression of 1040 genes, which represent 23.2% of the genome. The differentially expressed genes are primarily involved in amino acid metabolism, cell motility, and central carbon metabolism, indicating that the impact of glyphosate on the shikimate pathway also extends to other metabolic pathways. Expectedly, almost all genes encoding the proteins for the shikimate and specific aromatic amino acid pathways were downregulated after the addition of glyphosate. Furthermore, the expression of many energy- and metabolism-related genes was repressed. In contrast, glyphosate treatment induced the coordinated upregulation of at least 50 genes related to cell motility and chemotaxis. The reverse transcription-quantitative real-time PCR (RT-qPCR) data showed that the expression profiles of selected genes from the referred pathways were found to be consistent with the microarray data. The results suggest that the presence of glyphosate during growth induces metabolic starvation, an energy drain and other non-target effects. PMID:23247721

  8. 5-Imino-1,2-4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3? (GSK-3?) and phosphodiesterase 7 (PDE7) inhibitors: determination of blood-brain barrier penetration and binding to human serum albumin.

    Science.gov (United States)

    Pérez, Daniel I; Pistolozzi, Marco; Palomo, Valle; Redondo, Miriam; Fortugno, Cecilia; Gil, Carmen; Felix, Guy; Martinez, Ana; Bertucci, Carlo

    2012-04-11

    5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3? (GSK-3?) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood-brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood-brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug-protein system was studied by CD. The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA. PMID:22306656

  9. Effects of nitric oxide synthase inhibitors, L-NG-nitroarginine and L-NG-nitroarginine methyl ester, on responses to vasodilators of the guinea-pig coronary vasculature.

    OpenAIRE

    Vials, A.; Burnstock, G.

    1992-01-01

    1. The effects of L-NG-nitroarginine (L-NOARG) and L-NG-nitroarginine methyl ester (L-NAME) on vasodilatation induced by ATP, substance P, 5-hydroxytryptamine (5-HT), bradykinin and sodium nitroprusside (SNP) were examined in the guinea-pig coronary bed, by use of a Langendorff technique. The effects of these inhibitors of nitric oxide synthesis were assessed on their ability to inhibit both the amplitude and the area of the vasodilator response. 2. The vasodilator responses evoked by low dos...

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

    Science.gov (United States)

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

    2013-05-01

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

  11. The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

    OpenAIRE

    Nanaka Kikuchi; Shinjiro Ogita; Taiji Nomura; Yasuo Kato

    2012-01-01

    Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advant...

  12. Resistência de plantas aos herbicidas inibidores da acetolactato sintase Plant resistance to acetolactate synthase-inhibiting herbicides

    OpenAIRE

    Rizzardi, M. A.; Vidal, R. A.; Fleck, N. G.; Agostinetto, D.

    2002-01-01

    A resistência de plantas aos herbicidas é conseqüência, na maioria das vezes, de mutação ou da preexistência de genes que conferem resistência à população. No caso dos herbicidas inibidores da acetolactato sintase (ALS) ocorreram casos de resistência tanto em plantas daninhas quanto em culturas. Essa revisão foi realizada com o objetivo de discutir aspectos bioquímicos, genéticos e moleculares da resistência de plantas aos herbicidas inibidores da ALS, sendo destacados também...

  13. Insensitivity of acetolactate synthase to end-product inhibition in the mutant strains of a monensis producer Streptomyces cinnamonensis.

    Czech Academy of Sciences Publication Activity Database

    Kopecký, Jan; Janata, Ji?í

    Prague, 1999 - (Jen?, P.). s. 64 [Czech-Swiss Symposium on Advanced Biotechnology /1./. 04.09.1999-07.09.1999, Prague] Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EI - Biotechnology ; Bionics

  14. Hidropsia endolinfática experimental sob ação de inibidor da óxido nítrico sintase tipo II: avaliação com emissões otoacústicas e eletrococleografia Experimental endolymphatic hydrops under action of a type II nitric oxide synthase inhibitor: otoacoustic emissions evaluation and electrocochleography

    Directory of Open Access Journals (Sweden)

    Claudio Marcio Yudi Ikino

    2006-04-01

    Full Text Available No modelo experimental de hidropsia endolinfática há redução na amplitude das emissões otoacústicas produtos de distorção (EOAPD e elevação nos limiares eletrofisiológicos na eletrococleografia. Estudos mostraram que há expressão da óxido nítrico sintase tipo II (ONS II na cóclea com hidropsia, sugerindo a participação do óxido nítrico (ON na patogênese desta doença. O objetivo deste trabalho foi avaliar a ação de um inibidor da ONS II nas EOAPD e eletrococleografia em cobaias com hidropisia endolinfática experimental. MATERIAL E MÉTODOS: Foram estudadas 16 cobaias nas quais se induziu hidropsia endolinfática experimental por obliteração do ducto e saco endolinfático na orelha direita durante 16 semanas, divididas em dois grupos: oito cobaias recebendo um inibidor da ONS II, a aminoguanidina, por via oral e um grupo de oito cobaias como controle. Comparamos as amplitudes das EOAPD nas médias geométricas de freqüências de 1062, 2187, 4375 e 7000Hz, os limiares eletrofisiológicos nas freqüências de 1000, 2000, 4000 e 6000Hz e a relação entre os potenciais de somação e de ação (PS/PA entre os grupos. RESULTADOS: Não houve diferença significante nas EOAPD e na relação PS/PA entre os grupos. O grupo que recebeu a aminoguanidina apresentou menor elevação nos limiares eletrofisiológicos nas freqüências de 2000 (pIn experimental endolymphatic hydrops distortion-products otoacoustic emission (dpoae amplitudes decrease and there is elevation on electrocochleographic thresholds. Some authors found type ii nitric oxide synthase (nos ii expression in hydropic cochleas and they suggest nitric oxide (no may be involved in endolymphatic hydrops pathogenesis. The aim of this study was to evaluate the action of a nos ii inhibitor on dpoae and electrocochleography in experimental endolymphatic hydrops. MATERIAL E METHODS: endolymphatic hydrops was induced in 16 guinea pigs by obliterating the endolymphatic duct and sac in the right ear. They were divided in two groups: eigth guinea pigs under the action of aminoguanidine, a nos ii inhibitor and eigth control guinea pigs. We compared dpoae amplitudes at geometric means of frequencies 1062, 2187, 4375 and 7000 hz, compound action potential threshold at 1000, 2000, 4000 and 6000 hz and summating potential to action potential (sp/ap ratio between the groups during the postoperative observation period of 16 weeks. RESULTS: there were no significant changes in the dpoae amplitudes and in the sp/ap ratio. The group that received aminoguanidine had a lower degree of threshold increase at 2000 (p<0.05 And 6000 hz (p<0.05 In 12th postoperative week and at 1000 (p<0.05, 2000 (P<0.001, 4000 (P<0.001 And 6000 hz (p<0.001 At 16th postoperative week. CONCLUSIONS: nos ii inhibitor decreased the electrocochleography threshold elevation on experimental endolymphatic hydrops.

  15. An Indomethacin Analogue, N-(4-Chlorobenzoyl)-melatonin, is a Selective Inhibitor of Aldo-keto Reductase 1C3 (Type 2 3?-HSD, Type 5 17?-HSD, and Prostaglandin F Synthase), a Potential Target for the Treatment of Hormone Dependent and Hormone Independent Malignancies

    OpenAIRE

    Byrns, Michael C.; Steckelbroeck, Stephan; Penning, Trevor M.

    2007-01-01

    Aldo-keto reductase (AKR) 1C3 (type 2 3?-HSD, type 5 17?-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested...

  16. Nitric Oxide Synthase Inhibitors as Antidepressants

    DEFF Research Database (Denmark)

    Wegener, Gregers; Volke, Vallo

    2010-01-01

    Affective and anxiety disorders are widely distributed disorders with severe social and economic effects. Evidence is emphatic that effective treatment helps to restore function and quality of life. Due to the action of most modern antidepressant drugs, serotonergic mechanisms have traditionally been suggested to play major roles in the pathophysiology of mood and stress-related disorders. However, a few clinical and several pre-clinical studies, strongly suggest involvement of the nitric oxide (NO) signaling pathway in these disorders. Moreover, several of the conventional neurotransmitters, including serotonin, glutamate and GABA, are intimately regulated by NO, and distinct classes of antidepressants have been found to modulate the hippocampal NO level in vivo. The NO system is therefore a potential target for antidepressant and anxiolytic drug action in acute therapy as well as in prophylaxis. This paper reviews the effect of drugs modulating NO synthesis in anxiety and depression.

  17. Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function

    OpenAIRE

    Cheng, Yan; Wang, Miao; Yu, Ying; Lawson, John; Funk, Colin D.; Fitzgerald, Garret A.

    2006-01-01

    We investigated the mechanisms by which inhibitors of prostaglandin G/H synthase-2 (PGHS-2; known colloquially as COX-2) increase the incidence of myocardial infarction and stroke. These inhibitors are believed to exert both their beneficial and their adverse effects by suppression of PGHS-2–derived prostacyclin (PGI2) and PGE2. Therefore, the challenge remains to identify a mechanism whereby PGI2 and PGE2 expression can be suppressed while avoiding adverse cardiovascular events. Here, sele...

  18. Síntese e modificações de derivados heterocíclicos de D-arabinose: potenciais inibidores de glicose-6-fosfato isomerase e de glicosamina-6-fosfato sintase / Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

    Scientific Electronic Library Online (English)

    Renato Márcio Ribeiro, Viana; Maria Auxiliadora Fontes, Prado; Ricardo José, Alves.

    1710-17-01

    Full Text Available [...] Abstract in english The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(D-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from D-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the [...] opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethylphosphoryl chloride. The resulting 5-[D-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase.

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

    Science.gov (United States)

    Baker, S M; White, E E

    1996-05-01

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

  20. 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), a glycogen synthase kinase-3 inhibitor, displays therapeutic properties in a mouse model of pulmonary inflammation and fibrosis.

    Science.gov (United States)

    Gurrieri, Carmela; Piazza, Francesco; Gnoato, Marianna; Montini, Barbara; Biasutto, Lucia; Gattazzo, Cristina; Brunetta, Enrico; Cabrelle, Anna; Cinetto, Francesco; Niero, Raffaele; Facco, Monica; Garbisa, Spiridione; Calabrese, Fiorella; Semenzato, Gianpietro; Agostini, Carlo

    2010-03-01

    Glycogen synthase kinase (GSK)-3 modulates the production of inflammatory cytokines. Because bleomycin (BLM) causes lung injury, which is characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blocking GSK-3 activity with a specific inhibitor could affect the inflammatory and profibrotic cytokine network generated in the BLM-induced process of pulmonary inflammation and fibrosis. Thus, here we investigated the effects of the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) on a BLM-induced lung fibrosis model in mice. SB216763 prevented lung inflammation and the subsequent fibrosis when coadministered with BLM. Bronchoalveolar lavage fluid analysis of mice treated with BLM plus SB216763 revealed a significant reduction in BLM-induced alveolitis. Furthermore, SB216763 treatment was associated with a significantly lower production of inflammatory cytokines by macrophages. BLM-treated mice that received SB216763 developed alveolar epithelial cell damage and pulmonary fibrosis to a significantly lower extent compared with BLM-treated controls. These findings suggest that GSK-3 inhibition has a protective effect on lung fibrosis induced by BLM and candidate GSK-3 as a potential therapeutic target for preventing pulmonary fibrosis. PMID:19959748

  1. Tapentadol and nitric oxide synthase systems.

    Science.gov (United States)

    Bujalska-Zadro?ny, Magdalena; Woli?ska, Renata; G?si?ska, Emilia; Nagraba, ?ukasz

    2015-04-01

    Tapentadol, a new analgesic drug with a dual mechanism of action (?-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10?mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10?mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2?mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia. PMID:25485639

  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. Improving the acidic stability of Staphylococcus aureus ?-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues.

    Science.gov (United States)

    Zhang, Xian; Rao, Zhiming; Li, Jingjing; Zhou, Junping; Yang, Taowei; Xu, Meijuan; Bao, Teng; Zhao, Xiaojing

    2015-04-01

    The ?-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus ?-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K m and V max were 17.7 ?M and 2.06 mM min(-1), respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL(-1) of SaALDC activity, about 320 times higher comparing to 0.42 U mL(-1) of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host. PMID:25543264

  4. Dietary bioflavonoids inhibit Escherichia coli ATP synthase in a differential manner

    OpenAIRE

    Chinnam, Nagababu; Dadi, Prasanna K.; Sabri, Shahbaaz A.; Ahmad, Mubeen; Kabir, M. Anaul; Ahmad, Zulfiqar

    2010-01-01

    The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F1 or membrane bound F1FO E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were mori...

  5. 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.

  6. Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition.

    Czech Academy of Sciences Publication Activity Database

    Kopecký, Jan; Janata, Ji?í; Pospíšil, Stanislav; Felsberg, Jürgen; Spížek, Jaroslav

    1999-01-01

    Ro?. 266, - (1999), s. 162-166. ISSN 0006-291X R&D Projects: GA AV ?R KSK2020602 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology Impact factor: 3.161, year: 1999

  7. Structures of human constitutive nitric oxide synthases.

    Science.gov (United States)

    Li, Huiying; Jamal, Joumana; Plaza, Carla; Pineda, Stephanie Hai; Chreifi, Georges; Jing, Qing; Cinelli, Maris A; Silverman, Richard B; Poulos, Thomas L

    2014-10-01

    Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03?Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73?Å resolution. PMID:25286850

  8. Cytological and comparative proteomic analyses on male sterility in Brassica napus L. induced by the chemical hybridization agent monosulphuron ester sodium

    Science.gov (United States)

    Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility i...

  9. NOVEL HERBICIDES

    Science.gov (United States)

    Low-dose, high-potency herbicides are defined as those herbicides with a maximum label application rate of 0.5 pounds of active ingredient per acre. Several classes of chemicals fall into this category, including the acetolactate synthase (ALSase) inhibitor herbicides, imidazoli...

  10. Análise de crescimento de biótipos de amendoim-bravo (Euphorbia heterophylla) resistente e suscetível aos herbicidas inibidores da ALS / Growth analysis of wild poinsettia (Euphorbia heterophylla) biotypes resistant and susceptible to ALS inhibitor herbicides

    Scientific Electronic Library Online (English)

    A.M., Brighenti; D.L.P., Gazziero; E., Voll; F.S., Adegas; W.M.C., Val.

    2001-04-01

    Full Text Available A aplicação contínua de herbicidas do grupo químico das imidazolinonas, nas mesmas áreas de produção de soja, durante anos seguidos, no município de Cafelândia, PR, favoreceu a seleção de um biótipo resistente de amendoim-bravo (Euphorbia heterophylla) aos herbicidas inibidores da acetolactato sinta [...] se (ALS). Um estudo comparativo das características do crescimento do biótipo resistente e do suscetível foi realizado em casa de vegetação da Embrapa Soja, Londrina-PR, a fim de identificar diferenças no crescimento e no desenvolvimento das plantas e de seus órgãos. A produção de matéria seca total, a área foliar, a matéria seca dos caule, das raízes e das folhas, bem como a altura por planta, foram avaliadas em 13 vezes a intervalos regulares, iniciando aos 14 dias após a semeadura. A partir desses parâmetros, foram calculadas a taxa de crescimento relativo, a taxa assimilatória líquida, a razão de área foliar, a razão de peso foliar e a área foliar específica, que decrescem com a ontogenia das plantas de amendoim-bravo, sendo similares para ambos os biótipos. A matéria seca total acumulada pelas plantas e seus órgãos, a área foliar e a altura apresentaram comportamentos semelhantes para os biótipos resistente e suscetível. O ciclo vegetativo dos dois biótipos estudados não mostrou diferença significativa quanto ao crescimento e ao desenvolvimento. Abstract in english Repetitive spraying of imidazolinone herbicides year after year to control weeds in the soybean grown areas of Cafelândia, Paraná, Brazil, has favored the selection of an ALS (acetolactate synthase) inhibitor herbicide resistant biotype of wild poinsettia (Euphorbia heterophylla). A comparative stud [...] y of growth and development of wild poinsettia resistant and susceptible to ALS inhibitor herbicides was carried out in the greenhouse of the experimental station of Soybean Embrapa in Londrina, Paraná, Brazil. Total dry biomass yield, leaf area, shoot dry weight, leaf dry weight, root dry weight and height per plant were measured 13 times at 2 week intervals, starting 14 days after sowing. Relative growth rate, net assimilation rate, leaf area ratio, leaf weight ratio and specific leaf area decreased with plant ontogeny and behaved similarly in both biotypes. The total dry matter of the plants and their organs as well as the leaf area and plant height exhibited similar ranges of variability in both biotypes. There were no significant differences between biotypes both for growth and development characteristics.

  11. Análise de crescimento de biótipos de amendoim-bravo (Euphorbia heterophylla resistente e suscetível aos herbicidas inibidores da ALS Growth analysis of wild poinsettia (Euphorbia heterophylla biotypes resistant and susceptible to ALS inhibitor herbicides

    Directory of Open Access Journals (Sweden)

    A.M. Brighenti

    2001-04-01

    Full Text Available A aplicação contínua de herbicidas do grupo químico das imidazolinonas, nas mesmas áreas de produção de soja, durante anos seguidos, no município de Cafelândia, PR, favoreceu a seleção de um biótipo resistente de amendoim-bravo (Euphorbia heterophylla aos herbicidas inibidores da acetolactato sintase (ALS. Um estudo comparativo das características do crescimento do biótipo resistente e do suscetível foi realizado em casa de vegetação da Embrapa Soja, Londrina-PR, a fim de identificar diferenças no crescimento e no desenvolvimento das plantas e de seus órgãos. A produção de matéria seca total, a área foliar, a matéria seca dos caule, das raízes e das folhas, bem como a altura por planta, foram avaliadas em 13 vezes a intervalos regulares, iniciando aos 14 dias após a semeadura. A partir desses parâmetros, foram calculadas a taxa de crescimento relativo, a taxa assimilatória líquida, a razão de área foliar, a razão de peso foliar e a área foliar específica, que decrescem com a ontogenia das plantas de amendoim-bravo, sendo similares para ambos os biótipos. A matéria seca total acumulada pelas plantas e seus órgãos, a área foliar e a altura apresentaram comportamentos semelhantes para os biótipos resistente e suscetível. O ciclo vegetativo dos dois biótipos estudados não mostrou diferença significativa quanto ao crescimento e ao desenvolvimento.Repetitive spraying of imidazolinone herbicides year after year to control weeds in the soybean grown areas of Cafelândia, Paraná, Brazil, has favored the selection of an ALS (acetolactate synthase inhibitor herbicide resistant biotype of wild poinsettia (Euphorbia heterophylla. A comparative study of growth and development of wild poinsettia resistant and susceptible to ALS inhibitor herbicides was carried out in the greenhouse of the experimental station of Soybean Embrapa in Londrina, Paraná, Brazil. Total dry biomass yield, leaf area, shoot dry weight, leaf dry weight, root dry weight and height per plant were measured 13 times at 2 week intervals, starting 14 days after sowing. Relative growth rate, net assimilation rate, leaf area ratio, leaf weight ratio and specific leaf area decreased with plant ontogeny and behaved similarly in both biotypes. The total dry matter of the plants and their organs as well as the leaf area and plant height exhibited similar ranges of variability in both biotypes. There were no significant differences between biotypes both for growth and development characteristics.

  12. The ion channel of F-ATP synthase is the target of toxic organotin compounds.

    Science.gov (United States)

    von Ballmoos, Christoph; Brunner, Josef; Dimroth, Peter

    2004-08-01

    ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na(+)-translocating ATP synthase of Ilyobacter tartaricus or the H(+)-translocating counterpart of Escherichia coli with apparent Ki of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na+ ATP synthase, labeling is also suppressed in the presence of Na+ ions, suggesting an interference in Na+ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na+. TBT-Cl strongly inhibits Na+ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously. PMID:15277681

  13. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

    OpenAIRE

    Dozier, Jonathan K.; Distefano, Mark D.

    2011-01-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays use either radiolabeled substrates and are discontinuous, or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we ...

  14. Two distinct proton binding sites in the ATP synthase family.

    Science.gov (United States)

    von Ballmoos, Christoph; Dimroth, Peter

    2007-10-23

    The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes. PMID:17910472

  15. Proteasome inhibitors.

    Science.gov (United States)

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

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

    OpenAIRE

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

    1998-01-01

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

  17. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... synthase deficiency tend to be resistant (refractory) to treatment with antiseizure medications. GM3 synthase deficiency profoundly disrupts brain development. Most affected children have severe intellectual disability and do not develop skills such as reaching ...

  18. Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

    Science.gov (United States)

    Wittig, Ilka; Meyer, Bjoern; Heide, Heinrich; Steger, Mirco; Bleier, Lea; Wumaier, Zibiernisha; Karas, Michael; Schägger, Hermann

    2010-01-01

    Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function. PMID:20188060

  19. Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).

    Science.gov (United States)

    Manos-Turvey, Alexandra; Bulloch, Esther M M; Rutledge, Peter J; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2010-07-01

    Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate. PMID:20512795

  20. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    OpenAIRE

    Hargovan, Milan; Ferro, Albert

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibit...

  1. The ion channel of F-ATP synthase is the target of toxic organotin compounds

    OpenAIRE

    von Ballmoos, Christoph; Brunner, Josef; Dimroth, Peter

    2004-01-01

    ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na+-translocating ATP synthase of Ilyobacter tartaricus or the H+-translocating counterpart of Escherichia coli with apparent Ki of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl i...

  2. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs. PMID:17109150

  3. 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.

  4. Characterization of putative capsaicin synthase promoter activity.

    Science.gov (United States)

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

    2009-10-31

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

  5. 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.

  6. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    Science.gov (United States)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  7. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    Directory of Open Access Journals (Sweden)

    YoulaSTsantrizos

    2014-07-01

    Full Text Available The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP and geranylgeranyl pyrophosphate (GGPP are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  8. 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

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

    Science.gov (United States)

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

    2015-07-01

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

  10. Angiogenesis Inhibitors

    Science.gov (United States)

    ... Inhibitors: Breast cancer Colorectal cancer Esophageal cancer Gastrointestinal stromal tumor (GIST) Kidney (renal cell) cancer Liver (adult primary) cancer Lymphoma Melanoma Non-small cell lung cancer (NSCLC) Ovarian epithelial cancer Pancreatic cancer Prostate cancer Stomach (gastric) ...

  11. Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly.

    OpenAIRE

    Persson, K.; Schneider, G.; Jordan, D. B.; Viitanen, P. V.; Sandalova, T.

    1999-01-01

    Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, ...

  12. Análise comparativa do crescimento de biótipos de picão-preto (Bidens pilosa resistente e suscetível aos herbicidas inibidores da ALS Growth analysis of Bidens pilosa biotypes resistant and susceptible to ALS inhibitor herbicides

    Directory of Open Access Journals (Sweden)

    P.J. Christoffoleti

    2001-04-01

    Full Text Available A resistência de biótipos de plantas daninhas aos herbicidas inibidores da acetolactato sintase (ALS é causada pela insensibilidade desta enzima aos herbicidas que inibem sua atividade catalítica. A insensibilidade da enzima é decorrente de uma alteração estrutural, resultado da substituição de certos aminoácidos no sítio de ação do herbicida. Esta alteração na enzima pode eventualmente resultar, além da resistência ao herbicida, em modificações na taxa de crescimento da planta, fato este comprovado para os biótipos resistentes aos herbicidas inibidores do fotossistema II, os quais apresentam taxa de crescimento prejudicada pela alteração no sítio de ação sofrida pelo herbicida. Esta possível diminuição na taxa de crescimento da planta resistente tem conseqüências diretas na competitividade do biótipo e, portanto, na sua dinâmica dentro da população, afetando diretamente as estratégias de manejo da resistência. A presente pesquisa foi desenvolvida com o objetivo de comparar a taxa de crescimento de dois biótipos da planta daninha picão-preto (Bidens pilosa, sendo um resistente e um suscetível aos herbicidas inibidores da ALS. Um experimento foi montado em casa de vegetação, em vasos com capacidade de 5 L, sendo uma planta de cada biótipo por vaso, coletando-se a biomassa seca destas plantas e a área foliar semanalmente, iniciando-se 14 dias após o plantio. Os resultados de crescimento da biomassa e área foliar foram ajustados utilizando-se a função de Richards (log-logística. Desta análise, foram derivadas a taxa de crescimento absoluto (TCA, a taxa de crescimento relativo (TCR e a taxa de assimilação fotossintética líquida (TAL. O biótipo suscetível apresentou peso de biomassa seca superior ao resistente nas primeiras fases do crescimento, porém no final do ciclo o biótipo resistente igualou-se em tamanho de área foliar, pois apresentou, principalmente no início do ciclo de crescimento, TCA, TCR e TAL maiores que o suscetível. Dessa forma, concluiu-se que o biótipo de Bidens pilosa resistente aos herbicidas inibidores da ALS apresenta a mesma eficiência de produção de biomassa no final do ciclo. É provável que, quando em competição entre si e com as culturas, possua a mesma competitividade, sendo a dominância numérica de um biótipo sobre o outro decorrente apenas da pressão de seleção causada pelo herbicida.The resistance of weed biotypes to acetolactate synthase (ALS inhibitor herbicides is due to this enzyme's lack of sensitivity to ALS inhibitor herbicides, which inhibit its catalytic activity. ALS insensitivity results from a structural change in the aminoacid sequence, exactly in the site of action of these herbicides. Eventually this modification in the enzyme may result in a reduced plant growth rate. Such reduction was also observed in biotypes resistant to Photosystem II inhibitor herbicides. The possibility of a lower growth rate of the resistant plant may directly affect biotype competitiveness, its population dynamics and, as a consequence, resistance management strategies. The objective of this research was to compare the growth rates of both resistant and susceptible Bidens pilosa biotypes to ALS inhibitor herbicides. The experiment was conducted in a greenhouse, using one plant per pot of 5 L capacity. Four plants per biotype were harvested weekly, starting 14 days after planting, and the leaf area and dry biomass were measured. The Richards function fitted to the data enabled the derivation of absolute growth rate, relative growth rate and net assimilation rate. The susceptible biotype had a higher biomass accumulation during the early stages, with both biotypes having the same size, afterwards. The higher net assimilation rate of the resistant biotype during the early stages of growth was balanced by its lower size during the first four weeks of growth. It was concluded that both biotypes have the same size, being very likely that resistant and susceptible Bidens pilosa have the same competitiveness.

  13. Análise comparativa do crescimento de biótipos de picão-preto (Bidens pilosa) resistente e suscetível aos herbicidas inibidores da ALS / Growth analysis of Bidens pilosa biotypes resistant and susceptible to ALS inhibitor herbicides

    Scientific Electronic Library Online (English)

    P.J., Christoffoleti.

    2001-04-01

    Full Text Available A resistência de biótipos de plantas daninhas aos herbicidas inibidores da acetolactato sintase (ALS) é causada pela insensibilidade desta enzima aos herbicidas que inibem sua atividade catalítica. A insensibilidade da enzima é decorrente de uma alteração estrutural, resultado da substituição de cer [...] tos aminoácidos no sítio de ação do herbicida. Esta alteração na enzima pode eventualmente resultar, além da resistência ao herbicida, em modificações na taxa de crescimento da planta, fato este comprovado para os biótipos resistentes aos herbicidas inibidores do fotossistema II, os quais apresentam taxa de crescimento prejudicada pela alteração no sítio de ação sofrida pelo herbicida. Esta possível diminuição na taxa de crescimento da planta resistente tem conseqüências diretas na competitividade do biótipo e, portanto, na sua dinâmica dentro da população, afetando diretamente as estratégias de manejo da resistência. A presente pesquisa foi desenvolvida com o objetivo de comparar a taxa de crescimento de dois biótipos da planta daninha picão-preto (Bidens pilosa), sendo um resistente e um suscetível aos herbicidas inibidores da ALS. Um experimento foi montado em casa de vegetação, em vasos com capacidade de 5 L, sendo uma planta de cada biótipo por vaso, coletando-se a biomassa seca destas plantas e a área foliar semanalmente, iniciando-se 14 dias após o plantio. Os resultados de crescimento da biomassa e área foliar foram ajustados utilizando-se a função de Richards (log-logística). Desta análise, foram derivadas a taxa de crescimento absoluto (TCA), a taxa de crescimento relativo (TCR) e a taxa de assimilação fotossintética líquida (TAL). O biótipo suscetível apresentou peso de biomassa seca superior ao resistente nas primeiras fases do crescimento, porém no final do ciclo o biótipo resistente igualou-se em tamanho de área foliar, pois apresentou, principalmente no início do ciclo de crescimento, TCA, TCR e TAL maiores que o suscetível. Dessa forma, concluiu-se que o biótipo de Bidens pilosa resistente aos herbicidas inibidores da ALS apresenta a mesma eficiência de produção de biomassa no final do ciclo. É provável que, quando em competição entre si e com as culturas, possua a mesma competitividade, sendo a dominância numérica de um biótipo sobre o outro decorrente apenas da pressão de seleção causada pelo herbicida. Abstract in english The resistance of weed biotypes to acetolactate synthase (ALS) inhibitor herbicides is due to this enzyme's lack of sensitivity to ALS inhibitor herbicides, which inhibit its catalytic activity. ALS insensitivity results from a structural change in the aminoacid sequence, exactly in the site of acti [...] on of these herbicides. Eventually this modification in the enzyme may result in a reduced plant growth rate. Such reduction was also observed in biotypes resistant to Photosystem II inhibitor herbicides. The possibility of a lower growth rate of the resistant plant may directly affect biotype competitiveness, its population dynamics and, as a consequence, resistance management strategies. The objective of this research was to compare the growth rates of both resistant and susceptible Bidens pilosa biotypes to ALS inhibitor herbicides. The experiment was conducted in a greenhouse, using one plant per pot of 5 L capacity. Four plants per biotype were harvested weekly, starting 14 days after planting, and the leaf area and dry biomass were measured. The Richards function fitted to the data enabled the derivation of absolute growth rate, relative growth rate and net assimilation rate. The susceptible biotype had a higher biomass accumulation during the early stages, with both biotypes having the same size, afterwards. The higher net assimilation rate of the resistant biotype during the early stages of growth was balanced by its lower size during the first four weeks of growth. It was concluded that both biotypes have the same size, being very likely that resistant and susceptible Bidens pilosa

  14. Sucrose Synthase: Expanding Protein Function

    OpenAIRE

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

    2007-01-01

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

  15. 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

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

    Science.gov (United States)

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

    2003-09-15

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

  17. Regulation of neuronal nitric oxide synthase by histone, protamine, and myelin basic protein.

    Science.gov (United States)

    Hu, J; Fridlund, J; el-Fakahany, E E

    1995-04-01

    We examined the effects of endogenous basic proteins rich in the amino acid L-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively. PMID:7544448

  18. A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer-aided Drug Discovery.

    Science.gov (United States)

    Kim, Meekyum Olivia; Feng, Xinxin; Feixas, Ferran; Zhu, Wei; Lindert, Steffen; Bogue, Shannon; Sinko, William; de Oliveira, César; Rao, Guodong; Oldfield, Eric; McCammon, James Andrew

    2015-06-01

    With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis-farnesyl diphosphate synthase (cis-FPPS) and cis-decaprenyl diphosphate synthase (cis-DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis-FPPS, cis-DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis-DPPS, finding ~1 ?m inhibitors, and used the receiver operating characteristic-area under the curve (ROC-AUC) method to test the predictive power of X-ray and MD-derived cis-DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC50 ~0.5- to 20-?m inhibitor (depending on substrate) of cis-DPPS, a ~660-nm inhibitor of Rv3378c as well as a 4.8-?m inhibitor of cis-FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation. PMID:25352216

  19. Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep

    OpenAIRE

    Qu, Wei-min; Huang, Zhi-li; Xu, Xin-hong; Aritake, Kosuke; Eguchi, Naomi; Nambu, Fumio; Narumiya, Shu; Urade, Yoshihiro; Hayaishi, Osamu

    2006-01-01

    Prostaglandin (PG) D2 has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl4), a selective inhibitor of PGD synthase (PGDS), was shown to reduce promptly and effectively the amounts of sleep during the period of infusion. However, gene knockout (KO) mice of PGDS and prostaglandin D receptor (DP1R) showed essentially the same circadian profiles and daily amounts of s...

  20. Glycogen synthase kinase 3? induces apoptosis in cancer cells through increase of survivin nuclear localization

    OpenAIRE

    Li, Jiansha; Xing, Mingyou; Zhu, Min; Wang, Xi; Wang, Manxiang; Zhou, Sheng; Li, Naping; Wu, Renliang; Zhou, Muxiang

    2008-01-01

    Glycogen synthase kinase 3? (GSK3?) regulates numerous signaling pathways that control a wide range of cellular processes, including cell proliferation, differentiation, apoptosis and metabolism. We report a novel function of GSK3?: It interacts with the inhibitor-of-apoptosis protein (IAP) survivin to modulate its expression, thus regulating apoptosis in human lung cancer cells. A co-immunoprecipitation assay revealed that GSK3? can bind survivin. Activation of GSK3? induced translocati...

  1. Corrosion inhibitors

    International Nuclear Information System (INIS)

    In this paper, we briefly describe the characteristics, cost and electrochemical nature of the corrosion phenomena as well as some of the technologies that are currently employed to minimize its effect. The main subject of the paper however, deals with the description, classification and mechanism of protection of the so-called corrosion inhibitors. Examples of the use of these substances in different aggressive environments are also presented as means to show that these compounds, or their combination, can in fact be used as excellent and relatively cheap technologies to control the corrosion of some metals. In the last part of the paper, the most commonly used techniques to evaluate the efficiency and performance of corrosion inhibitors are presented as well as some criteria to make a careful and proper selection of a corrosion inhibitor technology in a given situation. (Author) 151 refs

  2. Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

    Scientific Electronic Library Online (English)

    Edivaldo Domingues, Velini; Maria Lúcia Bueno, Trindade; Elza, Alves; Ana Catarina, Catâneo; Celso Luis, Marino; Ivan de Godoy, Maia; Edson Seizo, Mori; Edson Luiz, Furtado; Iraê Amaral, Guerrini; Carlos Frederico, Wilcken.

    Full Text Available Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of w [...] eeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (https://forests.esalq.usp.br) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings.

  3. Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

    Directory of Open Access Journals (Sweden)

    Edivaldo Domingues Velini

    2005-01-01

    Full Text Available Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18 and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19 are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs in the FORESTs Genome Project database (https://forests.esalq.usp.br related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings.

  4. 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.

  5. Glycogen synthase kinase-3? inhibition of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum).

    Science.gov (United States)

    Yoshida, Jun; Nomura, Satomi; Nishizawa, Naoyuki; Ito, Yoshiaki; Kimura, Ken-ichi

    2011-01-01

    A new biological activity of 6-(methylsulfinyl)hexyl isothiocyanate derived from Wasabia japonica was discovered as an inhibitor of glycogen synthase kinase-3?. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3? at a K(i) value of 10.5 µM and showed ATP competitive inhibition. The structure-activity relationship revealed an inhibitory potency of methylsulfinyl isothiocyanate dependent on the alkyl chain length and the sulfoxide, sulfone, and/or the isothiocyanate moiety. PMID:21228474

  6. The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex.

    Science.gov (United States)

    Vain, Thomas; Crowell, Elizabeth Faris; Timpano, Hélène; Biot, Eric; Desprez, Thierry; Mansoori, Nasim; Trindade, Luisa M; Pagant, Silvère; Robert, Stéphanie; Höfte, Herman; Gonneau, Martine; Vernhettes, Samantha

    2014-06-19

    Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-?-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-?-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane. PMID:24948829

  7. Cyclophilin inhibitors.

    Science.gov (United States)

    Gallay, Philippe A

    2009-08-01

    The percentage of patients chronically infected with hepatitis C virus (HCV) who have reached sustained antiviral response has increased since the introduction of the pegylated interferon-alpha (pIFNa) and ribavirin (RBV) treatment. However, the current standard pIFNa/RBV therapy not only has a low success rate (about 50%) but is often associated with serious side effects. Thus, there is an urgent need for the development of new anti-HCV agents. Cyclophilin (Cyp) inhibitors are among the most promising of the new anti-HCV agents under development. Recent clinical studies demonstrate that Cyp inhibitors are potent anti-HCV drugs, with a novel mechanism of action and efficacy profiles that make them attractive candidates for combination with current and future HCV treatments. PMID:19628157

  8. Specificity of starch synthase isoforms from potato.

    Science.gov (United States)

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

    1999-12-01

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

  9. Stigmatella aurantiaca Sg a15 carries genes encoding type I and type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases: involvement of a type II synthase in aurachin biosynthesis.

    Science.gov (United States)

    Silakowski, B; Kunze, B; Müller, R

    2000-01-01

    3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases catalyse the first step of the shikimate pathway. Two unrelated DAHP synthase types have been described in plants and bacteria. Two type II (aroA(A2) and aroA(A5)) and one type I DAHP synthase gene (aroA001) were identified from the myxobacterium Stigmatella aurantiaca Sg a15. Inactivation of aroA(A5) leads to a mutant that is impaired in the biosynthesis of aurachins, which are electron transport inhibitors and contain an anthranilate moiety. Feeding of anthranilic acid to the mutant culture restores production of aurachins. Inactivation of aroA(A2) and aroA001 does not impair production of aurachins or other known secondary metabolites of S. aurantiaca Sg a15. PMID:10896221

  10. UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

    Directory of Open Access Journals (Sweden)

    Ramani Shilpa

    2010-08-01

    Full Text Available Abstract The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP synthase (EC 4.1.2.15 catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs. A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase.

  11. UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells.

    Science.gov (United States)

    Ramani, Shilpa; Patil, Nandadevi; Jayabaskaran, Chelliah

    2010-01-01

    The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase. PMID:20704760

  12. Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699

    International Nuclear Information System (INIS)

    A ?-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from rat liver. The structure of this ?-lactone, termed L-659,699, is (E,E)-11-[3-hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid. A partially purified preparation of cytoplasmic HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC50 of 0.12 ?M. The enzymes HMG-CoA reductase, ?-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, an fatty acid synthase were not inhibited to any extent by this compound. In cultured Hep G2 cells, the compound inhibited the incorporation of [14C]acetate into sterols with an IC50 of 6 ?M, while incorporation of [3H]mevalonate into sterols in these cells was not affected. The activity of HMG-CoA reductase in the cultured Hep G2 cells was induced in a dose-dependent manner by incubation with L-659,699. A 37-fold increase in reductase was observed after a 24-hr incubation with 62 ?M L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed

  13. Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L.

    Science.gov (United States)

    Berger, M G; Sprengart, M L; Kusnan, M; Fock, H P

    1986-06-01

    Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism. PMID:16664820

  14. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme. PMID:25514203

  15. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-05

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

  16. The impact of altered herbicide residues in transgenic herbicide-resistant crops on standard setting for herbicide residues

    OpenAIRE

    Kleter, G. A.; Unsworth, J. B.; Harris, C. A.

    2011-01-01

    The global area covered with transgenic (genetically modified) crops has rapidly increased since their introduction in the mid-1990s. Most of these crops have been rendered herbicide resistant, for which it can be envisaged that the modification has an impact on the profile and level of herbicide residues within these crops. In this article, the four main categories of herbicide resistance, including resistance to acetolactate-synthase inhibitors, bromoxynil, glufosinate and glyphosate, are r...

  17. Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

    OpenAIRE

    Cheng, Yufeng; Wang, Qian; Li, Zhanjie; Cui, Jianmin; Hu, Shengwu; Zhao, Huixian; Chen, Mingshun

    2013-01-01

    Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indica...

  18. A genomic approach to characterization of the Citrus terpene synthase gene family

    Scientific Electronic Library Online (English)

    Marcelo Carnier, Dornelas; Paulo, Mazzafera.

    Full Text Available Terpenes are a very large and structurally diverse group of secondary metabolites which are abundant in many essential oils, resins and floral scents. Additionally, some terpenes have roles as phytoalexins in plant-pathogen relationships, allelopathic inhibitors in plant-plant interactions, or as ai [...] rborne molecules of plant-herbivore multitrophic signaling. Thus the elucidation of the biochemistry and molecular genetics of terpenoid biosynthesis has paramount importance in any crop species. With this aim, we searched the CitEST database for clusters of expressed sequence tags (ESTs) coding for terpene synthases. Herein is a report on the identification and in silico characterization of 49 putative members of the terpene synthase family in diverse Citrus species. The expression patterns and the possible physiological roles of the identified sequences are also discussed.

  19. Dietary bioflavonoids inhibit Escherichia coli ATP synthase in a differential manner.

    Science.gov (United States)

    Chinnam, Nagababu; Dadi, Prasanna K; Sabri, Shahbaaz A; Ahmad, Mubeen; Kabir, M Anaul; Ahmad, Zulfiqar

    2010-06-01

    The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of 17 bioflavonoid compounds on purified F1 or membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC50 approximately 0.07 mM)>silymarin (IC50 approximately 0.11 mM)>baicalein (IC50 approximately 0.29 mM)>silibinin (IC50 approximately 0.34 mM)>rimantadin (IC50 approximately 2.0 mM)>amantidin (IC50 approximately 2.5 mM)>epicatechin (IC50 approximately 4.0 mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F1Fo membrane preparations and isolated purified F1. ATPase and growth assays suggested that the bioflavonoid compounds used in this study inhibited F1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase. PMID:20346967

  20. CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress

    OpenAIRE

    Tang, Yixin; Scheef, Elizabeth A.; Gurel, Zafer; Sorenson, Christine M.; Jefcoate, Colin R.; Sheibani, Nader

    2009-01-01

    We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung,...

  1. Arginase inhibition mediates renal tissue protection in diabetic nephropathy by a nitric oxide synthase 3-dependent mechanism

    OpenAIRE

    You, Hanning; gao, Ting; Cooper, Timothy K; Morris, Sidney M.; Awad, Alaa S.

    2013-01-01

    Recently we showed that pharmacological blockade or genetic deficiency of arginase-2 confers kidney protection in diabetic mouse models. Here we tested whether the protective effect of arginase inhibition is nitric oxide synthase-3 (eNOS)-dependent in diabetic nephropathy. Experiments were conducted in eNOS knockout and their wild type littermate mice using multiple low doses of vehicle or streptozotocin and treated with continuous subcutaneous infusion of vehicle or the arginase inhibitor S-...

  2. Inducible nitric oxide synthase is involved in the modulation of depressive behaviors induced by unpredictable chronic mild stress

    OpenAIRE

    Peng Yun-Li; Liu Yu-Ning; Liu Lei; Wang Xia; Jiang Chun-Lei; Wang Yun-Xia

    2012-01-01

    Abstract Background Experiences and inflammatory mediators are fundamental in the provocation of major depressive disorders (MDDs). We investigated the roles and mechanisms of inducible nitric oxide synthase (iNOS) in stress-induced depression. Methods We used a depressive-like state mouse model induced by unpredictable chronic mild stress (UCMS). Depressive-like behaviors were evaluated after 4 weeks of UCMS, in the presence and absence of the iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamid...

  3. Energy transduction in ATP synthase.

    Science.gov (United States)

    Elston, T; Wang, H; Oster, G

    1998-01-29

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit. This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model. PMID:9461222

  4. 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.

  5. Terpene synthases are widely distributed in bacteria

    Science.gov (United States)

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  6. Terpene synthases are widely distributed in bacteria.

    Science.gov (United States)

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-Ya, Kazuo; Omura, Satoshi; Cane, David E; Ikeda, Haruo

    2015-01-20

    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

  7. Molecular cloning of prostacyclin (PGI2) synthase

    International Nuclear Information System (INIS)

    PGI2 synthase is a hemoprotein which may be a cytochrome P450. To test this possibility, they have begun molecular cloning of PGI2 synthase. A cDNA library has been constructed in bacteriophage lambda-gt 10 using poly(A+) RNA prepared from cultured bovine endothelial cells. They are currently screening this library with synthetic 32P-labeled oligonucleotide probes. Synthesis of these probes is based on amino acid sequence data obtained with the holoenzyme purified by immunoaffinity chromatography and with tryptic peptides isolated by HPLC. The N-terminal sequence of bovine aortic PGI2 synthase is MSWAVVFGLLAALLLLLLLTRRRRRMPGERL. This N-terminal sequence shows significant (29% and 26%) homology with rabbit and rat phenobarbital(PB)-inducible P450s, respectively, but no significant sequence homologies (2 synthase and PB-inducible P450s differ in their amino acid compositions, particularly in their contents of tryptophan, cysteine and isoleucine. The sequences of three tryptic peptides have been determined. One pentapeptide contains one of the three cysteine residues present in PGI2 synthase; this peptide shows no homology with highly conserved cysteine peptides from cytochrome P-450s. Two other peptides (a penta- and a decapeptide) also show no homology with other P450s

  8. Arabidopsis CDS blastp result: AK109628 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109628 002-138-C02 At3g48560.1 acetolactate synthase, chloroplast / acetohydroxy-acid synthase ... (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 0.0 ...

  9. Arabidopsis CDS blastp result: AK242817 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242817 J090063G17 At3g48560.1 68416.m05302 acetolactate synthase, chloroplast / acetohydroxy-a ... cid synthase (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 0.0 ...

  10. Arabidopsis CDS blastp result: AK058963 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058963 001-020-C04 At3g48560.1 acetolactate synthase, chloroplast / acetohydroxy-acid synthase ... (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 2e-15 ...

  11. Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice

    OpenAIRE

    Sinz, Elizabeth H.; Kochanek, Patrick M.; Dixon, C. Edward; Clark, Robert S. B.; Carcillo, Joseph A.; Schiding, Joanne K.; Chen, Minzhi; Wisniewski, Stephen R.; Carlos, Timothy M.; Williams, Debra; Dekosky, Steven T.; Watkins, Simon C.; Marion, Donald W.; Billiar, Timothy R.

    1999-01-01

    Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days...

  12. Arginase Inhibition in Airways from Normal and Nitric Oxide Synthase 2-Knockout Mice Exposed to Ovalbumin

    OpenAIRE

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; O’roark, Erin M.; Kenyon, Nicholas J.; Last, Jerold A.

    2009-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize L-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N?-hydroxy-nor-L-arginine (nor-NOHA) significantly increased total L-arginine content in the airway compartment. We hypothesized that such an increase in L-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and ther...

  13. Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

    OpenAIRE

    Burnett, A. L.; Nelson, R. J.; Calvin, D. C.; Liu, J. X.; Demas, G. E.; Klein, S. L.; Kriegsfeld, L. J.; Dawson, V. L.; Dawson, T. M.; Snyder, S. H.

    1996-01-01

    BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mat...

  14. Acute Inhibition of GTP Cyclohydrolase 1 Uncouples Endothelial Nitric Oxide Synthase and Elevates Blood Pressure

    OpenAIRE

    Wang, Shuangxi; Xu, Jian; Song, Ping; Wu, Yong; Zhang, Junhua; Choi, Hyoung Chul; Zou, Ming-hui

    2008-01-01

    GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) dictating at least partly, the balance of nitric oxide (NO) and superoxide (O2•?) produced by this enzyme. The aim of this study was to determine the effect of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure in vivo. Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (DAHP...

  15. The structure of MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to be a salicylate synthase.

    Science.gov (United States)

    Harrison, Anthony J; Yu, Minmin; Gårdenborg, Therés; Middleditch, Martin; Ramsay, Rochelle J; Baker, Edward N; Lott, J Shaun

    2006-09-01

    The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis. PMID:16923875

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

    OpenAIRE

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryo...

  17. Producing dicarboxylic acids using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    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.

  18. Producing dicarboxylic acids using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Calmidazolium, a calmodulin inhibitor, inhibits endothelium-dependent relaxations resistant to nitro-L-arginine in the canine coronary artery.

    OpenAIRE

    Illiano, S.; Nagao, T.; Vanhoutte, P M

    1992-01-01

    1. The role of calmodulin in endothelium-dependent relaxations in the canine coronary artery, was investigated by use of the inhibitor of calmodulin, calmidazolium. 2. The endothelium-dependent relaxations to adenosine diphosphate (ADP) and nebivolol, a beta-adrenoceptor antagonist, in control solution, and to bradykinin in high potassium solution (to inhibit endothelium-dependent hyperpolarization), were abolished by nitro-L-arginine (30 microM), an inhibitor of nitro oxide-synthase. Calmida...

  20. Práticas de manejo e a resistência de Euphorbia heterophylla aos inibidores da ALS e tolerância ao glyphosate no Rio Grande do Sul Management practices x Euphorbia heterophylla resistance to ALS-inhibitors and tolerance to glyphosate in Rio Grande do Sul

    Directory of Open Access Journals (Sweden)

    L. Vargas

    2013-06-01

    Full Text Available A utilização intensiva do glyphosate nas lavouras de soja Roundup Ready® (RR no Rio Grande do Sul (RS, nos últimos anos, pode ter selecionado biótipos de leiteira (Euphorbia heterophylla resistentes ao herbicida. Esse cenário dificultará ainda mais o manejo da espécie, já que permanecem indícios da presença de biótipos resistentes também em herbicidas inibidores da acetolactato sintase (ALS. Assim, os objetivos deste trabalho foram avaliar a sensibilidade da leiteira a herbicidas inibidores da ALS e ao glyphosate, verificar a distribuição dos biótipos resistentes no RS e determinar os principais fatores agronômicos associados a falhas de controle. Para isso, amostras de sementes de plantas de leiteira foram coletadas em lavouras de soja RR localizadas em 56 municípios do Estado do RS. Por ocasião das coletas, os agricultores responderam a questionário que abordava o manejo das plantas daninhas na área. Usando-se as sementes coletadas, foram conduzidos dois experimentos em casa de vegetação: no primeiro, avaliou-se a resposta de 86 biótipos ao herbicida glyphosate, aplicado na dose de 2.160 g e.a. ha-1; e, no segundo, a resposta de 73 biótipos ao herbicida imazethapyr, aplicado na dose de 200 g i.a. ha-1. Os resultados obtidos evidenciam que todos os biótipos de leiteira avaliados são suscetíveis ao glyphosate, porém existem biótipos resistentes aos inibidores da ALS. As respostas do questionário indicam que práticas de manejo como uso de subdoses e/ou utilização intensiva do glyphosate e a ausência de rotação de culturas favorecem falhas no controle de leiteira pelo herbicida glyphosate em soja.The intensive use of glyphosate in Roundup Ready® (RR soybean fields in Rio Grande do Sul (RS, in recent years may have selected wild poinsettia (Euphorbia heterophylla biotypes resistant to the herbicide. This scenario will further complicate the management of this species, since evidence remains of the presence of herbicide resistant biotypes also in acetolactate synthase (ALS-inhibitors. Thus, the objectives of this work were to evaluate wild poinsettia's sensitivity to the ALS-inhibiting herbicides and glyphosate; to investigate the distribution of resistant biotypes in the state of RS;and to determine the main agronomic factors associated with control failures. Seeds of wild poinsettia plants that survived glyphosate applications were collected from RR soybean fields located in 56 municipalities in the state of RS. On the occasion, the farmers were interviewed through a questionnaire aiming to collect information on the management of the area. Using the seeds collected, two experiments were conducted under greenhouse conditions. The first evaluated the response of 86 biotypes to glyphosate, applied at the rate of 2.160 g ha-1 while the second experiment evaluated the response of the herbicide imazethapyr to 73 biotypes, applied at a dose of 200 g a.i. ha?1. The results show that all the wild poinsettia biotypes evaluated are susceptible to glyphosate, but some are resistant to ALS-inhibitors. The survey responses indicate that management practices such as the use of sub doses and/or intensive use of glyphosate, as well as lack of crop rotation favor failures in wild poinsettia control by glyphosate in soybean.

  1. Práticas de manejo e a resistência de Euphorbia heterophylla aos inibidores da ALS e tolerância ao glyphosate no Rio Grande do Sul / Management practices x Euphorbia heterophylla resistance to ALS-inhibitors and tolerance to glyphosate in Rio Grande do Sul

    Scientific Electronic Library Online (English)

    L., Vargas; M.A., Nohatto; D., Agostinetto; M.A., Bianchi; J.M., Paula; E., Polidoro; R.E., Toledo.

    2013-06-01

    Full Text Available A utilização intensiva do glyphosate nas lavouras de soja Roundup Ready® (RR) no Rio Grande do Sul (RS), nos últimos anos, pode ter selecionado biótipos de leiteira (Euphorbia heterophylla) resistentes ao herbicida. Esse cenário dificultará ainda mais o manejo da espécie, já que permanecem indícios [...] da presença de biótipos resistentes também em herbicidas inibidores da acetolactato sintase (ALS). Assim, os objetivos deste trabalho foram avaliar a sensibilidade da leiteira a herbicidas inibidores da ALS e ao glyphosate, verificar a distribuição dos biótipos resistentes no RS e determinar os principais fatores agronômicos associados a falhas de controle. Para isso, amostras de sementes de plantas de leiteira foram coletadas em lavouras de soja RR localizadas em 56 municípios do Estado do RS. Por ocasião das coletas, os agricultores responderam a questionário que abordava o manejo das plantas daninhas na área. Usando-se as sementes coletadas, foram conduzidos dois experimentos em casa de vegetação: no primeiro, avaliou-se a resposta de 86 biótipos ao herbicida glyphosate, aplicado na dose de 2.160 g e.a. ha-1; e, no segundo, a resposta de 73 biótipos ao herbicida imazethapyr, aplicado na dose de 200 g i.a. ha-1. Os resultados obtidos evidenciam que todos os biótipos de leiteira avaliados são suscetíveis ao glyphosate, porém existem biótipos resistentes aos inibidores da ALS. As respostas do questionário indicam que práticas de manejo como uso de subdoses e/ou utilização intensiva do glyphosate e a ausência de rotação de culturas favorecem falhas no controle de leiteira pelo herbicida glyphosate em soja. Abstract in english The intensive use of glyphosate in Roundup Ready® (RR) soybean fields in Rio Grande do Sul (RS), in recent years may have selected wild poinsettia (Euphorbia heterophylla) biotypes resistant to the herbicide. This scenario will further complicate the management of this species, since evidence remain [...] s of the presence of herbicide resistant biotypes also in acetolactate synthase (ALS)-inhibitors. Thus, the objectives of this work were to evaluate wild poinsettia's sensitivity to the ALS-inhibiting herbicides and glyphosate; to investigate the distribution of resistant biotypes in the state of RS;and to determine the main agronomic factors associated with control failures. Seeds of wild poinsettia plants that survived glyphosate applications were collected from RR soybean fields located in 56 municipalities in the state of RS. On the occasion, the farmers were interviewed through a questionnaire aiming to collect information on the management of the area. Using the seeds collected, two experiments were conducted under greenhouse conditions. The first evaluated the response of 86 biotypes to glyphosate, applied at the rate of 2.160 g ha-1 while the second experiment evaluated the response of the herbicide imazethapyr to 73 biotypes, applied at a dose of 200 g a.i. ha?1. The results show that all the wild poinsettia biotypes evaluated are susceptible to glyphosate, but some are resistant to ALS-inhibitors. The survey responses indicate that management practices such as the use of sub doses and/or intensive use of glyphosate, as well as lack of crop rotation favor failures in wild poinsettia control by glyphosate in soybean.

  2. Globe fringerush (Fimbristylis miliacea) cross resistance to als-inhibitor herbicides under field conditions in irrigated rice in the south of Brazil / Resistência cruzada de herbicidas inibidores da als em cuminho (Fimbristylis miliacea) sob condições de campo em lavouras de arroz irrigado no sul do Brasil

    Scientific Electronic Library Online (English)

    C.E., Schaedler; J.A., Noldin; D.S., Eberhardt; D., Agostinetto; N.R., Burgos.

    2013-12-01

    Full Text Available Herbicidas inibidores da ALS geralmente apresentam controle adequado de plantas daninhas em lavouras de arroz irrigado. Após anos consecutivos de uso, a espécie Cyperaceae cuminho (Fimbristylis miliacea) foi selecionada com resistência a herbicidas inibidores da ALS (acetolactato sintase). O cuminho [...] é uma das mais problemáticas plantas daninhas resistentes a herbicidas em arroz irrigado em Santa Catarina, Brasil. O objetivo desta pesquisa foi investigar a resistência cruzada aos inibidores da ALS em cuminho em condições de campo. Experimentos foram realizados em lavoura de arroz naturalmente infestada com cuminho resistente a ALS em Santa Catarina, nas safras 2008/09 e 2009/10. As unidades experimentais foram dispostas em delineamento de blocos casualizados, com cinco repetições consistindo de dois fatores (herbicida e dose) em arranjo fatorial 4 x 5. Os herbicidas inibidores da ALS foram bispyribac-sodium, ethoxysulfuron, pyrazosulfuron-etyl e penoxsulam. Plantas de cuminho com seis folhas foram pulverizados com doses de herbicida equivalentes a 0, 0,5, 1, 2 e 4X as doses recomendadas, com volume de calda de 200 L ha?1. Número de colmos, grãos cheios e estéril, estatura de planta, massa seca da parte aérea e produtividade de grãos foram avaliados na cultura do arroz. O controle de cuminho foi avaliado aos 28 e 70 dias após a aplicação do herbicida (DAA) e a massa seca da parte aérea 13 semanas após a aplicação do herbicida. A competição com cuminho reduziu o número de colmos e a produtividade de grãos de arroz. A população de cuminho nessa lavoura, foi resistente a todos os herbicidas inibidores da ALS testados. Penoxsulam apresentou maior atividade entre os tratamentos aos 28 e 70 DAA, porém o nível de controle foi de apenas 50 e 42%, respectivamente, no segundo ano de avaliação, não sendo suficiente para evitar perda de produtividade da cultura. Herbicidas alternativos e estratégias de controle são necessários para evitar perdas na produtividade das lavouras de arroz com infestação de cuminho resistente a herbicidas inibidores da ALS. Abstract in english ALS-inhibiting herbicides usually provide adequate weed control in irrigated rice fields. After consecutive years of use, the Cyperaceae species, globe fringerush (Fimbristylis miliacea) began to show resistance to ALS (acetolactate synthase) inhibitors. Globe fringerush is one of the most problemat [...] ic herbicide-resistant weeds in irrigated rice in the state of Santa Catarina in the South of Brazil. The objective of this research was to examine cross resistance of globe fringerush to ALS inhibitors, under field conditions. Two experiments were conducted in a rice field naturally infested with ALS-resistant globe fringerush in Santa Catarina, in the 2008/09 and 2009/10 cropping seasons. The experimental units were arranged in randomized complete block design, with five replicates, consisting of two factors (herbicide and dose) in a 4 x 5 factorial arrangement. ALS herbicides included bispyribac-sodium, ethoxysulfuron, pyrazosulfuron-ethyl and penoxsulam. Six-leaf globe fringerush was sprayed with herbicide doses of 0, 0.5, 1, 2 and 4X the recommended doses in a spray volume of 200 L ha-1. The number of rice culm, filled and sterile grains, plant height, dry shoot biomass and grain yield were recorded. Globe fringerush control was evaluated 28 and 70 days after herbicide application (DAA); shoots were harvested at 13 weeks after herbicide application and dry weight recorded. Competition with globe fringerush reduced the number of culm and rice grain yield. The globe fringerush biotype in this field was resistant to all ALS herbicides tested. Penoxsulam had the highest level of activity among treatments at 28 and 70 DAA, but the control level was only 50% and 42%, respectively, in the second year of assessment. This was not enough to prevent rice yield loss. Alternative herbicides and weed control strategies are necessary to avoid yield losses in rice fields infested with ALS-resistant biotypes of gl

  3. Proton pump inhibitors

    Science.gov (United States)

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by glands in ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This is a ...

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

    Science.gov (United States)

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

    2007-12-01

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

  5. The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

    Science.gov (United States)

    Zíková, Alena; Schnaufer, Achim; Dalley, Rachel A.; Panigrahi, Aswini K.; Stuart, Kenneth D.

    2009-01-01

    The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 ? subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought. PMID:19436713

  6. Characterization of a Chitin Synthase Encoding Gene and Effect of Diflubenzuron in Soybean Aphid, Aphis Glycines

    Directory of Open Access Journals (Sweden)

    Raman Bansal, M. A. Rouf Mian, Omprakash Mittapalli, Andy P. Michel

    2012-01-01

    Full Text Available Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR of chitin synthases. The quantitative real-time PCR (qPCR analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument; however, it had the highest expression in integument (~3.5 fold compared to gut. Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®, a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

  7. Organic corrosion inhibitors

    International Nuclear Information System (INIS)

    Adsorption of organic compounds on metallic electrodes is one of the main ways for its corrosion inhibition. The different classifications of corrosion inhibitors have been reviewed. Moreover, the most important factors in the action of organic corrosion inhibitors, metal charge surface and inhibitor structure are studied. From this, it is possible to propose the mechanisms of the inhibition. (author)

  8. Genomic organization of plant terpene synthases and molecular evolutionary implications.

    Science.gov (United States)

    Trapp, S C; Croteau, R B

    2001-06-01

    Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general. PMID:11404343

  9. Electrical power fuels rotary ATP synthase.

    Science.gov (United States)

    Dimroth, Peter; von Ballmoos, Christoph; Meier, Thomas; Kaim, Georg

    2003-12-01

    ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F(0) domain and present a mechanism for torque generation with the fundamental nature of the membrane potential as driving force in the core. PMID:14656431

  10. Nitric oxide synthase in the pineal gland

    OpenAIRE

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

    1996-01-01

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

  11. Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosynthesis.

    Science.gov (United States)

    Hezari, M; Lewis, N G; Croteau, R

    1995-10-01

    The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex series of reactions involving oxidations and side-chain acylations. Cyclization activity is located principally in yew stem bark and adhering cambium. The operationally soluble cyclization enzyme was partially purified (approximately 600-fold) by combination of anion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophoresis, in combination with gel permeation chromatography, allowed the identification of taxadiene synthase as a monomeric protein of molecular weight 79,000. In general properties (divalent metal ion requirement, kinetic constants, molecular weight), the taxadiene synthase of Pacific yew is similar to the diterpene cyclase abietadiene synthase involved in resin acid biosynthesis in other gymnosperms. However, in pH optimum and response to inhibitors, these two diterpene cyclases are distinctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synthase of lodgepole pine stem (constitutive) or of grand fir stem (wound-inducible) and the enzyme is not inducible to higher levels by stem wounding or elicitor treatment. PMID:7574719

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

    Science.gov (United States)

    Chi, Sulene L; Pizzo, Salvatore V

    2006-01-01

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

  13. The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory

    OpenAIRE

    Pogoryelov, Denys; Yu, Jinshu; Meier, Thomas; Vonck, Janet; Dimroth, Peter; Muller, Daniel J.

    2005-01-01

    The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically as...

  14. The Regulatory C-Terminal Domain of Subunit ? of FoF1 ATP Synthase Is Dispensable for Growth and Survival of Escherichia coli?†

    OpenAIRE

    Taniguchi, Naohiro; Suzuki, Toshiharu; Berney, Michael; Yoshida, Masasuke; Cook, Gregory M.

    2011-01-01

    The C-terminal domain of subunit ? of the bacterial FoF1 ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this i...

  15. Inhibition of Glycogen Synthase Kinase 3? Promotes Tight Junction Stability in Brain Endothelial Cells by Half-Life Extension of Occludin and Claudin-5

    OpenAIRE

    Ramirez, Servio H.; Fan, Shongshan; Dykstra, Holly; Rom, Slava; Mercer, Aaron; Reichenbach, Nancy L.; Gofman, Larisa; Persidsky, Yuri

    2013-01-01

    Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3? (GSK3?) inhibition has on primary human brain endothelial cells. Here we show that GSK3? inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electric...

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

    OpenAIRE

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

    2014-01-01

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

  17. The cellulose synthase superfamily in fully sequenced plants and algae

    OpenAIRE

    Xu Ying; Huang Jinling; Yin Yanbin

    2009-01-01

    Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is foun...

  18. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    OpenAIRE

    Srivastava Anurag; Shukla Nootan K; DattaGupta Siddartha; Sawhney Meenakshi; Kaur Jatinder; Ralhan Ranju

    2010-01-01

    Abstract Background We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signa...

  19. Thiol oxidation of mitochondrial F0-c subunits: a way to switch off antimicrobial drug targets of the mitochondrial ATP synthase.

    Science.gov (United States)

    Nesci, S; Ventrella, V; Trombetti, F; Pirini, M; Pagliarani, A

    2014-08-01

    A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents "the enzyme of life" of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the common drug binding site of the enzyme is somehow destabilized, thus weakening the enzyme-drug interactions and making the ATP synthase insensitive to drug inhibition. On these bases we hypothesize that the selective oxidation of these cysteine thiols can be exploited to desensitize the mitochondrial ATP synthase to drugs which target FO and maintain their inhibitory potency on bacterial ATP synthases. According to our hypothesis, this strategy could represent an intriguing tool to prevent adverse effects of antimicrobial drugs in mammals, thus enhancing the number of natural and synthetic compounds which can be used in therapy. To this aim studies should be addressed to the identification and formulation of compounds and/or treatments able to selectively oxidize the crucial cysteine thiols of c-subunits without affecting the overall functionality of the mitochondrial ATP synthase and other thiol containing proteins. PMID:24932580

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

    Science.gov (United States)

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

    2005-05-17

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

  1. Synthesis of 1,2[3H]-1,2-epoxy analogue of fructose-6P, an affinity label of Escherichia coli glucosamine-6P synthase

    International Nuclear Information System (INIS)

    1,2-anhydroglucitol-6P, a known inhibitor of glucose-6P isomerase, behaved as a fructose-6P site-directed irreversible inhibitor of bacterial glucosamine-6P synthase. The lack of reproducibility of the aldolase-mediated condensation of dihydroxyacetone phosphate and glycidaldehyde followed by borohydride reduction previously described prompted us to develop a chemical route to this compounds and its radiolabelled counterpart. The compound was synthesized in 13 steps from D-arabinose with a 6% overall yield. Tritium introduction was performed at step 11 (3 ? 4) allowing isolation of the title compound of high specific radioactivity. (author)

  2. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    Directory of Open Access Journals (Sweden)

    Srivastava Anurag

    2010-04-01

    Full Text Available Abstract Background We reported increased levels of Phosphatidyl Inositol synthase (PI synthase, (enzyme that catalyses phosphatidyl inositol (PI synthesis-implicated in intracellular signaling and regulation of cell growth in smokeless tobacco (ST exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC and premalignant lesions (leukoplakia, and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST exposure. Methods Tissue microarray (TMA Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Results Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000. Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005 and tobacco consumption (p = 0.03, OR = 9.0. Exposure of oral cell systems to smokeless tobacco (ST in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K and cyclin D1 levels. Conclusion Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco.

  3. Divinyl ether synthase gene and protein, and uses thereof

    Science.gov (United States)

    Howe, Gregg A. (East Lansing, MI); Itoh, Aya (Tsuruoka, JP)

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  4. Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

    Science.gov (United States)

    Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

    2010-01-01

    Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled ?-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane. PMID:20056103

  5. Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: effect of cold acclimation.

    Science.gov (United States)

    Rey, Benjamin; Roussel, Damien; Teulier, Loïc; Eyenga, Pierre; Degletagne, Cyril; Belouze, Maud; Duchamp, Claude

    2011-01-01

    We report the first evidence of a mitochondrial NO synthase (mtNOS) in bird skeletal muscle. In vitro, mtNOS activity stimulated by L-arginine reduced intermyofibrillar mitochondrial oxygen uptake and ATP synthesis rates, stimulated endogenous H(2)O(2) generation, but had no effect on oxidative phosphorylation efficiency. Arginine-induced effects were fully reversed by L-NAME, a known NOS inhibitor. When ducklings were cold exposed for 4 weeks, muscle mitochondria displayed an increased state 3 respiration, a reduced H(2)O(2) generation but no significant alteration in mtNOS activity. We conclude that mtNOS is expressed in avian skeletal muscle. PMID:21095190

  6. 18?-Glycyrrhetinic Acid Suppresses Cell Proliferation through Inhibiting Thromboxane Synthase in Non-Small Cell Lung Cancer

    OpenAIRE

    Huang, Run-yue; Chu, Yong-liang; Huang, Qing-chun; Chen, Xiu-min; Jiang, Ze-bo; Zhang, Xian; Zeng, Xing

    2014-01-01

    18?-glycyrrhetinic acid (18?-GA) is a bioactive component of licorice. The anti-cancer activity of 18?-GA has been studied in many cancer types, whereas its effects in lung cancer remain largely unknown. We first showed that 18?-GA effectively suppressed cell proliferation and inhibited expression as well as activity of thromboxane synthase (TxAS) in non-small cell lung cancer (NSCLC) cells A549 and NCI-H460. In addition, the administration of 18?-GA did not have any additional inhibitor...

  7. Involvement of neuronal nitric oxide synthase in N-methyl-N-nitrosourea-induced retinal degeneration in mice.

    Science.gov (United States)

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

    2015-03-01

    N-methyl-N-nitrosourea (MNU) is widely used to study the mechanism of retinal degenerative diseases (RDs) because of its selectivity of photoreceptor cell death. Many reports suggest that excessive nitric oxide (NO) plays a crucial role in neuronal cell death. We hypothesized that nitric oxide synthase (NOS)/NO are involved in photoreceptor cell death by MNU. We found that the levels of NO increased after MNU treatment. Furthermore, we demonstrated that neuronal NOS specific inhibitor attenuated photoreceptor cell death by MNU in mice. We believe that our findings might be a new target for the treatment of RDs. PMID:25837940

  8. Expression in Arabidopsis of a strawberry linalool synthase gene under the control of the inducible potato P12 promoter

    OpenAIRE

    Yang, L; Mercke, P.; van Loon, J.J.A.; Fang, Zhiyuan; Dicke, M.; Jongsma, M.A.

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FaNES1 linalool synthase (LIS) cDNA from strawberry with plastid targeting and a synthetic intron (LIS') was placed under the control of the wound inducible proteinase inhibitor 2 (PI2) promoter from potato. The construct pBin-PPI2-LIS' was transformed to Arabidopsis thaliana ecotype Columbia 0. Kanamycin resistant T0 seedlings were confirmed for the presence and transcription of the LIS' gene by PCR analysi...

  9. Dynamic Cycling with Hsp90 Stabilizes Neuronal Nitric Oxide Synthase Through Calmodulin-dependent Inhibition of Ubiquitination†

    OpenAIRE

    Peng, Hwei-ming; Morishima, Yoshihiro; Clapp, Kelly M.; Lau, Miranda; Pratt, William B.; Osawa, Yoichi

    2009-01-01

    NO production by neuronal nitric oxide synthase (nNOS) requires calmodulin and is enhanced by the chaperone Hsp90, which cycles dynamically with the enzyme. The proteasomal degradation of nNOS is enhanced by suicide inactivation and by treatment with Hsp90 inhibitors, the latter suggesting that dynamic cycling with Hsp90 stabilizes nNOS. Here, we use a purified ubiquitinating system containing CHIP (carboxyl terminus of Hsp70-interacting protein) as the E3 ligase to show that Hsp90 inhibits C...

  10. Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages.

    OpenAIRE

    Pahan, K.; Sheikh, F. G.; Namboodiri, A. M.; Singh, I.

    1997-01-01

    This study explores the role of mevalonate inhibitors in the activation of NF-kbeta and the induction of inducible nitric oxide synthase (iNOS) and cytokines (TNF-alpha, IL-1beta, and IL-6) in rat primary astrocytes, microglia, and macrophages. Lovastatin and sodium phenylacetate (NaPA) were found to inhibit LPS- and cytokine-mediated production of NO and expression of iNOS in rat primary astrocytes; this inhibition was not due to depletion of end products of mevalonate pathway (e.g., cholest...

  11. Treatment with the Hyaluronic Acid Synthesis Inhibitor 4-Methylumbelliferone Suppresses SEB-Induced Lung Inflammation

    OpenAIRE

    Uchakina, Olga N.; Hagele, Harriet F.; Mckallip, Robert J.

    2013-01-01

    Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use of the hyaluronic acid synthase inhibitor 4-methylumbelliferone (4-MU) on staphylococcal enterotoxin B (SEB) induced acute lung inflammation. Culturing SEB-activated immune cells with 4-M...

  12. Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

    OpenAIRE

    Cavazzuti, Antonio; Paglietti, Giuseppe; Hunter, William N.; Gamarro, Francisco; Piras, Sandra; Loriga, Mario; Alleca, Sergio; Corona, Paola; McLuskey, Karen; Tulloch, Lindsay; Gibellini, Federica; Ferrari, Stefania; Costi, Maria Paola

    2008-01-01

    Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determi...

  13. Glycogen synthase kinase-3: A promising therapeutic target for Fragile X Syndrome

    Directory of Open Access Journals (Sweden)

    RichardScottJope

    2011-11-01

    Full Text Available Recent advances in understanding the pathophysiological mechanisms contributing to Fragile X Syndrome (FXS have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP. FMRP may have several functions, but it is most well-established as an RNA-binding protein that regulates translation, and it is by this mechanism that FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3 may contribute to the therapeutic treatment of FXS is reviewed here. In the mouse model of FXS, which lacks FMRP expression (FX mice, GSK3 is hyperactive in several brain regions. Furthermore, significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in FXS patients also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS.

  14. Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase.

    Science.gov (United States)

    Asuncion Valenzuela, Malyn M; Castro, Imilce; Gonda, Amber; Diaz Osterman, Carlos J; Jutzy, Jessica M; Aspe, Jonathan R; Khan, Salma; Neidigh, Jonathan W; Wall, Nathan R

    2015-01-01

    New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma. PMID:25767396

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

    International Nuclear Information System (INIS)

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

  16. Inducible nitric oxide synthase haplotype associated with migraine and aura.

    Science.gov (United States)

    de O S Mansur, Thiago; Gonçalves, Flavia M; Martins-Oliveira, Alisson; Speciali, Jose G; Dach, Fabiola; Lacchini, Riccardo; Tanus-Santos, Jose E

    2012-05-01

    Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive NO amounts possibly contributing to migraine are derived from increased expression and activity of inducible NO synthase (iNOS). We tested the hypothesis that two functional, clinically relevant iNOS genetic polymorphisms (C(-1026)A-rs2779249 and G2087A-rs2297518) are associated with migraine with or without aura. We studied 142 healthy women without migraine (control group) and 200 women with migraine divided into two groups: 148 with migraine without aura (MWA) and 52 with aura (MA). Genotypes were determined by real-time polymerase chain reaction using the Taqman(®) allele discrimination assays. The PHASE 2.1 software was used to estimate the haplotypes. The A allele for the G2087A polymorphism was more commonly found in the MA group than in the MWA group (28 vs. 18%; P 0.05). The haplotype combining both A alleles for the two polymorphisms was more commonly found in the MA group than in the control group or in the MWA group (19 vs. 10 or 8%; P = 0.0245 or 0.0027, respectively). Our findings indicate that the G2087A and the C(-1026)A polymorphism in the iNOS gene affect the susceptibility to migraine with aura when their effects are combined within haplotypes, whereas the G2087A affects the susceptibility to aura in migraine patients. These finding may have therapeutic implications when examining the effects of selective iNOS inhibitors. PMID:22234503

  17. Glycogen synthase kinase 3? regulates urine concentrating mechanism in mice.

    Science.gov (United States)

    Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R; Kakade, Vijayakumar; Yu, Alan S L; Howard, Christiana; Rao, Reena

    2015-03-15

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3? and GSK3? isoforms. GSK3? has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3? has yet to be discerned. To investigate the role of GSK3? in urine concentration, we compared GSK3? knockout (GSK3?KO) mice with wild-type (WT) littermates. Under normal conditions, GSK3?KO mice had higher water intake and urine output. GSK3?KO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3?KO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3? in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3?KO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3? could play a crucial role in renal urine concentration and suggest that GSK3? might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus. PMID:25608967

  18. Structural classification and properties of ketoacyl synthases

    OpenAIRE

    Chen, Yingfei; Kelly, Erin E; Masluk, Ryan P; Nelson, Charles L.; Cantu, David C; Reilly, Peter J

    2011-01-01

    Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and t...

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

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% ?-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases. PMID:25230152

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

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes

    2014-01-01

    Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers.

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

    Science.gov (United States)

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

    2014-11-01

    Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers. PMID:25454716

  2. Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

    International Nuclear Information System (INIS)

    The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7,500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to subunit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degree C. The monomer is formed upon heating with SDS to 121 degree C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m-chlorophenylhydrazone. These resuyanide m-chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter

  3. Fgk3 glycogen synthase kinase is important for development, pathogenesis, and stress responses in Fusarium graminearum.

    Science.gov (United States)

    Qin, Jun; Wang, Guanghui; Jiang, Cong; Xu, Jin-Rong; Wang, Chenfang

    2015-01-01

    Wheat scab caused by Fusarium graminearum is an important disease. In a previous study, the FGK3 glycogen synthase kinase gene orthologous to mammalian GSK3 was identified as an important virulence factor. Although GSK3 orthologs are well-conserved, none of them have been functionally characterized in fungal pathogens. In this study, we further characterized the roles of FGK3 gene. The ?fgk3 mutant had pleiotropic defects in growth rate, conidium morphology, germination, and perithecium formation. It was non-pathogenic in infection assays and blocked in DON production. Glycogen accumulation was increased in the ?fgk3 mutant, confirming the inhibitory role of Fgk3 on glycogen synthase. In FGK3-GFP transformants, GFP signals mainly localized to the cytoplasm in conidia but to the cytoplasm and nucleus in hyphae. Moreover, the expression level of FGK3 increased in response to cold, H2O2, and SDS stresses. In the ?fgk3 mutant, cold, heat, and salt stresses failed to induce the expression of the stress response-related genes FgGRE2, FgGPD1, FgCTT1, and FgMSN2. In the presence of 80?mM LiCl, a GSK3 kinase inhibitor, the wild type displayed similar defects to the ?fgk3 mutant. Overall, our results indicate that FGK3 is important for growth, conidiogenesis, DON production, pathogenicity, and stress responses in F. graminearum. PMID:25703795

  4. Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.

    Science.gov (United States)

    Meier, Thomas; Matthey, Ulrich; von Ballmoos, Christoph; Vonck, Janet; Krug von Nidda, Tassilo; Kühlbrandt, Werner; Dimroth, Peter

    2003-01-10

    The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself. PMID:12488103

  5. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  6. Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2.

    Science.gov (United States)

    Eyler, Christine E; Wu, Qiulian; Yan, Kenneth; MacSwords, Jennifer M; Chandler-Militello, Devin; Misuraca, Katherine L; Lathia, Justin D; Forrester, Michael T; Lee, Jeongwu; Stamler, Jonathan S; Goldman, Steven A; Bredel, Markus; McLendon, Roger E; Sloan, Andrew E; Hjelmeland, Anita B; Rich, Jeremy N

    2011-07-01

    Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease. PMID:21729780

  7. Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate.

    Science.gov (United States)

    Yokoyama, Takeshi; Ostermann, Andreas; Mizuguchi, Mineyuki; Niimura, Nobuo; Schrader, Tobias E; Tanaka, Ichiro

    2014-04-01

    Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5?mm (?3.5?mm(3)) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4?Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported. PMID:24699741

  8. Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target

    DEFF Research Database (Denmark)

    Yang, Zhiping; Huang, Yuh-Chin T

    2014-01-01

    To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.

  9. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-02-26

    Covering: up to 2014The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions. PMID:25310997

  10. ATP synthase: a tentative structural model.

    Science.gov (United States)

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  11. Downregulation of the ?-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis.

    Science.gov (United States)

    Geisler, Daniela A; Päpke, Carola; Obata, Toshihiro; Nunes-Nesi, Adriano; Matthes, Annemarie; Schneitz, Kay; Maximova, Eugenia; Araújo, Wagner L; Fernie, Alisdair R; Persson, Staffan

    2012-07-01

    The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the ?-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the ?-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the ?-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development. PMID:22805435

  12. 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

  13. Instability of N-acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices.

    Science.gov (United States)

    Norred, W P; Riley, R T; Meredith, F I; Poling, S M; Plattner, R D

    2001-11-01

    Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides. It inhibits ceramide synthase, which is a proposed underlying mechanism responsible for the myriad of toxic endpoints observed. We previously reported that N-acetylation of FB1 prevents ceramide synthase inhibition, but cautioned that impure preparations of FA1 can contain a contaminant with the ability to inhibit ceramide synthase. We now report that FA1 spontaneously rearranges to O-acetylated analogs. These rearrangement products are putative inhibitors of ceramide synthase. Rat liver slices exposed to impure FA1 containing O-acetylated FB1 had sphinganine/sphingosine (Sa:So) ratios of 1.15-1.64. Control slices had Sa:So ratios of 0.07-0.24. Clean-up to remove the O-acetylated FB1 yielded purified FA1, which produced Sa:So ratios in liver slices of 0.08-0.18. After storage for approximately 1 year as either a dry powder in a desiccator, or as a dried film at 4 degrees C, the purified FA1 again contained O-acetylated FB1, and was capable of ceramide synthase inhibition. FA1 was most stable in neutral solution, but in acidic solution the equilibrium shifted towards the O-acetylated forms. FA1 in solid form also rearranged, but more slowly than in acid solution. As FA1 is considerably less cytotoxic than FB1, these results provide additional support for the conclusion that a primary amino group is necessary for both ceramide synthase inhibition and toxicity. PMID:11527566

  14. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    International Nuclear Information System (INIS)

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3?, 5?-adenosine diphosphate (3?, 5?-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3?, 5?-ADP is bound as the 3?, 5?-ADP part of CoA in the known structures of the CoA–AcpS and 3?, 5?-ADP–AcpS binary complexes. The position of the second 3?, 5?-ADP has never been described before. It is in close proximity to the first 3?, 5?-ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP

  15. Hypoxia-induced relaxation of porcine retinal arterioles in vitro depends on inducible NO synthase and EP4 receptor stimulation in the perivascular retina

    DEFF Research Database (Denmark)

    Hansen, Pernille OversØ; Kringelholt, Sidse

    2015-01-01

    PURPOSE: Hypoxia-induced relaxation of porcine retinal arterioles has been shown to be reduced during inhibition of prostaglandin synthesis and nitric oxide synthase (NOS). The purpose of this study was to identity the specific prostaglandin receptor(s) and source(s) of NO mediating this effect. METHODS: Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a myograph and were exposed to hypoxia in the presence of one of the following: the general NO synthase inhibitor L-NAME, the selective iNOS inhibitor 1400W, the selective nNOS inhibitor 7-nitroindazole, the general cyclooxygenase (COX) inhibitor ibuprofen or an antagonist to the FP- (AL 8810), DP- (BWA868C), EP1 - (SC-19220), EP2 - (PF-044189) or EP4 receptors (GW627368X). The experiments were repeated after removal of the perivascular retinal tissue. RESULTS: Hypoxia induced relaxation of retinal arterioles with preserved perivascular retinal tissue. This relaxation was significantly reduced in the presence of L-NAME, 1400W, ibuprofen and the EP4 receptor antagonist GW627368X. The simultaneous addition of L-NAME or 1400W in combination with ibuprofen, but not GW627368X, reduced hypoxia-induced vasorelaxation additively as compared to the effect of the compounds individually. CONCLUSION: Hypoxia-induced vasorelaxation of porcine retinal arterioles is mediated by inducible NOS and stimulation of EP4 receptors acting through separate pathways, but mechanisms unrelated to the studied prostaglandin receptors and NOS products are also involved.

  16. Alendronate induces gastric damage by reducing nitric oxide synthase expression and NO/cGMP/K(ATP) signaling pathway.

    Science.gov (United States)

    Silva, Renan O; Lucetti, Larisse T; Wong, Deysi V T; Aragão, Karoline S; Junior, Eudmar M A; Soares, Pedro M G; Barbosa, André Luiz R; Ribeiro, Ronaldo A; Souza, Marcellus H L P; Medeiros, Jand-Venes R

    2014-08-31

    Chronic use of alendronate has been linked to gastrointestinal tract problems. Our objective was to evaluate the role of the NO/cGMP/KATP signaling pathway and nitric oxide synthase expression in alendronate-induced gastric damage. Rats were either treated with the NO donor, sodium nitroprusside (SNP; 1, 3, and 10 mg/kg), or the NO synthase (NOS) substrate, L-arginine (L-Arg; 50, 100, and 200 mg/kg). Some rats were pretreated with either ODQ (a guanylate cyclase inhibitor; 10 mg/kg) or glibenclamide (KATP channels blocker; 10 mg/kg). In other experiments, rats were pretreated with L-NAME (non-selective NOS inhibitor; 10 mg/kg), 1400 W (selective inducible NOS [iNOS] inhibitor; 10 mg/kg), or L-NIO (a selective endothelial NOS [eNOS] inhibitor; 30 mg/kg). After 1 h, the rats were treated with alendronate (30 mg/kg) by gavage for 4 days. SNP and L-Arg prevented alendronate-induced gastric damage in a dose-dependent manner. Alendronate reduced nitrite/nitrate levels, an effect that was reversed with SNP or L-Arg treatment. Pretreatment with ODQ or glibenclamide reversed the protective effects of SNP and L-Arg. L-NAME, 1400 W, or L-NIO aggravated the severity of alendronate-induced lesions. In addition, alendronate reduced the expression of iNOS and eNOS in the gastric mucosa. Gastric ulcerogenic responses induced by alendronate were mediated by a decrease in NO derived from both eNOS and iNOS. In addition, our findings support the hypothesis that activation of the NO/cGMP/KATP pathway is of primary importance for protection against alendronate-induced gastric damage. PMID:24831353

  17. Nitric oxide in prepubertal rat ovary contribution of the ganglionic nitric oxide synthase system via superior ovarian nerve.

    Science.gov (United States)

    Casais, Marilina; Delgado, Silvia Marcela; Vallcaneras, Sandra; Sosa, Zulema; Rastrilla, Ana María

    2007-02-01

    Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. Considering the existence of the nitric oxide/ nitric oxide synthase system in the peripheral neural system and in the ovary, the aim of this work was to analyze if the liberation of NO in the ovarian compartment of prepubertal rats is of ovarian and/or ganglionic origin. The analysis is carried out from a physiological point of view using the experimental coeliac ganglion--Superior Ovarian Nerve--ovary model with and without ganglionic cholinergic stimulus Acetylcholine (Ach) 10(-6) M. Non selective and selective inhibitors of the synthase nitric oxide enzyme were added to the ovarian and ganglionic compartment, and the liberation of nitrites (soluble metabolite of the nitric oxide) in the ovarian incubation liquid was measured. We found that the non-selective inhibitor L-nitro-arginina methyl ester (L-NAME) in the ovarian compartment decreased the liberation of nitrites, and that Aminoguanidine (AG) in two concentrations in a non-dose dependent form provoked the same effect. The addition of Ach in ganglion magnified the effect of the inhibitors of the NOS enzyme. The most relevant results after the addition of inhibitors in ganglion were obtained with AG 400 and 800 microM. The inhibition was made evident with and without the joint action of Ach in ganglion. These data suggest that the greatest production of NO in the ovarian compartment comes from the ovary, mainly the iNOS isoform, though the coeliac ganglion also contributes through the superior ovarian nerve but with less quantity. PMID:17277728

  18. PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

    Science.gov (United States)

    BOOTH, LAURENCE; ROBERTS, JANE L.; CRUICKSHANKS, NICHOLA; TAVALLAI, SEYEDMEHRAD; WEBB, TIMOTHY; SAMUEL, PETER; CONLEY, ADAM; BINION, BRITTANY; YOUNG, HAROLD F.; POKLEPOVIC, ANDREW; SPIEGEL, SARAH; DENT, PAUL

    2015-01-01

    The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1?/XBP1 enhanced killing whereas knock down of eIF2?/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

  19. PDE5 inhibitors enhance celecoxib killing in multiple tumor types.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Cruickshanks, Nichola; Tavallai, Seyedmehrad; Webb, Timothy; Samuel, Peter; Conley, Adam; Binion, Brittany; Young, Harold F; Poklepovic, Andrew; Spiegel, Sarah; Dent, Paul

    2015-05-01

    The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1?/XBP1 enhanced killing whereas knock down of eIF2?/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

  20. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile. PMID:19454282

  1. Regulation of Th1 cells and experimental autoimmune encephalomyelitis (EAE) by glycogen synthase kinase-3

    Science.gov (United States)

    Beurel, Eléonore; Kaidanovich-Beilin, Oksana; Yeh, Wen-I; Song, Ling; Palomo, Valle; Michalek, Suzanne M.; Woodgett, James R.; Harrington, Laurie E.; Eldar-Finkelman, Hagit; Martinez, Ana; Jope, Richard S.

    2013-01-01

    Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the central nervous system, for which only limited therapeutic interventions are available. Since MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the present study, we tested if inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or ameliorate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3?, and Th1 cell production was inhibited in GSK3?-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts, significantly reduced the clinical symptoms of MOG35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode ameliorated clinical symptoms in a relapsing/remitting model of PLP139-151-induced EAE. Furthermore, deletion of GSK3? specifically in T cells was sufficient to ameliorate MOG35-55-induced EAE. These results demonstrate isoform-selective effects of GSK3 on T cell generation, therapeutic effects of GSK3 inhibitors in EAE, and that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS. PMID:23606540

  2. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    Science.gov (United States)

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-01

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed. PMID:14685317

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

    Science.gov (United States)

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

    2013-06-01

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

  4. Aliskiren enhances the protective effects of valsartan against cardiovascular and renal injury in endothelial nitric oxide synthase-deficient mice.

    Science.gov (United States)

    Yamamoto, Eiichiro; Kataoka, Keiichiro; Dong, Yi-Fei; Nakamura, Taishi; Fukuda, Masaya; Tokutomi, Yoshiko; Matsuba, Shinji; Nako, Hisato; Nakagata, Naomi; Kaneko, Takehito; Ogawa, Hisao; Kim-Mitsuyama, Shokei

    2009-09-01

    The protective effect of aliskiren, a direct renin inhibitor, against hypertensive cardiovascular and renal injury remains to be defined. This study was undertaken to examine the protective effects of the combination of aliskiren and valsartan, an angiotensin receptor blocker, against cardiovascular and renal injury. Endothelial NO synthase-deficient mice, subjected to cuff injury of femoral artery, were divided into 5 groups and were treated with the following: (1) vehicle; (2) aliskiren (25 mg/kg per day); (3) valsartan (8 mg/kg per day); (4) combined aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day); and (5) hydralazine (10 mg/kg per day) for 4 weeks. Aliskiren and valsartan alone markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, and coronary remodeling; prevented cuff injury-induced arterial intimal thickening; and reduced urinary albumin excretion, glomerular inflammation, and glomerulosclerosis in endothelial NO synthase-deficient mice. These beneficial effects of aliskiren and valsartan were associated with the significant attenuation of oxidative stress in these tissues. Hence, aliskiren and valsartan markedly exert the protective effects against cardiovascular and renal injury through the reduction of oxidative stress. Furthermore, compared with monotherapy with aliskiren or valsartan, the combination of a half dose of these drugs more greatly improved the above-mentioned cardiovascular and renal injuries of endothelial NO synthase-deficient mice, which were associated with greater attenuation of tissue oxidative stress by the combination therapy. Thus, the combination of aliskiren and valsartan exerts the synergistic organ-protective effects through synergistic attenuation of oxidative stress. The combination of aliskiren and valsartan seems to be a promising therapeutic strategy for hypertensive organ injury caused by endothelial NO synthase dysfunction. PMID:19597038

  5. The selectivity of protein kinase inhibitors: a further update

    Science.gov (United States)

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip

    2007-01-01

    The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214

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

    DEFF Research Database (Denmark)

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger

    2007-01-01

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

  7. Bacterial Na+-ATP synthase has an undecameric rotor

    OpenAIRE

    Stahlberg, Henning; Müller, Daniel J; Suda, Kitaru; Fotiadis, Dimitrios; Engel, Andreas; Meier, Thomas; Matthey, Ulrich; Dimroth, Peter

    2001-01-01

    Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F0 domain, which drives the extra-membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9–14 ?-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by ato...

  8. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    International Nuclear Information System (INIS)

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  9. Cloning and characterization of a mammalian pseudouridine synthase.

    OpenAIRE

    Chen, J.; J. R. Patton

    1999-01-01

    This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purif...

  10. Resistance to Integrase Inhibitors

    OpenAIRE

    Mathieu Métifiot; Christophe Marchand; Kasthuraiah Maddali; Yves Pommier

    2010-01-01

    Integrase (IN) is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses ...

  11. PDE5 inhibitors as therapeutics for heart disease, diabetes and cancer.

    Science.gov (United States)

    Das, Anindita; Durrant, David; Salloum, Fadi N; Xi, Lei; Kukreja, Rakesh C

    2015-03-01

    The phosphodiesterase 5 (PDE5) inhibitors, including sildenafil (Viagra™), vardenafil (Levitra™), and tadalafil (Cialis™) have been developed for treatment of erectile dysfunction. Moreover, sildenafil and tadalafil are used for the management of pulmonary arterial hypertension in patients. Since our first report showing the cardioprotective effect of sildenafil in 2002, there has been tremendous growth of preclinical and clinical studies on the use of PDE5 inhibitors for cardiovascular diseases and cancer. Numerous animal studies have demonstrated that PDE5 inhibitors have powerful protective effect against myocardial ischemia/reperfusion (I/R) injury, doxorubicin cardiotoxicity, ischemic and diabetic cardiomyopathy, cardiac hypertrophy, Duchenne muscular dystrophy and the improvement of stem cell efficacy for myocardial repair. Mechanistically, PDE5 inhibitors protect the heart against I/R injury through increased expression of nitric oxide synthases, activation of protein kinase G (PKG), PKG-dependent hydrogen sulfide generation, and phosphorylation of glycogen synthase kinase-3? - a master switch immediately proximal to mitochondrial permeability transition pore and the end effector of cardioprotection. In addition, PDE5 inhibitors enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs, including doxorubicin. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular and anti-cancer benefits. Despite mixed results of these clinical trials, there is a continuing strong interest by basic scientists and clinical investigators in exploring their new clinical uses. It is our hope that future new mechanistic investigations and carefully designed clinical trials would help in reaping additional benefits of PDE5 inhibitors for cardiovascular disease and cancer in patients. PMID:25444755

  12. Nitric oxide synthases and diabetic cardiomyopathy.

    Science.gov (United States)

    Khanna, Sanskriti; Singh, Gurinder Bir; Khullar, Madhu

    2014-12-01

    Cardiovascular complications associated with diabetes significantly contribute to high mortality and morbidity worldwide. The pathophysiology of diabetic cardiomyopathy (DCM), although extensively researched upon, is partially understood. Impairment in various signaling pathways including nitric oxide (NO) signaling has been implicated in the pathogenesis of diabetes induced myocardial damage. Nitric oxide synthases (NOS), the enzymes responsible for NO generation, play an important role in various physiological processes. Altered expression and activity of NOS have been implicated in cardiovascular diseases, however, the role of NOS and their regulation in the pathogenesis of DCM remain poorly understood. In the present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS. PMID:25153033

  13. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    OpenAIRE

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-?-bisabolene synthase from ginger rhizome, and ?-humulene synthase and ?-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identifi...

  14. New developments in cancer treatment with the novel thymidylate synthase inhibitor raltitrexed ('Tomudex').

    OpenAIRE

    Blackledge, G.

    1998-01-01

    Following the demonstration of efficacy, tolerability and quality-of-life benefits of raltitrexed ('Tomudex'), principally in advanced colorectal but also in other cancers, an extensive evaluation of combination therapy with other agents in patients with colorectal and other tumour types is being undertaken. This work has been prompted by preclinical observations of enhanced activity of raltitrexed when coadministered with other cytotoxic agents or radiotherapy and by preliminary results show...

  15. Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

    Czech Academy of Sciences Publication Activity Database

    Broží?ková, Carole; Mikulecká, Anna; Otáhal, Jakub

    2014-01-01

    Ro?. 63, ?. 5 (2014), s. 637-648. ISSN 0862-8408 R&D Projects: GA ?R(CZ) GAP303/10/0999; GA ?R(CZ) GPP304/11/P386; GA ?R(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : 7-nitroindazole * open field test * ladder rung walking test * brain excitability * blood gas analysis * rat Subject RIV: FH - Neurology Impact factor: 1.487, year: 2013

  16. Are anthocyanidins the immediate products of anthocyanidin synthase?

    OpenAIRE

    Turnbull, JJ; Sobey, WJ; Aplin, RT; Hassan, A.; Firmin, JL; Schofield, CJ; Prescott, AG

    2000-01-01

    Anthocyanidin synthase catalyses the in vitro conversion of its natural substrate, leucocyanidin, to cis- and transdihydroquercetin, quercetin and a small amount of cyanidin; incubation of trans-dihydroquercetin gave quercetin.

  17. Inducible nitric oxide synthase is crucial for plasma cell survival

    Science.gov (United States)

    Njau, Modesta N; Jacob, Joshy

    2015-01-01

    The viability of long-lived plasma cells is enhanced by the expression of inducible nitric oxide synthase, which relieves endoplasmic reticulum stress by triggering a response dependent on cGMP and protein kinase G. PMID:24549066

  18. Comparative study of Chalcone synthase promoters across plant families

    OpenAIRE

    Francisco Buitrago; Roberto Sierra; Alejandro Grajales; Luis Miguel Rodríguez; Pedro Jiménez; Adriana Bernal; Silvia Restrepo

    2009-01-01

    Estudio comparativo de promotores de la Chalcón Sintasa en diferentes familias de plantas In the post – genomic era the understanding of gene regulation has become a challenge and a research priority. In this research, we performed a comparative study of the regulator sequences of the chalcone synthase gene across plant families. Twenty-two sequences of chalcone synthase promoters were compared considering three regulator Cis elements: G-Box, H-Box and TATA Box. Our results show that...

  19. Purification and characterization of glutamate synthase from Azospirillum brasilense.

    OpenAIRE

    Ratti, S.; Curti, B.; Zanetti, G.; Galli, E.

    1985-01-01

    Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein...

  20. Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae.

    OpenAIRE

    Graf, R.; Mehmann, B; Braus, G H

    1993-01-01

    The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(...

  1. Role of endothelial nitric oxide synthase gene in vascular diseases

    OpenAIRE

    Akhter, M. S.; Biswas, Arijit; Saxena, Renu

    2013-01-01

    Abstract. The endothelial nitric oxide synthase (eNOS or NOS 3) is expressed in the endothelium where it produces Nitric Oxide (NO) from L-arginine. NO a powerful short lived vascular substance, plays a key role to maintain vascular homeostasis. Association of endothelial nitric oxide synthase gene variants and nitric oxide has been found in many vascular diseases chiefly stroke, coronary artery disease and hypertension. The variants have been associated with low plasma nitric oxide concentra...

  2. Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones.

    Science.gov (United States)

    Skouloubris, Stéphane; Djaout, Kamel; Lamarre, Isabelle; Lambry, Jean-Christophe; Anger, Karine; Briffotaux, Julien; Liebl, Ursula; de Reuse, Hilde; Myllykallio, Hannu

    2015-06-01

    ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation. PMID:26040760

  3. Calcium/calmodulin dependence of nitric oxide synthase from Viviparus ater

    Directory of Open Access Journals (Sweden)

    D Tagliazucchi

    2005-04-01

    Full Text Available The calcium ion dependence of soluble and particulate nitric oxyde synthase (NOS activity fromViviparus ater immunocytes was investigated. At a calcium ion concentration of 2 nM, the NOS activitymeasured by citrulline formation was 27.1 ± 2.2 and 9.3 ± 0.8 pmol/min/106cell for soluble andparticulate NOS, respectively. The increase in free calcium ion concentration to 300 nM increasesenzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 pmol/min/106cell, respectively. The 50 % activation of thecalcium-dependent activity is 91 and 97 nM Ca2+ for soluble and particulate enzymes. Trifluoperazine,an inhibitor of the calmodulin-dependent enzyme, partially inhibits both activities. Soluble NOS is fivetimes more sensitive than particulate NOS. The behaviour of both activities with three NOS inhibitors(7-nitroindazole, S-methylisothiourea sulphate, diphenyleneiodonium is very similar, with IC50 valuesthat are not significantly different. The calcium ion dependence of NOS activities, in a range of freecalcium ion variations, which are transiently observed in receptor-stimulated cells, suggests that nitricoxyde in V. ater immunocytes not only has a defensive role but also signalling relevance in crosstalkingbetween immunocytes and other cells.

  4. 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.

  5. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    OpenAIRE

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, wh...

  6. The Fused TrpEG from Streptomyces Venezuelae is an Anthranilate Synthase, Not a 2-Amino-2-deoxyisochorismate (ADIC) Synthase

    OpenAIRE

    Ashenafi, Meseret; Carrington, Renee; Collins, Alvin C.; Byrnes, W. Malcolm

    2008-01-01

    The chloramphenicol producer Streptomyces venezuelae contains an enzyme, SvTrpEG, that has a high degree of amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE of certain species of Pseudomonas. PhzE has the sequence signature of an anthranilate synthase, but recent evidence indicates that it catalyzes the production of 2-amino-2-deoxyisochorismate (ADIC), an intermediate in the two-step anthranilate synthase reaction, not anthranilate. In order to determine if SvTrpEG is...

  7. Chitin synthase I and chitin synthase II are not required for chitin synthesis in vivo in Saccharomyces cerevisiae.

    OpenAIRE

    Bulawa, C E; Osmond, B. C.

    1990-01-01

    In Saccharomyces cerevisiae, the polysaccharide chitin forms the primary division septum between mother cell and bud. Two related enzymes, chitin synthase I and chitin synthase II (UDP-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16), have been identified and their structural genes, CHS1 and CHS2, respectively, have been cloned and sequenced. Gene disruption experiments led to the conclusion that CHS2 is essential for cell division [Silverman, S.J., Sb...

  8. Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly.

    Science.gov (United States)

    Persson, K.; Schneider, G.; Jordan, D. B.; Viitanen, P. V.; Sandalova, T.

    1999-01-01

    Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 A resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four-stranded parallel beta-sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N-terminal beta-strand interacts with the beta-sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand beta1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the beta-hairpin connecting helices alpha4 and alpha5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface. PMID:10595538

  9. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    Science.gov (United States)

    Fujimoto, Masaru; Suda, Yasuyuki; Vernhettes, Samantha; Nakano, Akihiko; Ueda, Takashi

    2015-02-01

    The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3. PMID:25516570

  10. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  11. A New Class of Molecular Targeted Radioprotectors: GSK-3? Inhibitors

    International Nuclear Information System (INIS)

    Purpose: Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3? (GSK-3?)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury. Methods and Materials: A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3? inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of ?-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells. Results: Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3? inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-osis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3. Conclusions: Glycogen synthase kinase 3? small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3? inhibitors reduce deleterious consequences of intestinal irradiation and thereby improve quality of life during radiation therapy.

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

    Science.gov (United States)

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

    2003-07-25

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

  13. Resistance to Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Mathieu Métifiot

    2010-06-01

    Full Text Available Integrase (IN is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance.

  14. Cathepsin D inhibitors

    Directory of Open Access Journals (Sweden)

    M. Gacko

    2007-11-01

    Full Text Available Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirectproduct, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes withcathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme.Methods used to determine the activity and concentration of these inhibitors and their analytical, preparative and therapeuticapplications are discussed.

  15. Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

    OpenAIRE

    Blomqvist, K.; Nikkola, M; Lehtovaara, P; Suihko, M.L.; Airaksinen, U; Stråby, K B; Knowles, J K; Penttilä, M E

    1993-01-01

    The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena...

  16. Advances in mycobacterial isocitrate lyase targeting and inhibitors.

    Science.gov (United States)

    Krátký, M; Vinšová, J

    2012-01-01

    Isocitrate lyase plays a key role for survival of Mycobacterium tuberculosis in the latent form during a chronic stage of infection. This enzyme is important for M. tuberculosis during steady stage growth when it converts isocitrate to succinate and glyoxylate. Then, the glyoxylate is condensed with acetyl-CoA to form malate by malate synthase. The carbon conserving glyoxylate pathway has not been observed in mammals; therefore, it has been determined as a potential drug target for discovery of a new antituberculosis agent. Novel active molecules should shorten the duration of therapy, prevent resistance development and eliminate latent disease. The review summarizes recent progresses in isocitrate lyase inhibitors, overviews structural analogues of several metabolic intermediates (3-nitropropionate, 3-bromopyruvate, itaconate, itaconic anhydride), peptide inhibitors, and recently developed inhibitors with various chemical structures. The largest inhibitory activity against isocitrate lyase (IC(50) of 0.10 ± 0.01 ?M) and concomitantly a significant antimycobacterial activity has been presented by fluoroquinolone derivative 1-cyclopropyl-7-[3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-yl]-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, which has incorporated 3-nitropropionyl group as one of the structural analogue of succinate, a metabolic intermediate. PMID:23092127

  17. Substrate Ambiguity of 3-Deoxy-d-manno-Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae in the Context of Its Membership in a Protein Family Containing a Subset of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases

    OpenAIRE

    Subramaniam, Prem S.; Xie, Gang; Xia, Tianhui; Jensen, Roy A.

    1998-01-01

    3-Deoxy-d-manno-octulosonate 8-phosphate (KDOP) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing reactions. The genome of Neisseria gonorrhoeae contains one gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the two nonhomologous DAHP synthase families known, the N. gonorrhoeae protein belongs to the family I assemblage. KDOP synthase exhibited an ability to replace arabinose-5-P with either erythrose-4-P ...

  18. Neuronal nitric oxide synthase and N-methyl-D-aspartate neurons in experimental carbon monoxide poisoning

    International Nuclear Information System (INIS)

    We measured changes in nitric oxide (·NO) concentration in the cerebral cortex during experimental carbon monoxide (CO) poisoning and assessed the role for N-methyl-D-aspartate receptors (NMDARs), a glutamate receptor subtype, with progression of CO-mediated oxidative stress. Using microelectrodes, ·NO concentration was found to nearly double to 280 nM due to CO exposure, and elevations in cerebral blood flow, monitored as laser Doppler flow (LDF), were found to loosely correlate with ·NO concentration. Neuronal nitric oxide synthase (nNOS) activity was the cause of the ·NO elevation based on the effects of specific NOS inhibitors and observations in nNOS knockout mice. Activation of nNOS was inhibited by the NMDARs inhibitor, MK 801, and by the calcium channel blocker, nimodipine, thus demonstrating a link to excitatory amino acids. Cortical cyclic GMP concentration was increased due to CO poisoning and shown to be related to ·NO, versus CO, mediated guanylate cyclase activation. Elevations of ·NO were inhibited when rats were infused with superoxide dismutase and in rats depleted of platelets or neutrophils. When injected with MK 801 or 7-nitroindazole, a selective nNOS inhibitor, rats did not exhibit CO-mediated nitrotyrosine formation, myeloperoxidase (MPO) elevation (indicative of neutrophil sequestration), or impaired learning. Similarly, whereas CO-poisoned wild-type mice exhibited elevatioed wild-type mice exhibited elevations in nitrotyrosine and myeloperoxidase, these changes did not occur in nNOS knockout mice. We conclude that CO exposure initiates perivascular processes including oxidative stress that triggers activation of NMDA neuronal nNOS, and these events are necessary for the progression of CO-mediated neuropathology

  19. Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

    Directory of Open Access Journals (Sweden)

    Colpo Anna

    2010-10-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK-3 ? and ? are two serine-threonine kinases controlling insulin, Wnt/?-catenin, NF-?B signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3? and GSK-3? function in multiple myeloma (MM. Methods GSK-3 ? and ? expression and cellular localization were investigated by Western blot (WB and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 ? and ? isoforms. Survival signaling pathways were studied with WB analysis. Results GSK-3? and GSK-3? were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3? knock down decreased MM cell viability, while GSK-3? knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of ?-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3? knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. Conclusions These data suggest that in MM cells GSK-3? and ? i play distinct roles in cell survival and ii modulate the sensitivity to proteasome inhibitors.

  20. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan?s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  1. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.

    2014-01-01

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

  2. Are calcineurin inhibitors safer than mTOR inhibitors?

    OpenAIRE

    Yeh, Heidi; Markmann, James F.

    2012-01-01

    Isakova et al. report that kidney transplant recipients on mammalian target of rapamycin (mTOR) inhibitors do not have a lower risk of allograft failure but do have a higher risk of death than those on calcineurin inhibitors. Careful consideration is, therefore, required before converting to mTOR inhibitors to preserve renal function.

  3. Insect inhibitors of metalloproteinases.

    Science.gov (United States)

    Vilcinskas, Andreas; Wedde, Marianne

    2002-12-01

    Two types of peptidic metalloproteinase inhibitors have recently been identified in insects. A homologue of vertebrate tissue inhibitors of metalloproteinases (TIMPs) was found in the fruitfly Drosophila melanogaster which may contributes to regulation of a corresponding matrix metalloproteinase (MMP). The first member of MMPs from insects which shares similarity with vertebrate MMPs has also been cloned and characterized from Drosophila, suggesting conserved evolution of both MMPs and TIMPs. The first insect inhibitor of metalloproteinases (IMPI), which was identified in larvae of the greater wax moth, Galleria mellonella, shares no sequence similarity with known vertebrate or invertebrate proteins and represents the first non-TIMP-like inhibitor of metalloproteinases reported to date. In contrast to TIMPs, the IMPI is not active against MMPs but inhibits microbial metalloproteinases such as bacterial thermolysin. Insects may recognize such toxic metalloproteinases associated with invading pathogens by particular peptidic fragments that result from their nonregulated activity within the hemolymph. Metalloproteinases induce expression of the IMPI along with other antimicrobial proteins in course of humoral immune response of G. mellonella, thereby mediating regulation of metalloproteinase activity released within the hemolymph and inhibition of pathogen development as well. PMID:12665244

  4. Thymidine Phosphorylase Inhibitors.

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim

    Karachi : Bentham Science Publishers, 2011 - (Atta-ur-Rahman, F.; Choudhary, M.), s. 116-147 ISBN 978-1-60805-162-5 R&D Projects: GA MŠk 1M0508; GA AV ?R 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors * angiogenesis * cancer chemotherapy Subject RIV: CC - Organic Chemistry

  5. Corrosion inhibitor compositions

    International Nuclear Information System (INIS)

    A corrosion inhibitor compositon for hydrocarbon fuels consisting essentially of, by weight, (A) about 75% to 95% of at least one polymerized unsaturated aliphatic monocarboxylic acid, said unsaturated acid having 16 to 18 carbons per molecule, and (B) about 5% to 25% of at least one monoalkenylsuccinic acid in which the alkenyl group as 8 to 18 carbons

  6. The rice ent-KAURENE SYNTHASE LIKE 2 encodes a functional ent-beyerene synthase.

    Science.gov (United States)

    Tezuka, Daisuke; Ito, Akira; Mitsuhashi, Wataru; Toyomasu, Tomonobu; Imai, Ryozo

    2015-05-01

    The rice genome contains a family of kaurene synthase-like (OsKSL) genes that are responsible for the biosynthesis of various diterpenoids, including gibberellins and phytoalexins. While many OsKSL genes have been functionally characterized, the functionality of OsKSL2 is still unclear and it has been proposed to be a pseudogene. Here, we found that OsKSL2 is drastically induced in roots by methyl jasmonate treatment and we successfully isolated a full-length cDNA for OsKSL2. Sequence analysis of the OsKSL2 cDNA revealed that the open reading frame of OsKSL2 is mispredicted in the two major rice genome databases, IRGSP-RAP and MSU-RGAP. In vitro conversion assay indicated that recombinant OsKSL2 catalyzes the cyclization of ent-CDP into ent-beyerene as a major and ent-kaurene as a minor product. ent-Beyerene is an antimicrobial compound and OsKSL2 is induced by methyl jasmonate; these data suggest that OsKSL2 is a functional ent-beyerene synthase that is involved in defense mechanisms in rice roots. PMID:25824047

  7. Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae.

    Science.gov (United States)

    Basyuni, Mohammad; Oku, Hirosuke; Tsujimoto, Etsuko; Kinjo, Kazuhiko; Baba, Shigeyuki; Takara, Kensaku

    2007-10-01

    Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves. PMID:17803686

  8. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    Science.gov (United States)

    Balabaskaran Nina, Praveen; Dudkina, Natalya V; Kane, Lesley A; van Eyk, Jennifer E; Boekema, Egbert J; Mather, Michael W; Vaidya, Akhil B

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics. PMID:20644710

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

    Science.gov (United States)

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

    2012-01-01

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

  10. Cytotoxicity of mitochondria-targeted resveratrol derivatives: interactions with respiratory chain complexes and ATP synthase.

    Science.gov (United States)

    Sassi, Nicola; Mattarei, Andrea; Azzolini, Michele; Szabo', Ildiko'; Paradisi, Cristina; Zoratti, Mario; Biasutto, Lucia

    2014-10-01

    We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low ?M range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. "Capping" free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca(2+)] "spikes" in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore. PMID:24997425

  11. Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor.

    Science.gov (United States)

    Brabham, Chad; Lei, Lei; Gu, Ying; Stork, Jozsef; Barrett, Michael; DeBolt, Seth

    2014-11-01

    Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management. PMID:25077797

  12. Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death.

    Science.gov (United States)

    Yang, C-S; Matsuura, K; Huang, N-J; Robeson, A C; Huang, B; Zhang, L; Kornbluth, S

    2015-06-01

    Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer. PMID:25151963

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

    Directory of Open Access Journals (Sweden)

    Nassar Marwa

    2006-10-01

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

  14. Kava ichthyosis: a nitric oxide synthase inhibition?

    Directory of Open Access Journals (Sweden)

    Barguil Yann

    2014-03-01

    Full Text Available Purpose: Kava is a traditional Pacific beverage made from the root of Piper methysticum. It is mainly used for its sedative properties due to lipophilic lactones called kavalactones. Various preparations or medications made from this plant can be purchased via the internet. Kava action mechanisms include cell membrane stabilisation, inhibition of intracellular Ca2+ increase and enzyme inactivation. Chronic or heavy kava consumption results in the skin taking on a scaly aspect. Biologically, an isolated increase in serum gamma-glutamyltransferase is apparent. Cases of sudden death after heavy kava sessions have occurred in Australia, and nine cases in New Caledonia were reported by our forensic laboratory during the 2000?mid-2013 period. No clear explanation has been given. We describe the possible action mechanism. Methods: We monitored 116 heavy kava drinkers. A multiple-probe drug cocktail was used on six other volunteers, all heavy and chronic kava drinkers, before and after kava abstinence to carry out CYP450 phenotyping. Results: The heavy chronic drinkers showed an isolated increase in GGT without any biological or clinical abnormality other than scaly skin. With the multiple-probe drug cocktail an inhibition of the CYP1A2 isoenzyme was demonstrated. In kava dermopathy a lack of epithelial nitric oxide production leading to an increased S-nitroso-glutathione degradation by the epithelial gamma-glutamyltransferase should be considered. Conclusions: As there are close structural and functional similarities between nitric oxide synthase (NOS and CYP1A2, and as we have formerly demonstrated that kava inhibits CYP1A2, an inhibition of NOS in chronic kava drinkers must be studied to see if ichthyosis can be explained, and if high blood GGT level is a reflection of epithelial cell GGT activity. Furthermore, a decrease in NO bioavailability can cause myocardial and vascular dysfunctions and hypercoagulability, leading to acute coronary syndrome or ischemic stroke. This mechanism should be explored in cases of “post-kava session sudden death”.

  15. The Fused TrpEG from Streptomyces Venezuelae is an Anthranilate Synthase, Not a 2-Amino-2-deoxyisochorismate (ADIC) Synthase

    Science.gov (United States)

    Ashenafi, Meseret; Carrington, Renee; Collins, Alvin C.; Byrnes, W. Malcolm

    2012-01-01

    The chloramphenicol producer Streptomyces venezuelae contains an enzyme, SvTrpEG, that has a high degree of amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE of certain species of Pseudomonas. PhzE has the sequence signature of an anthranilate synthase, but recent evidence indicates that it catalyzes the production of 2-amino-2-deoxyisochorismate (ADIC), an intermediate in the two-step anthranilate synthase reaction, not anthranilate. In order to determine if SvTrpEG is likewise an ADIC synthase, we have cloned the gene for SvTrpEG, expressed the recombinant enzyme in Escherichia coli, and purified the enzyme. Analysis of the SvTrpEG-catalyzed reaction mixture using UV-visible spectrophotometry, fluorescence spectrometry, and high-performance liquid chromatography shows that the product of the reaction is anthranilate, not ADIC. Our results therefore reveal that, despite its sequence similarity to PhzE, SvTrpEG is an anthranilate synthase, not an ADIC synthase. PMID:18646313

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

    OpenAIRE

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

    1988-01-01

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

  17. Unique rotary ATP synthase and its biological diversity.

    Science.gov (United States)

    von Ballmoos, Christoph; Cook, Gregory M; Dimroth, Peter

    2008-01-01

    F1F0 ATP synthases convert energy stored in an electrochemical gradient of H+ or Na+ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore under diverse environmental conditions. The ATP synthase of each particular cell is confronted with specific challenges, imposed by the specific environment, and thus by necessity must adapt to these conditions for optimal operation. Examples of these adaptations include diverse mechanisms for regulating the ATP hydrolysis activity of the enzyme, the utilization of different coupling ions with distinct ion binding characteristics, different ion-to-ATP ratios reflected by variations in the size of the rotor c ring, the mode of ion delivery to the binding sites, and the different contributions of the electrical and chemical gradients to the driving force. PMID:18573072

  18. Acetylcholinesterase inhibitors attenuate angiogenesis

    OpenAIRE

    Miyazaki, Ryohei; Ichiki, Toshihiro; Hashimoto, Toru; Ikeda, Jiro; Kamiharaguchi, Aya; Narabayashi, Eriko; Matsuura, Hirohide; Takeda, Kotaro; Sunagawa, Kenji

    2012-01-01

    Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimer's disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to de...

  19. Osteocompatibility of Biofilm Inhibitors

    OpenAIRE

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A.

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), f...

  20. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

    OpenAIRE

    Houghton, P.J.; Germain, G S; Hazelton, B J; Pennington, J W; Houghton, J A

    1989-01-01

    GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphat...

  1. Divergence of multimodular polyketide synthases revealed by a didomain structure

    OpenAIRE

    Zheng, Jianting; Gay, Darren C.; Demeler, Borries; White, Mark A.; Keatinge-clay, Adrian T.

    2012-01-01

    The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 Å resolution structure of the didomain comprised of the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ~600 Å2 interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do no...

  2. Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

    Science.gov (United States)

    Reichau, Sebastian; Jiao, Wanting; Walker, Scott R.; Hutton, Richard D.; Baker, Edward N.; Parker, Emily J.

    2011-01-01

    Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined. PMID:21454647

  3. Relaxation of rat thoracic aorta induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation.

    Science.gov (United States)

    Moritoki, H; Hisayama, T; Takeuchi, S; Kondoh, W; Imagawa, M

    1994-03-01

    1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), each inhibited the endothelium-dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 microM) and cyclo-oxygenase inhibitor, indomethacin (100 microM for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3-10 microM) and W-7 (100 microM) also abolished CPA-induced relaxation. 5 CPA (10 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA-stimulated increase in the levels of cyclic GMP. 7 In Ca(2+)-free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca(2+)-ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation. PMID:7517325

  4. Homology modeling and Molecular docking studies of AS1 (Anthranilate synthase component I (TrpE model of Mycobacterium tuberculos

    Directory of Open Access Journals (Sweden)

    Naresh Kumar K

    2013-07-01

    Full Text Available The emergence of multi-drug resistant (MDR strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE, whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. Anthranilate synthase is an interesting target enzyme for antimicrobial activity due to its presence in microorganisms for the synthesis of the essential amino acid tryptophan.  In the present study three compounds Cannabigerolic acid, cannabinolic acid and adhumulone from Cannabis sativa have been used for insilio docking studies.  Inhibitory studies (invitro of these compounds against Microorganism have reported earlier.  Our approach is to find out the compounds inhibiting the AS1 of MTB by insilico docking and also find out compounds having similar pharmacophore characters from ZINC database so that those compounds can be procured of synthesized in laboratory and used for AS1 inhibitor studies.  This study shows that AS can be used as a target enzyme to investigate the mode of action of our compounds in MTB.

  5. Loop residues and catalysis in OMP synthase

    DEFF Research Database (Denmark)

    Wang, Gary P.; Hansen, Michael Riis

    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 in kcat/KM for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in kcat/KM, respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of kcat, with minor effects on kcat/KM. Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. ?102?106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 104-fold reduction of kcat but almost unaltered KD values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1?-3H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1?-3H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p)]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.

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

    Science.gov (United States)

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

    1997-05-01

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

  7. Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

    International Nuclear Information System (INIS)

    The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-phosphate, is therefore an interesting target in the fight against microorganisms. In this work is described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC50 in the mmol L-1 range. (author)

  8. Downregulation of the ?-Subunit Reduces Mitochondrial ATP Synthase Levels, Alters Respiration, and Restricts Growth and Gametophyte Development in Arabidopsis[W][OA

    Science.gov (United States)

    Geisler, Daniela A.; Päpke, Carola; Obata, Toshihiro; Nunes-Nesi, Adriano; Matthes, Annemarie; Schneitz, Kay; Maximova, Eugenia; Araújo, Wagner L.; Fernie, Alisdair R.; Persson, Staffan

    2012-01-01

    The mitochondrial ATP synthase (F1Fo complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the ?-subunit links the membrane-embedded Fo part to the matrix-facing central stalk of F1. We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the ?-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the ?-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development. PMID:22805435

  9. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel (Maryland); (GWU); (Georgia)

    2012-05-24

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

  10. Endothelium-dependent vascular hyporesponsiveness without detection of nitric oxide synthase induction in aortas of cirrhotic rats.

    Science.gov (United States)

    Weigert, A L; Martin, P Y; Niederberger, M; Higa, E M; McMurtry, I F; Gines, P; Schrier, R W

    1995-12-01

    The present experiments were designed to test if induction of nitric oxide synthase (NOS) plays a role in the systemic vasodilation observed in hepatic cirrhosis. Because endotoxin levels are elevated in cirrhosis, and endotoxin stimulates inducible nitric oxide synthase (iNOS) expression in several cell lines, aortas of carbon tetrachloride-induced cirrhotic rats with ascites were evaluated for iNOS expression. Endotoxin-treated rats were studied as positive controls. Phenylephrine contraction was decreased in aortic rings with endothelium from both endotoxin-treated and cirrhotic rats as compared with controls. However, after endothelium denudation, the reduced contractility persisted in endotoxin-treated rats but disappeared in cirrhotic rats. L-Nitro-arginine-methylester (L-NAME), a nonselective inhibitor of NOS, potentiated the phenylephrine contraction of aortic rings with and without endothelium from endotoxin-treated rats but only rings with endothelium from cirrhotic rats. Moreover, aminoguanidine (AG), a preferential inhibitor of iNOS, did not affect phenylephrine contraction of rings with or without endothelium from cirrhotic rats but reversed the blunted response in endotoxin-treated rats. Northern analysis detected iNOS RNA (mRNA) expression in aortas of endotoxin-treated rats but did not detect it from cirrhotic rats. In summary, although several previous studies provide evidence for in vivo overproduction of nitric oxide in cirrhosis, the present results do not support iNOS induction as the source of nitric oxide in aortas of cirrhotic rats. Rather, because the aortic vascular hyporesponsiveness in cirrhosis is endothelium-dependent, overexpression or overstimulation of the endothelial constitutive isoform of NOS appears to be involved. PMID:7489998

  11. Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization.

    Science.gov (United States)

    Wang, Yung Lin; Chow, Sih Yao; Lin, Yi Ting; Hsieh, Yu Chiao; Lee, Guan Chiun; Liaw, Shwu Huey

    2014-12-01

    Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (?/?)8 barrel, subdomain B, a C-terminal ? domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the -1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis. PMID:25478833

  12. Biochemische Untersuchungen an der Chlorophyll Synthase aus Avena sativa

    OpenAIRE

    Schmid, Heidi

    2003-01-01

    Die Chlorophyll Synthase ist ein äußerst hydrophobes Transmembranprotein und sitzt in der Thylakoidmembran im Chloroplasten. Sie verestert Chlorophyllid und Phytylpyrophosphat zu Chlorophyll. In dieser Arbeit sind Untersuchungen zur Lage der Transmembranbereiche, zum aktiven Zentrum bzw. zu essentiellen Aminosäuren und zum Reaktionsmechanismus dargestellt.

  13. Crystallization of ?1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    International Nuclear Information System (INIS)

    ?1-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. ?1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å3 Da?1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

  14. Bacterial selenocysteine synthase--structural and functional properties.

    Science.gov (United States)

    Tormay, P; Wilting, R; Lottspeich, F; Mehta, P K; Christen, P; Böck, A

    1998-06-15

    Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur. PMID:9688279

  15. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  16. 1L-myo-inositol-1-phosphate synthase.

    Science.gov (United States)

    Majumder, A L; Johnson, M D; Henry, S A

    1997-09-01

    1L-myo-Inositol-1-phosphate synthase catalyzes the conversion of D-glucose 6-phosphate to 1L-myo-inositol-1-phosphate, the first committed step in the production of all inositol-containing compounds, including phospholipids, either directly or by salvage. The enzyme exists in a cytoplasmic form in a wide range of plants, animals, and fungi. It has also been detected in several bacteria and a chloroplast form is observed in alga and higher plants. The enzyme has been purified from a wide range of organisms and its active form is a multimer of identical subunits ranging in molecular weight from 58,000 to 67,000. The activity of the synthase is stimulated by NH4Cl and inhibited by glucitol 6-phosphate and 2-deoxyglucose 6-phosphate. Structural genes (INO1) encoding the 1L-myo-inositol-1-phosphate synthase subunit have been isolated from several eukaryotic microorganisms and higher plants. In baker's yeast, Saccharomyces cerevisiae, the transcriptional regulation of the INO1 gene has been studied in detail and its expression is sensitive to the availability of phospholipid precursors as well as growth phase. The regulation of the structural gene encoding 1L-myo-inositol-1-phosphate synthase has also been analyzed at the transcriptional level in the aquatic angiosperm, Spirodela polyrrhiza and the halophyte, Mesembryanthemum crystallinum. PMID:9370339

  17. Neuronal nitric oxide synthase supports Renin release during sodium restriction through inhibition of phosphodiesterase 3

    DEFF Research Database (Denmark)

    Sällström, Johan; Jensen, Boye L

    2010-01-01

    BACKGROUND: Mice with targeted deletion of neuronal nitric oxide (NO) synthase (nNOS?(/)?) display inability to increase plasma renin concentration (PRC) in response to sodium restriction. nNOS has a distinct expression at the macula densa (MD), and in the present study, it was tested whether nNOS supports renin release by cyclic guanosine monophosphate (cGMP)-mediated inhibition of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase 3 (PDE3) in juxtaglomerular (JG) cells. METHODS: The experiments were performed in conscious nNOS?(/)? and wild types after 10 days on a low-sodium diet by acute treatment with the PDE3-inhibitor milrinone, the PDE5 inhibitor zaprinast, or vehicle, using a crossover study protocol. PRC was measured with the antibody-trapping technique and blood pressure with telemetry. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were estimated by measurements of inulin- and para-amino hippuric acid (PAH) clearances, respectively. RESULTS: The basal PRC was reduced innNOS?(/)? compared to the wild types. Administration of milrinone caused a more pronounced PRC increase in nNOS?(/)?, resulting in normalized renin levels, whereas PDE5 inhibition did not affect PRC in any genotype. The blood pressure was similar in both genotypes, and milrinone did not affect blood pressure compared to vehicle. GFR and RPF were similar at baseline and were reduced by milrinone. CONCLUSIONS: The present study provides in vivo evidence supporting the view that NO, selectively derived from nNOS, mediates renin release during sodium restriction by inhibiting PDE3, which would increase renin release by elevating cAMP levels in the JG cells.

  18. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    LENUS (Irish Health Repository)

    Cathcart, Mary-Clare

    2012-02-01

    BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced\\/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC.

  19. Allicin protects rat cortical neurons against mechanical trauma injury by regulating nitric oxide synthase pathways.

    Science.gov (United States)

    Zhou, Yue-fei; Li, Wen-tao; Han, Hong-cheng; Gao, Da-kuan; He, Xiao-sheng; Li, Liang; Song, Jin-ning; Fei, Zhou

    2014-01-01

    Allicin, a small molecule that is responsible for the typical smell and most of the functions of garlic, possesses a broad spectrum of pharmacological activities and is considered to have therapeutic potential in many pathologic conditions. In the present study, we investigated the potential protective effect of allicin in an in vitro model of traumatic brain injury (TBI) using primary cultured rat cortical neurons. We found that allicin treatment significantly reduced mechanical trauma-induced lactate dehydrogenase (LDH) release and inhibited apoptotic neuronal death in a dose-dependent manner. These protective effects were observed even if allicin treatment was delayed to 2h after injury. Allicin significantly decreased the expression of inducible nitric oxide synthase (iNOS) and increased the phosphorylation of endothelial NOS (eNOS) but had no effect on neuronal NOS (nNOS) expression. Allicin-induced protection in cortical neurons was augmented by iNOS and nNOS antagonists and was partly reversed by blocking eNOS phosphorylation. In addition, allicin treatment inhibited the TBI-induced activation of ERK and further enhanced the phosphorylation of Akt in TBI-injured neurons. The Akt inhibitor LY294002 attenuated the allicin-induced increase in eNOS expression and phosphorylation, whereas the ERK inhibitor PD98059 had opposite effects on the expression of iNOS and eNOS. Pretreatment with LY294002 or PD98059 partly prevented or further enhanced allicin-induced neuroprotection, respectively. Collectively, these data demonstrate that allicin treatment may be an effective therapeutic strategy for traumatic neuronal injury and that the potential underlying mechanism involves Akt- and ERK-mediated regulation of NOS pathways. PMID:24184006

  20. Inhibition of glycogen synthase kinase-3 reduces L-DOPA-induced neurotoxicity

    International Nuclear Information System (INIS)

    The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), used for the treatment of Parkinson's disease, remains controversial. Although there are many reports suggesting that long-term treatment of L-DOPA causes neuronal death, an increasing body of recent evidence has proposed that L-DOPA might be neuroprotective rather than neurotoxic. We investigated the effect of L-DOPA on neuronally differentiated PC12 (nPC12) cells by treating cells with various concentrations of L-DOPA for 24 h. We also studied whether glycogen synthase kinase (GSK)-3 activation is related to L-DOPA-induced neurotoxicity by simultaneously treating cells with several concentrations of L-DOPA and a GSK-3 inhibitor for 24 h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, cell counting kit-8, and DAPI staining all showed that L-DOPA decreased nPC12 cell viability at high concentrations. In addition, 100 ?M L-DOPA treatment significantly increased the activity of GSK-3 and death signals including cytochrome c, activated caspase-3 and cleaved PARP, and decreased survival signals including heat shock transcription factor-1 in a concentration-dependent manner. Treatment with GSK-3 inhibitor VIII or lithium chloride prevented L-DOPA-induced cell death. Together, these results suggest that L-DOPA induces neuronal cell death at high concentrations and that the neurotoxic effect of L-DOPA might be mediated in part by GSK-3 activation by GSK-3 activation

  1. Effects of nitric oxide synthase inhibition on sympathetically-mediated tachycardia

    Science.gov (United States)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aim of the present study was to determine whether inhibition of nitric oxide (NO) synthesis directly alters the tachycardia produced by sympathetically-derived norepinephrine. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 50 micromol/kg, i.v.), produced a marked rise in mean arterial blood pressure. This pressor response was associated with a fall in heart rate which involved the withdrawal of cardiac sympathetic nerve activity. The NO-donor, sodium nitroprusside (5 microg/kg, i.v.), produced a pronounced fall in mean arterial blood pressure but only a minor increase in heart rate. The beta-adrenoceptor agonist, isoproterenol (0.5 micromol/kg, i.v.), and the membrane-permeable cAMP analogue, 8-(4-chlorophenylthiol)-cAMP (10 micromol/kg, i.v.), produced falls in mean arterial blood pressure and pronounced increases in heart rate. The indirectly acting sympathomimetic agent, tyramine (0.5 mg/kg, i.v.), produced a pressor response and a tachycardia. The effects of sodium nitroprusside, tyramine, isoproterenol and 8-(4-chlorophenylthiol)-cAMP on mean arterial blood pressure were not markedly affected by L-NAME. However, the tachycardia produced by these agents was considerably exaggerated in the presence of this NO synthesis inhibitor. These findings suggest that L-NAME potentiates the tachycardia produced by sympathetically-derived norepinephrine. The increased responsiveness to norepinephrine may involve (i) a rapid up-regulation of cardiac beta1-adrenoceptors and cAMP signaling in cardiac pacemaker cells due to the loss of the inhibitory influence of cardiac NO, and (ii) the up-regulation of beta1-adrenoceptor-mediated signal transduction processes in response to the L-NAME-induced withdrawal of cardiac sympathetic nerve activity.

  2. Toxicity of checkpoint inhibitors.

    Science.gov (United States)

    Schmerling, Rafael Aron

    2014-09-01

    Immunologic checkpoint inhibitor is a new class of antineoplastic drugs which mechanism of action depends on the interaction with the immune system. The first in class was ipilimumab, anti-CTLA4, and soon the anti-PD1 (Programmed Death 1) and anti-PDL1 are expected to be available. Although the profile of adverse events is unique, they are predictable and, by complying with the guidelines available, the management of these drugs is safe in the great majority of patients. Here, it is provided a review of adverse events and their management. PMID:25841457

  3. ACE INHIBITORS: A COMPREHENSIVE REVIEW

    Directory of Open Access Journals (Sweden)

    Pradeep Kumar Arora* and Ashish Chauhan

    2013-02-01

    Full Text Available Hypertension is a chronic increase in blood pressure, characterized as primary and secondary hypertension. The disorder is associated with various risk factors like obesity, diabetes, age, lack of exercise etc. Hypertension is being treated since ancient times by Ayurvedic, Chinese and Unani medicine. Now various allopathic drugs are available which include diuretics, calcium channel blockers, ?-blockers, ?-blockers, vasodilators, central sympatholytics and ACE-inhibitors. Non-pharmacological treatments include weight reduction, dietary sodium reduction, increased potassium intake and reduction in alcohol consumption. ACE-inhibitors are widely used in the treatment of hypertension by inhibiting the angiotensin converting enzyme responsible for the conversion of angiotensin I to angiotensin II (responsible for vasoconstriction. Various structure activity relationship studies led to the synthesis of ACE-inhibitors, some are under clinical development. This comprehensive review gives various guidelines on classification of hypertension, hypertension therapy including ancient, pharmacological, non-pharmacological therapies, pharmacoeconomics, historical perspectives of ACE, renin, renin angiotensin system (circulating vs local RAS, mechanism of ACE inhibitors, and development of ACE inhibitors. Review also emphasizes on the recent advancements on ACE inhibitors including drugs in clinical trials, computational studies on ACE-inhibitors, peptidomimetics, dual, natural, multi-functional ACE inhibitors, and conformational requirements for ACE-inhibitors.

  4. Glycogen metabolic genes are involved in trehalose-6-phosphate synthase-mediated regulation of pathogenicity by the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Badaruddin, Muhammad; Holcombe, Lucy J; Wilson, Richard A; Wang, Zheng-Yi; Kershaw, Michael J; Talbot, Nicholas J

    2013-01-01

    The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a ?agl1?gph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae. PMID:24098112

  5. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Daijun Zhou, Qiang Mei, Han Luo, Bo Tang, Peiwu Yu

    2012-01-01

    Full Text Available Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC, but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls, 24(8,263 cases and 12,033 controls, 12(3,758 cases and 5,646 controls, and 13 (5,511 cases and 7,265 controls studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR C677T and A1289C, methione synthase reductase (MTRR A66G, methionine synthase (MTR A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96, while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18. Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89 and Caucasians (OR = 0.84, 95%CI 0.76-0.93 in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36. No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results.

  6. Mechanism-oriented redesign of an isomaltulose synthase to an isomelezitose synthase by site-directed mutagenesis.

    Science.gov (United States)

    Görl, Julian; Timm, Malte; Seibel, Jürgen

    2012-01-01

    An isomelezitose synthase was redesigned out of the sucrose isomerase from Protaminobacter rubrum for the synthesis of isomelezitose (6-O(F)-glucosylsucrose), a potential nutraceutical. The variants F297A, F297P, R333K, F321A_F319A and E428D catalyze the formation of isomelezitose in up to 70 % yield. PMID:22124943

  7. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1?3),(1?4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  8. Wound-induced ethylene synthesis and expression and formation of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia-lyase, and peroxidase in wounded mesocarp tissue of Cucurbita maxima.

    Science.gov (United States)

    Kato, M; Hayakawa, Y; Hyodo, H; Ikoma, Y; Yano, M

    2000-04-01

    1-Aminocyclopropane-1-carboxylate (ACC) synthase was rapidly induced in mesocarp tissue of Cucurbita maxima after wounding in the cut surface layer in 1 mm thickness (ca. 9 cells) (first layer) in both the enzyme activity and the levels of transcript. This led to a rapid accumulation of ACC and hence ethylene production. In the inside tissue (1-2 mm) (second layer), no significant induction of ACC synthase was observed, which resulted in a low level of ACC, although ethylene was evolved at a much lower rate than the first one. In contrast to ACC synthase, ACC oxidase was induced markedly in both the first and second layers and the development of its activity and the levels of mRNA remained high until later stages. It was considered that wound ethylene was closely associated with the development of ACC oxidase, since 2,5-norbornadiene (NBD), an inhibitor of ethylene action, substantially suppressed it. Phenylalanine ammonia-lyase (PAL) greatly increased in activity after wounding similarly to that of ACC synthase, in which increase in PAL activity occurred predominantly in the first layer. Induction of peroxidase activity after wounding had a close correlation in profile with that of ACC oxidase in that marked increases in the activity were observed in both the first and second layers and were strongly suppressed by NBD application. Four peroxidase isozymes were found by PAGE, among which a fraction was newly detected after wounding. PMID:10845457

  9. Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera.

    Science.gov (United States)

    Ferriols, Victor Marco Emmanuel N; Yaginuma, Ryoko; Adachi, Masao; Takada, Kentaro; Matsunaga, Shigeki; Okada, Shigeru

    2015-01-01

    The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom. PMID:25996801

  10. Role of Glycogen Synthase Kinase-3? in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Xanthi Antoniou

    2010-01-01

    Full Text Available The phosphorylation of Amyloid Precursor Protein (APP at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK, glycogen synthase kinase-3? (GSK-3? and cyclin-dependent kinase 5 (Cdk5 can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 ?M for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3? activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3? reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3? signaling pathway.

  11. Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bulfer, Stacie L.; Scott, Erin M.; Couture, Jean-François; Pillus, Lorraine; Trievel, Raymond C.; (Michigan); (UCSD)

    2010-01-12

    Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed {alpha}/{beta} topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

  12. Cardiac myocyte neuronal nitric oxide synthase. New therapeutic target in heart failure?

    Science.gov (United States)

    Loyer, X; Samuel, J L; Heymes, C

    2005-12-01

    Although nitric oxide-dependent regulation of contractile function is altered in the diseased and failing heart, several aspects of nitric oxide (NO) signalling in the myocardium remain poorly understood. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NO synthase isoforms (NOS) as compared to the use of mouse models genetically deficient or overexpressing the NOS thought to be responsible for the increase in NO production in heart failure (mainly NOS2 and NOS3). In recent years, identification of the neuronal NOS (NOS1) isoform in cardiac myocytes and the recognition of the importance of its subcellular localisation have greatly advanced the understanding of the critical role of NOS1-derived NO in the control of myocardial contractility both in the normal and failing heart. The challenge is now to confirm these emerging findings on the critical role of NOS1-derived NO in human cardiac physiology and hopefully translate them into therapy. PMID:16435605

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

    Science.gov (United States)

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

    2015-01-01

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

  14. Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

    DEFF Research Database (Denmark)

    Graebe, Martin; Brond, Lone

    2004-01-01

    The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that l-NAME treatment dose dependently deteriorated cumulative sodium balance by reducing urinary sodium excretion. Five weeks after CBL, renal clearance studies were performed, followed by Western blotting of the electroneutral type 3 sodium/proton exchanger (NHE3) and the Na-K-ATPase present in proximal tubules. Untreated CBL rats showed a decreased proximal reabsorption with a concomitant reduction of NHE3 and Na-K-ATPase levels, indicating that tubular segments distal to the proximal tubules were responsible for the increased sodium reabsorption. l-NAME-treated CBL rats showed an increased proximal reabsorption measured by the lithium clearance method and showed a marked increase in NHE3 and Na-K-ATPase protein levels. Our results show that chronic l-NAME treatment exacerbates the sodium retention found in CBL rats by a significant increase in proximal tubular reabsorption.

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Effect of tetrahydrobiopterin on nitric oxide synthase-containing cells in the rat hippocampus.

    Science.gov (United States)

    Koshimura, Kunio; Murakami, Yoshio; Tanaka, Junko; Yamamoto, Masahiro; Kato, Yuzuru

    2004-10-01

    We have observed that tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS), acts as a self-protection factor against nitric oxide (NO) toxicity in PC12 cells. To further investigate the self-protection action of BH4 in vivo, the effect of deletion of endogenous BH4 on NO-producing cells was examined in the rat hippocampus. Following the peripheral infusion of 50 mM 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, using a miniosmotic pump for 14 days, BH4 content in the hippocampus decreased as compared with the control group administered with vehicle solution, which had no effect on brain BH4 content. When the rats were administered with 50 mM DAHP and 10 mM BH4, the DAHP-induced decrease in BH4 content was prevented. The extracellular concentration of NO metabolites remained unchanged following DAHP administration, suggesting that DAHP-induced decrease in BH4 content had no effect on NO production. The number of NOS-positive cells decreased following DAHP administration in the hippocampal regions, while the number of NOS-negative cells remained unchanged. The DAHP-induced decrease in the NOS-positive cell number was prevented by the administration of 10 mM BH4 and DAHP. These results suggest that endogenous BH4 may affect NOS-positive cell number in the rat hippocampus. PMID:15380323

  17. 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.

  18. Modeling studies with Helicobacter pylori octaprenyl pyrophosphate synthase reveal the enzymatic mechanism of trans-prenyltransferases.

    Science.gov (United States)

    Zhang, Jinyong; Zhang, Xiaoli; Zhang, Rui; Wu, Chao; Guo, Ying; Mao, Xuhu; Guo, Gang; Zhang, Ying; Wang, Da-Cheng; Li, Defeng; Zou, Quanming

    2012-12-01

    Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8 ? resolution and refined to 2.0 ? resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg(2+) mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP. PMID:22982238

  19. Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress

    Directory of Open Access Journals (Sweden)

    Roberta Venè

    2014-09-01

    Full Text Available Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenylretinamide (4HPR, the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11-dien-28-oic acid methyl ester, arsenic trioxide (As2O3, and phenylethyl isothiocyanate (PEITC, while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3? (GSK3?. The antioxidant N-acetylcysteine decreased GSK3? phosphorylation and poly(ADP-ribose polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3? phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1, and transient elevation of intracellular glutathione (GSH in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3? or transfection with the non-phosphorylatable GSK3?-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3? phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3? phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance.

  20. Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.

    Science.gov (United States)

    Dodbele, Samantha; Martinez, Christina D; Troutman, Jerry M

    2014-08-01

    Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes. PMID:25020247

  1. Inhibitory effect of organotin compounds on rat neuronal nitric oxide synthase through interaction with calmodulin

    International Nuclear Information System (INIS)

    Organotin compounds, triphenyltin (TPT), tributyltin, dibutyltin, and monobutyltin (MBT), showed potent inhibitory effects on both L-arginine oxidation to nitric oxide and L-citrulline, and cytochrome c reduction catalyzed by recombinant rat neuronal nitric oxide synthase (nNOS). The two inhibitory effects were almost parallel. MBT and TPT showed the highest inhibitory effects, followed by tributyltin and dibutyltin; TPT and MBT showed inhibition constant (IC50) values of around 10 ?M. Cytochrome c reduction activity was markedly decreased by removal of calmodulin (CaM) from the complete mixture, and the decrease was similar to the extent of inhibition by TPT and MBT. The inhibitory effect of MBT on the cytochrome c reducing activity was rapidly attenuated upon dilution of the inhibitor, and addition of a high concentration of CaM reactivated the cytochrome c reduction activity inhibited by MBT. However, other cofactors such as FAD, FMN or tetrahydrobiopterin had no such ability. The inhibitory effect of organotin compounds (100 ?M) on L-arginine oxidation of nNOS almost vanished when the amount of CaM was sufficiently increased (150-300 ?M). It was confirmed by CaM-agarose column chromatography that the dissociation of nNOS-CaM complex was induced by organotin compounds. These results indicate that organotin compounds disturb the interaction between CaM and nNOS, thereby inhibiting electron transfer from the reductase domain to cytochrome c and the oxygase domain to cytochrome c and the oxygenase domain

  2. 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.

  3. Theaflavins inhibit the ATP synthase and the respiratory chain without increasing superoxide production.

    Science.gov (United States)

    Li, Bo; Vik, Steven B; Tu, Youying

    2012-08-01

    Four dietary polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (TF3), have been isolated from black tea, and their effects on oxidative phosphorylation and superoxide production in a model system (Escherichia coli) have been examined. The esterified theaflavins were all potent inhibitors of the membrane-bound adenosine triphosphate (ATP) synthase, inhibiting at least 90% of the activity, with IC(50) values in the range of 10-20 ?M. ATP-driven proton translocation was inhibited in a similar fashion, as was the purified F(1)-ATPase, indicating that the primary site of inhibition was in the F(1) sector. Computer modeling studies supported this interpretation. All four theaflavins were also inhibitory towards the electron transport chain, whether through complex I (NDH-1) or the alternative NADH dehydrogenase (NDH-2). Inhibition of NDH-1 by TF3 appeared to be competitive with respect to NADH, and this was supported by computer modeling studies. Rates of superoxide production during NADH oxidation by each dehydrogenase were measured. Superoxide production was completely eliminated in the presence of about 15 ?M TF3, suggesting that inhibition of the respiratory chain by theaflavins does not contribute to superoxide production. PMID:21924889

  4. Glycogen Synthase Kinase 3? Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

    Directory of Open Access Journals (Sweden)

    Liang Ma

    2013-08-01

    Full Text Available Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3? (GSK3?, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3? inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3? by either lithium chloride (LiCl or specific GSK3? inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952 and type II (ARK1 endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3? activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  5. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    OpenAIRE

    C?olovic?, Mirjana B.; Krstic?, Danijela Z.; Lazarevic?-pas?ti, Tamara D.; Bondz?ic?, Aleksandra M.; Vasic?, Vesna M.

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are appl...

  6. Proteinaceous alpha-araylase inhibitors.

    DEFF Research Database (Denmark)

    Svensson, Birte; Fukuda, Kenji

    2004-01-01

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous a-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function.

  7. Proteinase inhibitors in Brazilian leguminosae

    Directory of Open Access Journals (Sweden)

    C. A. M. Sampaio

    1991-01-01

    Full Text Available Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil, were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000, Torresea cearensis (Mr = 13,000, Bauhinia pentandra (Mr = 20,000 and Bauhinia bauhinioides (Mr = 20,000. E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

  8. Arabidopsis transcriptional responses differentiating closely related chemicals (herbicides) and cross-species extrapolation to Brassica

    Science.gov (United States)

    Using whole genome Affymetrix ATH1 GeneChips we characterized the transcriptional response of Arabidopsis thaliana Columbia 24 hours after treatment with five different herbicides. Four of them (chloransulam, imazapyr, primisulfuron, sulfometuron) inhibit acetolactate synthase (A...

  9. A Common Genetic Basis for Cross-Sensitivity to Mesotrione and Nicosulfuron in Sweet Corn Hybrid Cultivars and Inbreds Grown Throughout North America

    Science.gov (United States)

    In previous research, the sweet corn inbred line Cr1 was observed to be sensitive to multiple postemergence herbicides, including four acetolactate synthase (ALS)-inhibiting herbicides, three 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides, a growth regulator herbicide combination, ...

  10. Sirtuin activators and inhibitors.

    Science.gov (United States)

    Villalba, José M; Alcaín, Francisco J

    2012-01-01

    Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD(+) for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection, and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activities. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex(®) , SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. PMID:22730114

  11. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    Energy Technology Data Exchange (ETDEWEB)

    Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  12. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario (UIUC); (JHU-MED)

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

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

    Science.gov (United States)

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

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in trigg