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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

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

    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

  3. Plant availability and phytotoxicity of soil bound residues of herbicide ZJ0273, a novel acetolactate synthase potential inhibitor.

    Han, Ailiang; Yue, Ling; Li, Zheng; Wang, Haiyan; Wang, Yue; Ye, Qingfu; Lu, Long; Gan, Jay

    2009-11-01

    The plant availability and phytotoxicity of soil bound residues (BR) of herbicide ZJ0273, a novel acetolactate synthase (ALS) potential inhibitor, to rice (Oryza sativa L.) and corn (Zea mays L.) was investigated in three different soils including a Fluvio-marine yellow loamy soil (S(1)), a Red clayey soil (S(2)), and a Coastal saline soil (S(3)), using (14)C-labeling tracer and bioassay techniques. When soils were amended with BR at 0.6, 1.2 and 1.8 nmol g(-1), dose-dependent and significant inhibition was observed for rice seedlings within 14d after treatment, but no significant inhibition occurred to corn seedlings in the same treatment. Radioactive analysis of soil extracts following sequential extractions showed that the (14)C labeled residues of ZJ0273 were released from the amended soil BR upon planting. For example, when amended with 1.8 nmol g(-1), about 68.3%, 57.0%, and 61.1%, respectively, of the added BR were released in S(1), S(2), and S(3) planted with rice seedlings, whereas 38.9%, 32.7% and 32.6% became available for uptake in the corresponding soils planted with corn seedlings. The released compounds were identified as ZJ0273 and its degradation products M1 and M2, with M2 as the primary component. Bioassay on rice showed that concentration for 50% inhibition (IC(50)) of ZJ0273, M1, and M2 were 33.16, 1.93 and 0.49 microM, respectively. Therefore, BR formed after application of ZJ0273 may become available for plant uptake during rice cultivation and lead to phytotoxic effects, and the phytotoxicity is mainly caused by the release of the biologically active metabolite M2. This knowledge is valuable for designing crop rotation practices so that crop injury and yield losses due to carry-over herbicide phytotoxicity may be avoided. PMID:19732936

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

    Muhitch, Michael J.

    1988-01-01

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

  5. Transformation of carrots with mutant acetolactate synthase for Orobanche (broomrape) control.

    Aviv, Dvora; Amsellem, Ziva; Gressel, Jonathan

    2002-12-01

    Parasitic Orobanche spp are major constraints to vegetable crop production in the Mediterranean basin (to eastern Europe) and in localized places in India, China and the USA. Transgenic target-site herbicide resistance (eg, to acetolactate synthase inhibitors) allows for movement of unmetabolized herbicide through the crop to the photosynthate sink in the parasite, as well as through the soil. We report the successful engineering of a mutant acetolactate synthase (ALS) gene into carrot, allowing control of broomrape already in heterozygotes of the first back-crossed generation, by imazapyr, an imidazolinone ALS inhibitor. It is expected that homozygotes will have higher levels of resistance. PMID:12476991

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

    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...

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

    Annemarie C. Van Wely; Nader Soltani; Darren E. Robinson; David C. Hooker; Mark B. Lawton; Sikkema, Peter H.

    2014-01-01

    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 year...

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

    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.

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

    D.L.P. Gazziero

    2003-08-01

    Full Text Available A extenso das reas com seleo de populaes de plantas daninhas resistentes a herbicidas tem aumentado rapidamente no Brasil nos ltimos anos, sendo citado como causa principal desta seleo a recomendao inadequada de produtos. Com o objetivo de avaliar a eficcia de controle de plantas daninhas atravs de herbicidas, com diferentes mecanismos de ao, sobre plantas de Bidens subalternans, foi conduzido o presente trabalho, que envolveu um experimento de casa de vegetao e dois de campo, com as culturas de milho e soja. A pesquisa foi realizada a partir de populaes de plantas de Bidens subalternans com suspeita de resistncia aos herbicidas inibidores da ALS encontradas em rea de produo comercial nas quais ocorriam falhas de controle atravs desses herbicidas. Os resultados permitiram confirmar a seleo de populaes resistentes aos herbicidas inibidores da acetolactato sintase (ALS e encontrar alternativas para o manejo destas populaes, por meio do uso de produtos com mecanismo de ao diferenciado, tanto para a cultura da soja quanto para a do milho. Produtos inibidores da protoporfirinognio oxidase (PROTOX, da fotossntese e da diviso celular, aplicados isoladamente ou em misturas, controlaram adequadamente o bitipo 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. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    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.

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

    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

  12. Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death.

    Zhao, Lun; Jing, Xue; Chen, Li; Liu, Yingjun; Su, Yanan; Liu, Tingting; Gao, Changbin; Yi, Bin; Wen, Jing; Ma, Chaozhi; Tu, Jinxing; Zou, Jitao; Fu, Tingdong; Shen, Jinxiong

    2015-12-01

    Tribenuron-methyl (TM) is a powerful sulfonylurea herbicide that inhibits branched-chain amino acid (BCAA) biosynthesis by targeting the catalytic subunit (CSR1) of acetolactate synthase (ALS). Selective induction of male sterility by foliar spraying of TM at low doses has been widely used for hybrid seed production in rapeseed (Brassica napus); however, the underlying mechanism remains unknown. Here, we report greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems after TM application. Constitutive or anther-specific expression of csr1-1D (a CSR1 mutant) eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis. The results of TM daub-stem experiments, combined with the observations of little TM accumulation in anthers and reversion of TM-induced male sterility by targeted expression of the TM metabolism gene Bel in either the mesophyll or phloem, suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem. Microscopy and immunoblotting revealed that autophagy, a bulk degradation process induced during cell death, was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS. Moreover, TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA. These data suggested that TM was polar-transported to anthers, resulting in BCAA starvation via anther-specific ALS inhibition and, ultimately, autophagic cell death in anthers. PMID:26362932

  13. Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms 1

    Christopher, John T.; Powles, Stephen B.; Holtum, Joseph A. M.

    1992-01-01

    WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-14C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS. PMID:16653216

  14. Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis.

    Endo, Masaki; Osakabe, Keishi; Ichikawa, Hiroaki; Toki, Seiichi

    2006-03-01

    Precise modification of plant genomes via gene targeting (GT) is important for the study of gene function in vivo. A reliable GT system using the protoporphyrinogen oxidase (PPO) gene in Arabidopsis was reported 4 years ago; however, there are no subsequent successful reports of GT in Arabidopsis. A previous study showed ectopic gene targeting (EGT) of the endogenous gene in two-thirds of GT plants, which was an obstacle to efficient true gene targeting (TGT). The endogenous acetolactate synthase (ALS) gene is involved in the biosynthesis of branched chain amino acids in plants and is the site of action of several herbicides. To confirm the generality of the GT system in Arabidopsis, and to characterize the EGT event in plants in detail, we converted ALS from a herbicide (imazapyr)-susceptible to a -resistant form by GT. We obtained two imazapyr-resistant plants following GT. One of the targeting events was TGT while the other was EGT. After detailed Southern blotting, PCR and nucleotide sequence analysis of the EGT plant, we determined the genomic position and structure of the ectopically targeted site. Based on our findings, we discuss the possible mechanisms of EGT in plants. PMID:16418231

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

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

    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. Averaged ov...

  16. Occurrence, genetic control and evolution of non-target-site based resistance to herbicides inhibiting acetolactate synthase (ALS) in the dicot weed Papaver rhoeas.

    Scarabel, Laura; Pernin, Fanny; Dlye, Christophe

    2015-09-01

    Non-target-site resistance (NTSR) to herbicides is a major issue for the chemical control of weeds. Whilst predominant in grass weeds, NTSR remains largely uninvestigated in dicot weeds. We investigated the occurrence, inheritance and genetic control of NTSR to acetolactate synthase (ALS) inhibitors in Papaver rhoeas (corn poppy) using progenies from plants with potential NTSR to the imidazolinone herbicide imazamox. NTSR to imazamox was inherited from parents over two successive generations. NTSR to tritosulfuron (a sulfonylurea) was observed in F1 generations and inherited in F2 generations. NTSR to florasulam (a triazolopyrimidine) emerged in F2 generations. Our findings suggest NTSR was polygenic and gradually built-up by accumulation over generations of loci with moderate individual effects in single plants. We also demonstrated that ALS alleles conferring herbicide resistance can co-exist with NTSR loci in P. rhoeas plants. Previous research focussed on TSR in P. rhoeas, which most likely caused underestimation of NTSR significance in this species. This may also apply to other dicot species. From our data, resistance to ALS inhibitors in P. rhoeas appears complex, and involves well-known mutant ALS alleles and a set of unknown NTSR loci that confer resistance to ALS inhibitors from different chemical families. PMID:26259184

  17. Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

    Mathesius, C A; Barnett, J F; Cressman, R F; Ding, J; Carpenter, C; Ladics, G S; Schmidt, J; Layton, R J; Zhang, J X Q; Appenzeller, L M; Carlson, G; Ballou, S; Delaney, B

    2009-12-01

    Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology. PMID:19682528

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

    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 Ala₁₂₂ to Val and Ala₁₂₂ 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 ¹⁴C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. ¹⁴C-Penoxsulam translocation out of the treated leaf (≤2%) was similar among all biotypes. ¹⁴C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. ¹⁴C-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

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

    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...

  20. Herbicide resistance in Aster squamatus conferred by a less sensitive form of acetolactate synthase.

    Osuna, Maria D; Fischer, Albert J; De Prado, Rafael

    2003-11-01

    A biotype of Aster squamatus (Sprengel) Hieronymus with suspected resistance to the ALS-inhibiting herbicide imazapyr was detected in a chicken farm in the province of Seville, Spain, which had been treated once a year with imazapyr for 10 years. Resistance to imazapyr in this biotype was studied using dose-response experiments, absorption and translocation assays, metabolism studies and ALS activity assays. The rate of imazapyr required to inhibit A squamatus growth by 50% (ED50) was 15 times higher for the R (resistant) than for the S (susceptible) biotype. Cross-resistance existed for the ALS-inhibitors imazamox, imazethapyr, amidosulfuron, nicosulfuron, rimsulfuron, triasulfuron and tribenuron, but not for bensulfuron. Control of A squamatus using alternative herbicides was poor with clopyralid, intermediate with quinclorac, amitrole and MCPA, and excellent with 2,4-D, glufosinate and glyphosate. Absorption of [14C]imazapyr increased over time for both the R and S biotypes, and translocation from the treated leaf to shoots and roots was similar in both biotypes, with most of the radioactivity remaining in the treated leaf. No metabolites of imazapyr were detected in either biotype. Sensitivity of the ALS enzyme (target site) to imazapyr was lower for the R biotype (I50(R) = 4.28 x I50(S)). The mechanism of imazapyr resistance in this R biotype appears to be an altered ALS conferring decreased sensitivity to imazapyr at the whole-plant level. PMID:14620047

  1. Evolution of herbicide resistance in weeds: initial frequency of target site-based resistance to acetolactate synthase-inhibiting herbicides in Lolium rigidum.

    Preston, C; Powles, S B

    2002-01-01

    The frequency of individuals resistant to two acetolactate synthase (ALS)-inhibiting herbicides in three previously untreated populations of Lolium rigidum was determined. The frequency of individuals resistant to the sulfonylurea herbicide sulfometuron-methyl varied from 2.2 x 10(-5) to 1.2 x 10(-4) and the frequency of individuals resistant to the imidazolinone herbicide imazapyr varied from 1 x 10(-5) to 5.8 x 10(-5) depending on the population. Application of sulfometuron-methyl selected individuals with a herbicide-insensitive ALS, which was also cross-resistant to imazapyr. The high initial frequency of individuals resistant to ALS-inhibiting herbicides in L. rigidumpopulations never previously exposed to these herbicides helps explain the rapid evolution of herbicide resistance in this species once ALS-inhibiting herbicides were used. PMID:11813100

  2. Temperature-dependent acetoin production by Pyrococcus furiosus is catalyzed by a biosynthetic acetolactate synthase and its deletion improves ethanol production.

    Nguyen, Diep M N; Lipscomb, Gina L; Schut, Gerrit J; Vaccaro, Brian J; Basen, Mirko; Kelly, Robert M; Adams, Michael W W

    2016-03-01

    The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100°C by fermenting sugars to acetate, carbon dioxide and molecular hydrogen as the major end products. The organism has recently been exploited to produce biofuels using a temperature-dependent metabolic switch using genes from microorganisms that grow near 70°C. However, little is known about its metabolism at the lower temperatures. We show here that P. furiosus produces acetoin (3-hydroxybutanone) as a major product at temperatures below 80°C. A novel type of acetolactate synthase (ALS), which is involved in branched-chain amino acid biosynthesis, is responsible and deletion of the als gene abolishes acetoin production. Accordingly, deletion of als in a strain of P. furiosus containing a novel pathway for ethanol production significantly improved the yield of ethanol. These results also demonstrate that P. furiosus is a potential platform for the biological production of acetoin at temperatures in the 70-80°C range. PMID:26721637

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

    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

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

    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

  5. Farnesyl diphosphate synthase inhibitors with unique ligand-binding geometries.

    Liu, Yi-Liang; Cao, Rong; Wang, Yang; Oldfield, Eric

    2015-03-12

    Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis. PMID:25815158

  6. Protein inhibitor of neuronal nitric oxide synthase interacts with protein kinase A inhibitors.

    Yu, Jianqiang; Yu, Long; Chen, Zheng; Zheng, Lihua; Chen, Xiaosong; Wang, Xiang; Ren, Daming; Zhao, Shouyuan

    2002-03-28

    Protein kinase A (PKA) and neuronal nitric oxide synthase (nNOS) are important signaling molecules. It is well known that PKA can specifically phosphorylate nNOS. But the underlying molecular mechanism is still obscure. Our data indicate that the protein inhibitor of nNOS (PIN) binds to protein kinase A inhibitors (PKIs), which suggests that PKIs, together with PIN, might mediate the phosphorylation of nNOS by PKA. PMID:11978406

  7. Fatty acid synthase inhibitors isolated from Punica granatum L

    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)

  8. Fatty acid synthase inhibitors isolated from Punica granatum L

    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)

  9. Glucosylceramide synthase inhibitors differentially affect expression of glycosphingolipids.

    Alam, Shahidul; Fedier, André; Kohler, Reto S; Jacob, Francis

    2015-04-01

    Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration. PMID:25715344

  10. 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

    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.

  11. Biochemistry: Acetohydroxyacid Synthase

    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.

  12. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    Hsueh-Fen Juan

    2011-08-01

    Full Text Available Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitordesigns.

  13. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    McMillan, Kirk; Adler, Marc; Douglas S. Auld; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation ...

  14. Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.

    Magellan, Hervé; Boccara, Martine; Drujon, Thierry; Soulié, Marie-Christine; Guillou, Catherine; Dubois, Joëlle; Becker, Hubert F

    2013-09-01

    Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties. PMID:23886809

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

    Zhang, Wei; CHEN, Chao; Cao, Ruikai; Maurmann, Leila; LI, PING

    2014-01-01

    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 analog...

  16. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents. PMID:21730159

  17. High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors

    High-quality crystals of human haematopoietic prostaglandin D synthase in complex with novel inhibitors were obtained in microgravity. Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D2, an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC50) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC50 value of 50 nM extended to 1.1 Å resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein

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

    Ribas A. Vidal; 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 sintase (ALS) em vári...

  19. Temporal Phosphoproteome Dynamics Induced by an ATP Synthase Inhibitor Citreoviridin.

    Hu, Chia-Wei; Hsu, Chia-Lang; Wang, Yu-Chao; Ishihama, Yasushi; Ku, Wei-Chi; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-12-01

    Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 ? and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies. PMID:26503892

  20. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies. PMID:11827032

  1. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-01-01

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N-acyl-l-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acy...

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

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

    2009-01-01

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

  3. Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor.

    Montgomery, Heather J; Perdicakis, Basil; Fishlock, Dan; Lajoie, Gilles A; Jervis, Eric; Guy Guillemette, J

    2002-06-01

    Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited. PMID:11937350

  4. Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases.

    Adada, Mohamad; Luberto, Chiara; Canals, Daniel

    2016-05-01

    Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann-Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics. PMID:26200918

  5. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia.

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2015-11-25

    The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  6. Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

    Skovpen, Yulia V; Conly, Cuylar J T; Sanders, David A R; Palmer, David R J

    2016-02-17

    Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development. PMID:26836694

  7. Induction by inhibitors of nitric oxide synthase of hyperresponsiveness in the human nasal airway

    Turner, P.J.; Maggs, J R L; J. C. Foreman

    2000-01-01

    The effects of inhibitors of nitric oxide synthase (NOS) on the responsiveness of the human nasal airway were investigated, by measuring the nasal response to histamine and bradykinin.Repeated intranasal administration of NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA), 1 μmol per nostril every 30 min for 6 h, increased the nasal obstruction induced by histamine, 50–500 μg, and bradykinin, 200 μg per nostril. A single administration of L-NAME, 1 μmol per nostril...

  8. Blockade of tolerance to morphine but not to kappa opioids by a nitric oxide synthase inhibitor.

    Kolesnikov, Y A; Pick, C.G.; Ciszewska, G; Pasternak, G W

    1993-01-01

    The nitric oxide synthase inhibitor NG-nitro-L-arginine (NO2Arg) blocks morphine tolerance in mice. After implantation of morphine pellets the analgesic response decreases from 100% on the first day to 0% on the third. Coadministration of NO2Arg along with the pellets markedly retards the development of tolerance; 60% of mice are analgesic after 3 days, and 50% of mice are analgesic after 5 days. In a daily injection paradigm the analgesic response to morphine is reduced from 60% to 0% by 5 d...

  9. Identification of a Glycogen Synthase Kinase-3β Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

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

    2011-01-01

    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-3β (GSK-3β) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3β. We designed a series of second generation b...

  10. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  11. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  12. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases

    Christensen, Quin H.; Grove, Tyler L.; Booker, Squire J; Greenberg, E. Peter

    2013-01-01

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these i...

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

    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 signifi cant potential application value in the treatment and prevention of metabolic syndromes.

  14. Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

    Marzinzik, Andreas L; Amstutz, Ren; Bold, Guido; Bourgier, Emmanuelle; Cotesta, Simona; Glickman, J Fraser; Gtte, Marjo; Henry, Christelle; Lehmann, Sylvie; Hartwieg, J Constanze D; Ofner, Silvio; Pell, Xavier; Roddy, Thomas P; Rondeau, Jean-Michel; Stauffer, Frdric; Stout, Steven J; Widmer, Armin; Zimmermann, Johann; Zoller, Thomas; Jahnke, Wolfgang

    2015-11-01

    Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases. PMID:26381451

  15. Improvement of Dolichol-linked Oligosaccharide Biosynthesis by the Squalene Synthase Inhibitor Zaragozic Acid*

    Haeuptle, Micha A.; Welti, Michael; Troxler, Heinz; Hlsmeier, Andreas J.; Imbach, Timo; Hennet, Thierry

    2011-01-01

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc2Man5 in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

  16. Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

    Haeuptle, Micha A; Welti, Michael; Troxler, Heinz; Hlsmeier, Andreas J; Imbach, Timo; Hennet, Thierry

    2011-02-25

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

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

    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.

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

    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 μl 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.

  19. Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

    Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

    2015-10-01

    Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade. PMID:26397189

  20. Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive rats.

    Han, Jie; Jiang, Dong-Mei; Ye, Yang; Du, Chang-Qing; Yang, Jian; Hu, Shen-Jiang

    2016-05-01

    Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium‑dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar‑Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration‑dependent manner. The Ang II‑induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium‑dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in SHRs. PMID:27035426

  1. Nitric Oxide Synthase Inhibitor Improves De Novo and Long-Term l-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats

    Padovan-Neto, Fernando Eduardo; Echeverry, Marcela Bermúdez; Chiavegatto, Silvana; Del-Bel, Elaine

    2011-01-01

    Inhibitors of neuronal and endothelial nitric oxide synthase decrease l-3,4-dihidroxifenilalanine (l-DOPA)-induced dyskinesias in rodents. The mechanism of nitric oxide inhibitor action is unknown. The aims of the present study were to investigate the decrease of l-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA)-lesioned rats by nitric oxide inhibitors following either acute or chronic treatment. The primary findings of this study were that NG-nitro-l-Arginine, an inhibitor of endothelial and neuronal nitric oxide synthase, attenuated AIMs induced by chronic and acute l-DOPA. In contrast, rotational behavior was attenuated only after chronic l-DOPA. The 6-OHDA lesion and the l-DOPA treatment induced a bilateral increase (1.5 times) in the neuronal nitric oxide synthase (nNOS) protein and nNOS mRNA in the striatum and in the frontal cortex. There was a parallel increase, bilaterally, of the FosB/ΔFosB, primarily in the ipsilateral striatum. The exception was in the contralateral striatum and the ipsilateral frontal cortex, where chronic l-DOPA treatment induced an increase of approximately 10 times the nNOS mRNA. Our results provided further evidence of an anti-dyskinetic effect of NOS inhibitor. The effect appeared under l-DOPA acute and chronic treatment. The l-DOPA treatment also revealed an over-expression of the neuronal NOS in the frontal cortex and striatum. Our results corroborated findings that l-DOPA-induced rotation differs between acute and chronic treatment. The effect of the NOS inhibitor conceivably relied on the l-DOPA structural modifications in the Parkinsonian brain. Taken together, these data provided a rationale for further evaluation of NOS inhibitors in the treatment of l-DOPA-induced dyskinesia. PMID:21713068

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

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

    2009-05-01

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

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

    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 β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α 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 β-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.

  4. Quinazoline thymidylate synthase inhibitors: methods for assessing the contribution of polyglutamation to their in vitro activity.

    Jackman, A L; Kimbell, R; Brown, M; Brunton, L; Boyle, F T

    1995-10-01

    Many quinazoline thymidylate synthase (TS) inhibitors undergo intracellular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibitory activity towards TS (IC50 0.001-2 microM) and the growth of L1210 cells (IC50 0.005-10 microM), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L1210: RD1694) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were validated using a series of quinazoline-based TS inhibitors with well-defined activity for TS, folypolyglutamate synthetase (FPGS) and the reduced-folate cell membrane carrier (RFC). Short-exposure growth-inhibition assays or the measurement of TS activity in situ after various incubation times, followed by different lengths of time in drug-free medium, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since only the growth-inhibitory potency of polyglutamated analogues is significantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyglutamation of a particular compound than isolated FPGS assays alone. PMID:7495479

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

    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.

  6. Repositioning Proton Pump Inhibitors as Anticancer Drugs by Targeting the Thioesterase Domain of Human Fatty Acid Synthase

    Fako, Valerie E.; Wu, Xi; Pflug, Beth; Liu, Jing-Yuan; Zhang, Jian-Ting

    2014-01-01

    Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of free fatty acids, is up-regulated in many cancers. FASN is essential for cancer cell survival and contributes to drug resistance and poor prognosis. However, it is not expressed in most nonlipogenic normal tissues. Thus, FASN is a desirable target for drug discovery. Although different FASN inhibitors have been identified, none has successfully moved into clinical use. In this study, using in silico screening of an FD...

  7. Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain[S

    Larsen, Scott D; Wilson, Michael W.; Abe, Akira; Shu, Liming; George, Christopher H; Kirchhoff, Paul; Showalter, H. D. Hollis; Xiang, Jianming; Keep, Richard F.; Shayman, James A

    2012-01-01

    Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of co...

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

    C.A.A. Perotti

    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 g/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<0.02. The coinjection of LPS and 7-NI was followed by a significant (P<0.02 hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

  9. Pharmacodynamic comparison of LY3023703, a novel microsomal prostaglandin e synthase 1 inhibitor, with celecoxib.

    Jin, Y; Smith, C L; Hu, L; Campanale, K M; Stoltz, R; Huffman, L G; McNearney, T A; Yang, X Y; Ackermann, B L; Dean, R; Regev, A; Landschulz, W

    2016-03-01

    To assess the safety, tolerability, and pharmacology of LY3023703, a microsomal prostaglandin E synthase 1 (mPGES1) inhibitor, a multiple ascending dose study was conducted. Forty-eight subjects received LY3023703, celecoxib (400 mg), or placebo once daily for 28 days. Compared with placebo, LY3023703 inhibited ex vivo lipopolysaccharide-stimulated prostaglandin E2 (PGE2 ) synthesis 91% and 97% on days 1 and 28, respectively, after 30-mg dosing, comparable to celecoxib's effect (82% inhibition compared to placebo). Unlike celecoxib, which also inhibited prostacyclin synthesis by 44%, LY3023703 demonstrated a maximal increase in prostacyclin synthesis of 115%. Transient elevations of serum aminotransferase were observed in one subject after 30-mg LY3023703 dosing (10 upper limit of normal (ULN)), and one subject after 15-mg dosing (about 1.5 ULN). Results from this study suggest that mPGES1 inhibits inducible PGE synthesis without suppressing prostacyclin generation and presents a novel target for inflammatory pain. PMID:26351780

  10. Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain.

    Larsen, Scott D; Wilson, Michael W; Abe, Akira; Shu, Liming; George, Christopher H; Kirchhoff, Paul; Showalter, H D Hollis; Xiang, Jianming; Keep, Richard F; Shayman, James A

    2012-02-01

    Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate. PMID:22058426

  11. Synthesis and biological evaluation of novel phosphoramidate derivatives of coumarin as chitin synthase inhibitors and antifungal agents.

    Ji, Qinggang; Ge, Zhiqiang; Ge, Zhixing; Chen, Kaizhi; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Yang, Xiaolan; Liao, Fei

    2016-01-27

    A series of novel phosphoramidate derivatives of coumarin have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their chitin synthase inhibition activity and antimicrobial activity in vitro. The bioactive assay manifested that most of the target compounds exhibited good efficacy against CHS and a variety of clinically important fungal pathogens. In particular, compound 7t with IC50 of 0.08 mM against CHS displayed stronger efficiency than the reference Polyoxin B with IC50 of 0.16 mM. In addition, the apparent Ki values of compound 7t was 0.096 mM while the Km of Chitin synthase prepared from Candida tropicalis was 3.86 mM for UDP-N-acetylglucosamine, and the result of the Ki showed that the compounds was a non-competitive inhibitor of the CHS. As far as the antifungal activity is concerned, compounds 7o, 7r and 7t were highly active against Aspergillus flavus with MIC values in the range of 1 μg/mL to 2 μg/Ml while the results of antibacterial screening showed that these compounds have negligible actions to the tested bacteria. These results indicated that the design of these compounds as antifungal agents was rational. PMID:26647304

  12. Molecular docking analysis of selected Clinacanthus nutans constituents as xanthine oxidase, nitric oxide synthase, human neutrophil elastase, matrix metalloproteinase 2, matrix metalloproteinase 9 and squalene synthase inhibitors

    Radhakrishnan Narayanaswamy

    2016-01-01

    Full Text Available Background: Clinacanthus nutans (Burm. f. Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO, nitric oxide synthase (NOS, human neutrophil elastase (HNE, matrix metalloproteinase (MMP 2 and 9, and squalene synthase (SQS using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET, and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0 toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS.

  13. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    Sebastián, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. PMID:27131621

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

    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

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

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

    1986-05-01

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

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

    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.

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

    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

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

    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 (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

  19. Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

    The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time

  20. Nitric oxide synthase inhibitor improves de novo and long-term L-DOPA-induced dyskinesia in hemiparkinsonian rats

    Marcela Bermdez Echeverry

    2011-06-01

    Full Text Available Inhibitors of neuronal and endothelial nitric oxide synthase decrease l-3,4-dihidroxifenilalanine (L-DOPA-induced dyskinesias in rodents. The mechanism of nitric oxide inhibitor action is unknown. The aims of the present study were to investigate the decrease of L-DOPA-induced abnormal involuntary movements in 6-hydroxydopamine (6-OHDA-lesioned rats by nitric oxide inhibitors following either acute or chronic treatment. The primary findings of this study were that NG-nitro-L-Arginine, an inhibitor of endothelial and neuronal nitric oxide synthase, attenuated abnormal involuntary movements induced by chronic and acute L-DOPA. In contrast, rotational behavior was attenuated only after chronic L-DOPA. L-DOPA improved stepping test performance, and its chronic administration did not alter open field behavior. Our results indicated a correlation between apomorphine-induced rotation and the decrease in the number of adjusting steps performed with the contralateral forepaw in the 6-OHDA-lesioned rats.The 6-OHDA lesion and the L-DOPA treatment induced a bilateral increase (1.5 times in the nNOS protein and nNOS mRNA in the striatum and in the frontal cortex. There was a parallel increase, bilaterally, of the FosB/?FosB, primarily in the ipsilateral striatum. The exception was in the contralateral striatum and the ipsilateral frontal cortex, where chronic L-DOPA treatment induced an increase of approximately 10 times the nNOS mRNA. Our results provided further evidence of an anti-dyskinetic effect of NOS inhibitor. The effect appeared under L-DOPA acute and chronic treatment. The L-DOPA treatment also revealed an over-expression of the neuronal NOS in the frontal cortex and striatum. Our results corroborated findings that L-DOPA-induced rotation differs between acute and chronic treatment. The effect of the NOS inhibitor conceivably relied on the L-DOPA structural modifications in the parkinsonian brain. Taken together, these data provided a rationale for further evaluati

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

    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

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

    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.

  3. Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment

    Ana Martinez; Carmen Gil; Perez, Daniel I.

    2011-01-01

    Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The s...

  4. Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement

    DeBolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Melo, Carlos V.; Ross, Loretta; Cutler, Sean R.; Somerville, Christopher; Bonetta, Dario

    2007-01-01

    Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of bot...

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

    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. Synthesis and enzymatic evaluation of 2- and 4-aminothiazole-based inhibitors of neuronal nitric oxide synthase

    Graham R. Lawton

    2009-06-01

    Full Text Available Highly potent and selective inhibitors of neuronal nitric oxide synthase (nNOS possessing a 2-aminopyridine group were recently designed and synthesized in our laboratory and were shown to have significant in vivo efficacy. In this work, analogs of our lead compound possessing 2- and 4-aminothiazole rings in place of the aminopyridine were synthesized. The less basic aminothiazole rings will be less protonated at physiological pH than the aminopyridine ring, and so the molecule will carry a lower net charge. This could lead to an increased ability to cross the blood-brain barrier thereby increasing the in vivo potency of these compounds. The 2-aminothiazole-based compound was less potent than the 2-aminopyridine-based analogue. 4-Aminothiazoles were unstable in water, undergoing tautomerization and hydrolysis to give inactive thiazolones.

  7. The radioprotective effect of L-NAME inhibitor of NO-synthase in Chinese hamster cells in culture

    Radioprotective effect of L-NAME - one of the inhibitors of NO-synthase - was estimated by the yield of the aberrant anaphases after exposure of Chinese hamster cells to different doses of γ-rays and β-particles. Decrease of the frequency of radiation-induced chromosome aberrations was observed during LNAME cell treatment before irradiation (1-4 h) only. 3 Gy dose without LNAME and 6 Gy dose with L-NAME were equieffective ones. The treatment of cells with L-NAME decreased the level of SH-groups in cells and decreased fluorescence intensity of DNA-ethidium bromide complex during flow cytometry. Results obtained indicate the involvement of NO-dependent mechanism of the realization of the radiation-induced damage to the hereditary cell structure. Optimal conditions for the realization of the conceivable mechanism of radioprotective effect of L-NAME

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

    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 Nω-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 μM and 100% inhibition at the highest dose tested (30 μM. 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 μM, respectively. 2-AMP did not show any dose-dependent response, but was highly effective (57% inhibition at an intermediate dose of 30 μM and an IC50 of 25 μM. 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.

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

    Sadowski, Martin C.; Pouwer, Rebecca H.; Jennifer H. Gunter; 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...

  10. Effects of an endogenous nitric oxide synthase inhibitor on phorbol myristate acetate-induced acute lung injury in rats.

    Lin, Hen I; Chu, Shi Jye; Wang, David; Chen, Hsing I; Hsu, Kang

    2003-01-01

    1. In the present study, we determined whether the endogenous nitric oxide (NO) synthase (NOS) inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME) could ameliorate the acute lung injury (ALI) induced by phorbol myristate acetate (PMA) in rat isolated lung. 2. Typical ALI was induced successfully by PMA during 60 min of observation. At 2 micro g/kg, PMA elicited a significant increase in microvascular permeability (measured using the capillary filtration coefficient Kfc), lung weight gain, lung weight/bodyweight ratio, pulmonary arterial pressure (PAP) and protein concentration of bronchoalveolar lavage fluid. 3. Pretreatment with the NOS inhibitor l-NAME (5 mmol/L) significantly attenuated ALI. None of the parameters reflective of lung injury showed significant increase, except for PAP (P < 0.001). The addition of l-arginine (4 mmol/L) blocked the protective effective of l-NAME. Pretreatment with l-arginine exacerbated PMA-induced lung injury. 4. These data suggest that l-NAME significantly ameliorates ALI induced by PMA in rats, indicating that endogenous NO plays a key role in the development of lung oedema in PMA-induced lung injury. PMID:12859432

  11. Food-Related Compounds That Modulate Expression of Inducible Nitric Oxide Synthase May Act as Its Inhibitors

    Jesus Olivero-Verbel

    2012-07-01

    Full Text Available Natural compounds commonly found in foods may contribute to protect cells against the deleterious effects of inflammation. These anti-inflammatory properties have been linked to the modulation of transcription factors that control expression of inflammation-related genes, including the inducible nitric oxide synthase (iNOS, rather than a direct inhibitory action on these proteins. In this study, forty two natural dietary compounds, known for their ability to exert an inhibitory effect on the expression of iNOS, have been studied in silico as docking ligands on two available 3D structures for this protein (PDB ID: 3E7G and PDB ID: 1NSI. Natural compounds such as silibinin and cyanidin-3-rutinoside and other flavonoids showed the highest theoretical affinities for iNOS. Docking affinity values calculated for several known iNOS inhibitors significatively correlated with their reported half maximal inhibitory concentrations (R = 0.842, P < 0.0001, suggesting the computational reliability of the predictions made by our docking simulations. Moreover, docking affinity values for potent iNOS inhibitors are of similar magnitude to those obtained for some studied natural products. Results presented here indicate that, in addition to gene expression modulation of proteins involved in inflammation, some chemicals present in food may be acting by direct binding and possible inhibiting actions on iNOS.

  12. Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

    Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase-esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase-esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase-esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase-esterase activities

  13. Transport of nitric oxide synthase inhibitors through cationic amino acid carriers in human erythrocytes.

    Forray, M I; Angelo, S; Boyd, C A; Devés, R

    1995-12-22

    The interaction of arginine analogues, which are known to inhibit nitric oxide synthase, with two cationic amino acid transporters of human erythrocytes (systems y+ and y+L) was studied. Arginine and relevant analogues [NG-monomethyl-L-arginine (L-NMMA); NG-monomethyl-D-arginine (D-NMMA) and NG-nitro-L-arginine (L-NOARG)] were found to inhibit labeled lysine influx into intact erythrocytes. As expected, the pattern of inhibition reflected the contribution of the two distinct transport systems. All analogues showed a higher affinity for system y+L than for system y+. The half-saturation (inhibition) constants estimated for systems y+ and y+L (+/- SEM) were (microM): L-arginine, 55.7 +/- 5.4 and 2.4 +/- 0.1; L-NMMA, 151 +/- 13 and 7.5 +/- 0.5; D-NMMA, 2660 +/- 404 and 269 +/- 25; L-NOARG, 9414 +/- 169 and 594 +/- 35. The transport properties of the analogues were investigated using an assay based on the trans-stimulation of lysine efflux. The addition of saturating concentrations of unlabeled analogues to the external medium stimulated efflux of labeled lysine through systems y+L and y+, showing that the analogues can enter the cell through these pathways. PMID:8849321

  14. Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica.

    An, Hyo-Jin; Nugroho, Agung; Song, Byong-Min; Park, Hee-Juhn

    2015-01-01

    Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic acid were isolated and identified by NMR spectroscopy. The structure of isoeugenin was determined as 7-hydroxy-5-methoxy-2-methylchromone by the 2D-NMR technique. Among the four compounds, isoeugenin has the lowest IC50 value on the inhibition of NO production in LPS-activated macrophage RAW264.7 cells (IC50, 9.33 ?g/mL). In addition, isoeugenin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines mRNA levels. Taken together, these results suggest that the anti-inflammatory activity of isoeugenin is associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells. Accordingly, our results suggest that the new chromone isoegenin should be considered a potential treatment for inflammatory disease. PMID:26633331

  15. The impact of asymmetric dimethylarginine (ADAMA), the endogenous nitric oxide (NO) synthase inhibitor, to the pathogenesis of gastric mucosal damage.

    Szlachcic, Aleksandra; Krzysiek-Maczka, Gracjana; Pajdo, Robert; Targosz, Aneta; Magierowski, Marcin; Jasnos, Katarzyna; Drozdowicz, Danuta; Kwiecien, Slawomir; Brzozowski, Tomasz

    2013-01-01

    This review was designed to provide an update on the role of asymmetric arginine (ADMA), the endogenous inhibitor of nitric oxide (NO) synthase in the pathophysiology of the upper gastrointestinal (GI) tract. Numerous studies in the past confirmed that NO is a multifunctional endogenous gas molecule involved in most of the body organs' functional and metabolic processes including the regulation of gastrointestinal (GI) secretory functions, motility, maintenance of GI integrity, gastroprotection and ulcer healing. NO is metabolized from L-arginine by enzymatic reaction in the presence of constitutive NO synthase. In upper GI tract, NO acts as a potent vasodilator known to increase gastric mucosa blood flow, regulates the secretion of mucus and bicarbonate, inhibits the gastric secretion and protects the gastric mucosa against the damage induced by a variety of damaging agents and corrosive substances. In contrast, ADMA first time described by Vallance and coworkers in 1992, is synthesized by the hydrolysis of proteins containing methylated arginine amino acids located predominantly within the nucleus of cells. This molecule has been shown to competitively inhibit NO synthase suggesting its regulatory role in the functions of vascular endothelial cells and systemic circulation in humans and experimental animals. Nowadays, ADMA is a potentially important risk factor for coronary artery diseases and a marker of cardiovascular risk. Increased plasma levels of ADMA have been documented in several conditions that are characterized by endothelial dysfunction, including hypertension, hypercholesterolemia, hyperglycemia, renal failure and tobacco exposure. The role of ADMA in other systems including GI-tract has been so far less documented. Nevertheless, ADMA was shown to directly induce oxidative stress and cell apoptosis in gastric mucosal cells in vitro and to contribute to the inflammatory reaction associated with major human pathogen to gastric mucosa, Helicobacter pylori (H.pylori). Infection of gastric mucosa with this germ or H. pylori water extract led to marked increase in the plasma concentration of ADMA and significantly inhibited bicarbonate secretion, considered as one of the important components of upper GI-tract defense system. When administered to rodents, ADMA aggravated gastric mucosal lesions injury induced by cold stress, ethanol and indomethacin and this worsening effect on gastric lesions was accompanied by the significant increase in the plasma level of ADMA. This exaggeration of gastric lesions by ADMA was coincided with the inhibition of NO, the suppression of gastric blood flow and excessive release of proinflammatory cytokine TNF-α. This metabolic analog of L-arginine applied to rats was exposed to water immersion and restraint stress and ischemia-reperfusion, causing an elevation of plasma levels of ADMA and gastric MDA content, which is the marker of lipid peroxidation. These effects, including the rise in the plasma levels of ADMA in rats with stress and ischemia-reperfusion-induced gastric lesions, were attenuated by concomitant treatment with L-arginine, the substrate for NO-synthase, and superoxide dismutase (SOD), a reactive oxygen metabolite scavenger added to ADMA. We conclude that ADMA could be considered as an important factor contributing to the pathogenesis of gastric mucosal damage and inflammatory reaction in H. pylori-infected stomach due to inhibition of NO, suppression of GI microcirculation, and the proinflammatory and proapoptotic actions of this arginine analog. PMID:22950506

  16. Differential effects of serotonin reuptake inhibitors on erectile responses, NO-production, and neuronal NO synthase expression in rat corpus cavernosum tissue

    Angulo, Javier; Peiró, Concepción; Sanchez-Ferrer, Carlos F; Gabancho, Sonia; Cuevas, Pedro; Gupta, Sandeep; Tejada, Iñigo Sáenz de

    2001-01-01

    Increased incidence of impotence is associated with some selective serotonin-reuptake-inhibitors (SSRIs), but the pathophysiological mechanism is unknown. Paroxetine and citalopram are extensively used SSRIs, but only paroxetine has been shown to inhibit nitric oxide synthase (NOS) activity. NO is a key mediator of penile erection. Thus, the aim of this study was to determine the effects of paroxetine and citalopram on erectile function and NO production, in a rat model. Application of cavern...

  17. EFFECTS OF THE NITRIC OXIDE SYNTHASE INHIBITOR L-NAME ON RECOGNITION AND SPATIAL MEMORY DEFICITS PRODUCED BY DIFFERENT NMDA RECEPTOR ANTAGONIST IN THE RAT

    Boultadakis, Antonios; Pitsikas, Nikolaos

    2010-01-01

    Abstract There is consistent experimental evidence that non-competitive antagonists of the NMDA receptor, such as ketamine, MK-801, and phencyclidine (PCP) impair cognition and produce psychotomimetic effects in rodents. Nitric oxide (NO) is considered as an intracellular messenger in the brain. The implication of NO in learning and memory is well documented. The present study was designed to investigate the ability of the NO synthase (NOS) inhibitor L-NAME to antagonize recognitio...

  18. Effects of the Nitric Oxide Synthase Inhibitor -NAME on Recognition and Spatial Memory Deficits Produced by Different NMDA Receptor Antagonists in the Rat

    Boultadakis, Antonios; Pitsikas, Nikolaos

    2010-01-01

    There is consistent experimental evidence that noncompetitive antagonists of the N-methyl--aspartate (NMDA) receptor, such as ketamine, MK-801, and phencyclidine (PCP), impair cognition and produce psychotomimetic effects in rodents. Nitric oxide (NO) is considered as an intracellular messenger in the brain. The implication of NO in learning and memory is well documented. This study was designed to investigate the ability of the NO synthase inhibitor -NAME to antagonize recognition and spatia...

  19. Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change*s

    Li, Yi-Ching; Chiang, Chia-Wang; Yeh, Hui-Chun; Hsu, Pei-Yung; Whitby, Frank G; Wang, Lee-Ho; Chan, Nei-Li

    2008-01-01

    Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H2. PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-s...

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

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

    2015-01-01

    Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5α-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats w...

  1. Pharmacodynamic Target Evaluation of a Novel Oral Glucan Synthase Inhibitor, SCY-078 (MK-3118), Using an In Vivo Murine Invasive Candidiasis Model

    Lepak, Alexander J.; Marchillo, Karen; Andes, David R.

    2014-01-01

    Echinocandins inhibit the synthesis of β-1,3-d-glucan in Candida and are the first-line therapy in numerous clinical settings. Their use is limited by poor oral bioavailability, and they are available only as intravenous therapies. Derivatives of enfumafungin are novel orally bioavailable glucan synthase inhibitors. We performed an in vivo pharmacodynamic (PD) evaluation with a novel enfumafungin derivative, SCY-078 (formerly MK-3118), in a well-established neutropenic murine model of invasiv...

  2. Lapatinib, a Dual EGFR and HER2 Tyrosine Kinase Inhibitor, Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

    Kim, Hwang-Phill; Yoon, Young-Kwang; Kim, Jin-Won; HAN, SAE-WON; Hur, Hyung-Seok; Park, Jinah; LEE, JU-HEE; Oh, Do-Youn; Im, Seock-Ah; BANG, YUNG-JUE; KIM, TAE-YOU

    2009-01-01

    Background Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated. Methodology and Principal Findings In this study, we demon...

  3. Counteraction by nitric oxide synthase inhibitor of neurochemical alterations of dopaminergic system in 6-OHDA-lesioned rats under L-DOPA treatment.

    Del-Bel, Elaine; Padovan-Neto, Fernando Eduardo; Szawka, Raphael Escorsim; da-Silva, Célia Aparecida; Raisman-Vozari, Rita; Anselmo-Franci, Janete; Romano-Dutra, Angélica Caroline; Guimaraes, Francisco Silveira

    2014-01-01

    Nitric oxide synthase inhibitors reduce L-3, (Del-Bel et al., Cell Mol Neurobiol 25(2):371-392, 2005) 4-dihydroxyphenylalanine (L-DOPA)-induced abnormal motor effects subsequent to depletion of dopaminergic neurons in rodents and non-human primates. The present study used quantitative high-performance liquid chromatography to analyze, for the first time, dopamine metabolism in striatum of rats in order to elucidate the mechanism of action of the nitric oxide synthase inhibitors. Adult male Wistar rats received unilateral microinjection of saline (sham) or 6-hydroxydopamine (6-OHDA-lesioned) in the medial forebrain bundle. Past 3 weeks, rats were treated during 21 days with L-DOPA/benserazide (30 mg/kg/7.5 mg/kg, respectively, daily). On the 22nd day rats received an intraperitoneal (i.p.) injection of either vehicle or 7-nitroindazole, a preferential neuronal nitric oxide synthase inhibitor before L-DOPA. Abnormal involuntary movements and rotarod test were assessed as behavioral correlate of motor responses. Lesion intensity was evaluated through tyrosine hydroxylase immunohystochemical reaction. Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and an extent of dopamine striatal tissue levels/dopamine metabolism were measured in the striatum. Lesion with 6-OHDA decreased dopamine, DOPAC, and DOPAC/dopamine ratio in the lesioned striatum. L-DOPA treatment induced abnormal involuntary movements and increased DOPAC/dopamine ratio (nearly five times) in the lesioned striatum. L-DOPA-induced dyskinesia was mitigated by 7-nitroindazole, which also decreased dopamine turnover, dopamine and DOPAC levels. Our results revealed an almost two times increase in dopamine content in the non-lesioned striatum of 6-OHDA-lesioned rats. Reduction of striatal DOPAC/dopamine ratio in dyskinetic rats may suggest an increase in the dopamine availability. Our data confirm contribution of nitrergic transmission in the pathogenesis of L-DOPA-induced dyskinesia with potential utilization of nitric oxide synthase inhibitors for treatment. PMID:23807548

  4. Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents.

    Ge, Zhiqiang; Ji, Qinggang; Chen, Chunyan; Liao, Qin; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Liao, Fei

    2016-04-01

    A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC50 of 0.10 mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC50 of 0.18 mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4 μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents. PMID:25815669

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

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

    2015-01-01

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

  6. Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform.

    Southan, G J; Zingarelli, B; O'Connor, M; Salzman, A L; Szabó, C

    1996-02-01

    1. The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2. AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 microM (EC50 values for NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine were 159 and > 1000 microM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may complete with L-arginine for its binding site on NOS. 3. The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4. Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5. These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6. In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than NG-methyl-L-arginine and NG-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7. AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8. High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9. In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10. In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS. PMID:8646406

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

    N. Novaretti

    2010-11-01

    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 interfere 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 < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS. The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson’s disease.

  8. Synthesis of Potent Inhibitors of ?-Ketoacyl-Acyl Carrier Protein Synthase III as Potential Antimicrobial Agents

    Song Li

    2012-04-01

    Full Text Available Mycobacterium tuberculosis FabH, an essential enzyme in the mycolic acid biosynthetic pathway, is an attractive target for novel anti-tubercolosis agents. Structure-based design and synthesis of 1-(4-carboxybutyl-4-(4-(substituted benzyloxyphenyl-1H-pyrrole-2-carboxylic acid derivatives 7ah, a subset of eight potential FabH inhibitors, is described in this paper. The Vilsmeier-Haack reaction was employed as a key step. The structures of all the newly synthesized compounds were identified by IR, 1H-NMR, 13C-NMR, ESI-MS and HRMS. The alamarBlue microassay was employed to evaluate the compounds 7ah against Mycobacterium tuberculosis H37Rv. The results demonstrate that the compound 7d possesses good in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Minimum Inhibitory Concentration value [MIC], 12.5 g/mL.These compounds may prove useful in the discovery and development of new anti-tuberculosis drugs.

  9. The Discovery of Potentially Selective Human Neuronal Nitric Oxide Synthase (nNOS Inhibitors: A Combination of Pharmacophore Modelling, CoMFA, Virtual Screening and Molecular Docking Studies

    Guanhong Xu

    2014-05-01

    Full Text Available Neuronal nitric oxide synthase (nNOS plays an important role in neurotransmission and smooth muscle relaxation. Selective inhibition of nNOS over its other isozymes is highly desirable for the treatment of neurodegenerative diseases to avoid undesirable effects. In this study, we present a workflow for the identification and prioritization of compounds as potentially selective human nNOS inhibitors. Three-dimensional pharmacophore models were constructed based on a set of known nNOS inhibitors. The pharmacophore models were evaluated by Pareto surface and CoMFA (Comparative Molecular Field Analysis analyses. The best pharmacophore model, which included 7 pharmacophore features, was used as a search query in the SPECS database (SPECS, Delft, The Netherlands. The hit compounds were further filtered by scoring and docking. Ten hits were identified as potential selective nNOS inhibitors.

  10. 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.

    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

  11. Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors.

    Li, Ya-Li; Qi, Xiang-Yu; Jiang, Hui; Deng, Xiao-Dong; Dong, Yan-Ping; Ding, Ting-Bo; Zhou, Lu; Men, Peng; Chu, Yong; Wang, Ren-Xiao; Jiang, Xian-Cheng; Ye, De-Yong

    2015-09-15

    Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 μM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 μM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors. PMID:26314925

  12. Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.

    Cabrera-Salazar, Mario A; Deriso, Matthew; Bercury, Scott D; Li, Lingyun; Lydon, John T; Weber, William; Pande, Nilesh; Cromwell, Mandy A; Copeland, Diane; Leonard, John; Cheng, Seng H; Scheule, Ronald K

    2012-01-01

    Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases. PMID:22912851

  13. S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid: a model for potential bioreductively activated prodrugs for inhibitors of nitric oxide synthase (NOS) activity.

    Ulhaq, S; Naylor, M A; Chinje, E C; Threadgill, M D; Stratford, I J

    1997-01-01

    Treatment of 1,1-dimethylethyl S-(2-1,1-dimethylethoxycarbonylamino)-5-bromopentanoate with 1-potassio-2-nitroimidazole, followed by deprotection, afforded S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid, which was reduced to S-2-amino-5-(2-aminoimidazol-1-yl)pentanoic acid. This aminoimadazole inhibited rat brain nitric oxide synthase (NOS) activity 3.2 times more potently than did the nitro analogue. Thus S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid is a potent prodrug which may be bioreductively activated to a NOS inhibitor in hypoxic solid tumours. PMID:9051114

  14. Effects of Nerve Growth Factor and Nitric Oxide Synthase Inhibitors on Amyloid Precursor Protein mRNA Levels and Protein Stability

    MacKinnon, Janet C.; Huether, Patricia; Kalisch, Bettina E

    2012-01-01

    We determined previously that nitric oxide (NO) modulates the nerve growth factor (NGF)-mediated increases in amyloid precursor protein (APP) levels in PC12 cells. To elucidate potential mechanisms, the effects of NGF and NO synthase (NOS) inhibitors on APP mRNA levels and protein stability were evaluated. Surprisingly, treatment of PC12 cells with NGF resulted in decreased levels of APP695 and APP751/770 mRNA. Therefore, the effect of NGF on APP protein stability was examined using the trans...

  15. The Effect of Neuronal Nitric Oxide Synthase Inhibitor 7-Nitroindazole on the Cell Death Induced by Zinc Administration in the Brain of Rats

    GÖKÇE, M. Fatih; BAĞIRICI, Faruk; DEMİR, Şerif

    2009-01-01

    Aim: To evaluate the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) on hippocampal cell loss induced by zinc hemisulfate salt (ZnSO4·7H2O) treatment. Materials and Methods: Rats were divided into 3 groups (n = 21; 7 rats in each group): Control, Zinc, and Zinc+7-NI. In the Zinc and Zinc+7-NI (pretreatment) groups, ZnSO4·7H2O was applied intracortically to the left sensory motor cortex. The 7-NI (50 mg/kg) was injected into the third group (Zinc+7-NI) in...

  16. Evaluation of BM-573, a novel TXA(2) synthase inhibitor and receptor antagonist, in a porcine model of myocardial ischemia-reperfusion

    Kolh, Philippe; Rolin, S.; Tchana-Sato, Vincent; Petein, M.; Ghuysen, Alexandre; LAMBERMONT, Bernard; Hanson, Julien; Magis, David; P. Segers; Masereel, B; D'ORIO, Vincenzo; Dogné, Jean-Michel

    2006-01-01

    Aims: To investigate whether BM-573 (N-tert-butyl-N'-[2-(4'-methylphenylam\\ino)-5-nitro-benzenesulfonyl]urea), an original combined thromboxane A(2) synthase inhibitor and receptor antagonist, prevents reperfusion injury in acutely ischemic pigs. Methods: Twelve animals were randomly divided in two groups: a control group (n = 6) intravenously infused with vehicle, and a BM-573-treated group (n = 6) infused with BM-573 (10 mg kg(-1) h(-1)). In both groups, the left anterior descending (LAD) c...

  17. Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase.

    Fishlock, Dan; Perdicakis, Basil; Montgomery, Heather J; Guillemette, J Guy; Jervis, Eric; Lajoie, Gilles A

    2003-03-20

    Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity. PMID:12614872

  18. Elevation of radiolabelled thymidine uptake in RIF-1 fibrosarcoma and HT29 colon adenocarcinoma cells after treatment with thymidylate synthase inhibitors

    We recently showed an increase in tumour uptake of 2-[11C]thymidine in patients with gastrointestinal malignancies after thymidylate synthase (TS) inhibition. To understand the phenomenon in more detail, we investigated whether TS inhibition by different TS inhibitors leads to a dose- and time-dependent change in the uptake of radiolabelled thymidine, and whether radiotracer uptake is related to changes in cell viability resulting from treatment. RIF-1 and HT29 cells were treated with the TS inhibitors 5-fluorouracil (5-FU) and AG337 (nolatrexed dihydrochloride), as well as cisplatin as control. The cell viability and net accumulation of [3H]thymidine after a 1-h pulse was determined at different times after drug treatment. In both cell lines, [3H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [3H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [3H]thymidine uptake in RIF-1 and HT29 cells at all time points. Cell viability was compromised only after 24 h. Using two types of TS inhibitor, we have shown an increase in [3H]thymidine uptake, in a dose-dependent manner, a few hours after TS inhibition when the cell viability was not compromised. This effect was not seen with a non-TS inhibitor. These findings suggest that 2-[11C]thymidine positron emission tomography can be used to study TS inhibition in vivo at early time points when cell viability is not compromised and may therefore be helpful in the development of new TS inhibitors and in differentiating between patients with tumours sensitive to TS inhibitors and those unlikely to respond. (orig.)

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

    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

    Roč. 124, č. 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

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

    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...

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

    Kopecký, Jan; Pospíšil, Stanislav; Janata, Jiří

    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

  2. Catechol-based substrates of chalcone synthase as a scaffold for novel inhibitors of PqsD.

    Allegretta, Giuseppe; Weidel, Elisabeth; Empting, Martin; Hartmann, Rolf W.

    2015-01-01

    A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the ac...

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

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

    2004-01-01

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

  4. In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity

    Sunil L Harer

    2014-01-01

    Full Text Available Purpose: The imidazopyridine moiety is important pharmacophore that has proven to be useful for a number of biologically relevant targets, also reported to display antibacterial, antifungal, antiviral properties. Riboflavin biosynthesis involving catalytic step of Lumazine synthase is absent in animals and human, but present in microorganism, one of marked advantage of this study. Still, this path is not exploited as antiinfective target. Here, we proposed different interactions between [1H,3H] imidazo[4,5-b] pyridine test ligands and target protein Lumazine synthase (protein Data Bank 2C92, one-step synthesis of title compounds and further evaluation of them for in vitro antimicrobial activity. Materials and Methods: Active pocket of the target protein involved in the interaction with the test ligands molecules was found using Biopredicta tools in VLifeMDS 4.3 Suite. In-silico docking suggests H-bonding, hydrophobic interaction, charge interaction, aromatic interaction, and Vanderwaal forces responsible for stabilizing enzyme-inhibitor complex. Disc diffusion assay method was used for in vitro antimicrobial screening. Results and Discussion: Investigation of possible interaction between test ligands and target lumazine synthase of Mycobacterium tuberculosis suggested 1i and 2f as best fit candidates showing hydrogen bonding, hydrophobic, aromatic and Vanderwaal′s forces. Among all derivatives 1g, 1j, 1k, 1l, 2a, 2c, 2d, 2e, 2h, and 2j exhibited potent activities against bacteria and fungi compared to the standard Ciprofloxacin and Fluconazole, respectively. The superiority of 1H imidazo [4,5-b] pyridine compounds having R′ = Cl >No 2 > NH 2 at the phenyl/aliphatic moiety resident on the imidazopyridine, whereas leading 3H imidazo[4,5-b] pyridine compounds containing R/Ar = Cl > No 2 > NH 2> OCH 3 substituents on the 2 nd position of imidazole.

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

    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-α 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 TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα 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 TNFα-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.

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

    Highlights: •EV-077 reduced TNF-α 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 TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα 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 TNFα-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

  7. Comparative Pharmacophore Modeling and QSAR Studies for Structural Requirements of some Substituted 2-Aminopyridine Derivatives as Inhibitors of Nitric Oxide Synthases.

    Sharma, Mukesh C

    2015-06-01

    The present study is an attempt in this direction seeking for the development and comparison of QSAR models of substituted 2-aminopyridine derivatives as inhibitors of nitric oxide synthases by different feature selection methods. The QSAR study was carried out on V-life Molecular Design Suite software, and the derived best QSAR model was derived by partial component regression method. The statistically significant best model with high correlation coefficient ([Formula: see text]) was selected for further study. The model was further validated by means of crossed squared correlation coefficient ([Formula: see text] and [Formula: see text]) which shows model has good predictive ability. The best 3D-QSAR model showed [Formula: see text] and standard error = 0.1954. The predictive ability of the resultant model was evaluated using a test set molecules and the predicted [Formula: see text] The results reveal that the acceptor, donor, aliphatic, and aromatic pharmacophore properties are favorable contour sites for both the activities. The two-dimensional and k-nearest-neighbor contour plots were required for further understanding of the relationship between structural features of substituted 2-aminopyridine derivatives and their activities which should be applicable to design newer potential inducible nitric oxide synthases. PMID:26202943

  8. Radiosynthesis and preliminary PET evaluation of glycogen synthase kinase 3? (GSK-3?) inhibitors containing [(11)C]methylsulfanyl, [(11)C]methylsulfinyl or [(11)C]methylsulfonyl groups.

    Kumata, Katsushi; Yui, Joji; Xie, Lin; Zhang, Yiding; Nengaki, Nobuki; Fujinaga, Masayuki; Yamasaki, Tomoteru; Shimoda, Yoko; Zhang, Ming-Rong

    2015-08-15

    Three compounds 1-3 containing methyl-sufanyl, sufinyl, or sulfonyl groups are strong inhibitors of glycogen synthase kinase 3? (GSK-3?), an enzyme associated with Alzheimer's disease. We labeled 1-3 with (11)C for a positron emission tomography (PET) brain imaging study. A novel thiophenol precursor 4 for radiosynthesis was prepared by reacting sulfoxide 2 with trifluoroacetic anhydride. [(11)C]1 was synthesized by reacting 4 with [(11)C]methyl iodide in 52 5% radiochemical yield (n = 5, based on [(11)C]CO2, corrected for decay). Oxidation of [(11)C]1 with Oxone produced [(11)C]2 and [(11)C]3, respectively. PET with [(11)C]1 and [(11)C]3 showed 2 fold higher brain uptake of radioactivity in a mouse model of cold water stress in which GSK-3? expression was increased, than in the controls. PMID:26067173

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

    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)

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

    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.

  11. 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

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    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 T. 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 compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

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

    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.

  13. Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.

    Choi, Kyoung-Jae; Yu, Yeon Gyu; Hahn, Hoh Gyu; Choi, Jung-Do; Yoon, Moon-Young

    2005-08-29

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics. PMID:16111681

  14. Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

    Bruno, Annalisa; Di Francesco, Luigia; Coletta, Isabella; Mangano, Giorgina; Alisi, Maria Alessandra; Polenzani, Lorenzo; Milanese, Claudio; Anzellotti, Paola; Ricciotti, Emanuela; Dovizio, Melania; Di Francesco, Andrea; Tacconelli, Stefania; Capone, Marta L; Patrignani, Paola

    2010-01-01

    Abstract Inhibitors of microsomal prostaglandin(PG)E synthase-1(mPGES-1) are being developed for the relief of pain. Redirection of the PGH2 substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH2 metabolism in lipopolysaccharide(LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE2, thromboxane(TX)B2, PG...

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

    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

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

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

    2012-04-24

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

  17. A General Method for Selection of ?-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

    Curic, Mirjana; Stuer-Lauridsen, Birgitte; Renault, Pierre; Nilsson, Dan

    1999-01-01

    The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the ?-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototro...

  18. The selective neuronal nitric oxide synthase inhibitor 7-nitroindazole has acute analgesic but not cumulative effects in a rat model of peripheral neuropathy

    Henry JL

    2011-03-01

    Full Text Available Liliane J Dableh, James L HenryDepartment of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, CanadaAbstract: Chronic neuropathic pain that may arise from various nerve injuries or insults remains notoriously difficult to manage. The neuronal isoform of the enzyme nitric oxide synthase (nNOS has been shown to be involved in the spinal transmission of nociception in animal models of chronic pain. The aim of this study is to evaluate the effect of single dose and repeated administration of a selective nNOS inhibitor. Rats were unilaterally implanted with a 2-mm polyethylene cuff around the sciatic nerve. Paw withdrawal thresholds were measured using von Frey filament stimulation. Rats were given 10, 20, or 30 mg/kg of 7-nitroindazole (7-NI, or vehicle, on days 2, 5, and 7 after model induction, respectively. Paw withdrawal thresholds were measured before and at 30 and 60 min after injection. 7-NI significantly increased paw withdrawal thresholds at 60 min at the 20 and 30 mg/kg dosages. In the second part of this study, rats were given 20 mg/kg 7-NI daily for five days starting immediately after cuff implantation (days 0 to 4, and the cuff was removed on day 4. Withdrawal thresholds were measured intermittently over a 24-day observation period. No differences in withdrawal thresholds were observed between drug and vehicle-treated rats. Therefore, early and repeated administration of 7-NI did not affect the development or progression of the model. In conclusion, inhibition of nNOS had an analgesic but not a pre-emptive effect in this model of peripheral neuropathic pain.Keywords: neuronal nitric oxide synthase, nitric oxide, 7-nitroindazole, neuropathic pain, peripheral nerve injury, nociception

  19. 16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana.

    Roy, Arnab; Roberts, Frank G; Wilderman, P Ross; Zhou, Ke; Peters, Reuben J; Coates, Robert M

    2007-10-17

    The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(II)- and Pb(IV)-induced cyclizations onto the Delta12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC50 = 1 x 10-7 and 1 x 10-6 M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl diphosphate substrate (Km = 3 x 10-7 M). Large enhancements of binding affinities (IC50 = 4 x 10-9 and 2 x 10-8 M) were observed in the presence of 1 mM pyrophosphate, which is consistent with a tightly bound ent-beyeranyl+/pyrophosphate- ion pair intermediate in the cyclization-rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 x 10-5 M) exhibited by ent-beyeran-16-exo-yl diphosphate (11) and its failure to undergo bridge rearrangement to kaurene appear to rule out the covalent diphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-16-yl carbocation with potential applications as an active site probe for the various ent-diterpene cyclases and as a novel, selective inhibitor of gibberellin biosynthesis in plants. PMID:17892288

  20. Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla Dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida; Pereira, Maristela

    2015-09-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. PMID:26124176

  1. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    Skov, Sren; Pedersen, Marianne Terndrup; Andresen, Lars; Straten, Per Thor; Woetmann, Anders; Odum, Niels

    2005-01-01

    We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...... small interfering RNA or by different inhibitors showed that GSK-3 activity is essential for the induced MICA/B expression. We thus present evidence that cancer cells which survive the direct induction of cell death by HDAC inhibitors become targets for NKG2D-expressing cells like NK cells, gammadelta T...... apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3 by...

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

    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-α/β 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-α/β 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.

  3. Single dose of inducible nitric oxide synthase inhibitor induces prolonged inflammatory cell accumulation and fibrosis around injured tendon and synovium.

    Darmani, Homa; Crossan, James C; Curtis, Adam

    2004-06-01

    The aim of the current study was to investigate the effect of inhibition of nitric oxide (NO) production after injury on inflammatory cell accumulation and fibrosis around digital flexor tendon and synovium. A standard crush injury was applied to the flexor tendons of the middle digit of the hindpaw and the overlying muscle and synovium of female Wistar rats. Thirty animals received an intraperitoneal injection of either isotonic saline or N(G)-nitro-l-arginine methyl ester (L-NAME; 5 mg/kg) immediately following the crush injury, and five animals were then sacrificed at various intervals and the paws processed for histology. Another group of five animals was sacrificed after 3 days for nitrite determinations. The results showed that nitrite production and hence NO synthase activity is doubled at the acute phase of tendon wound healing, and we can prevent this by administering a single dose of L-NAME immediately after injury. The incidence and severity of fibrocellular adhesions between tendon and synovium was much more marked in animals treated with L-NAME. Treatment with L-NAME elicited a chronic inflammatory response characterised by a persistent and extraordinarily severe accumulation of large numbers of inflammatory cells in the subcutaneous tissues, in muscle and in tendon. These findings indicate that in the case of injured tendon and synovium, NO could act to protect the healing tissue from an uncontrolled inflammatory response. PMID:15223606

  4. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

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

    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/μCT imaging. GSK-3 inhibitors caused β-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/μCT. 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

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

    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/μCT imaging. GSK-3 inhibitors caused β-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/μCT. 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.

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

    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.

  8. Design, synthesis and evaluation of novel quinazoline-2,4-dione derivatives as chitin synthase inhibitors and antifungal agents.

    Ji, Qinggang; Yang, Dan; Wang, Xin; Chen, Chunyan; Deng, Qiao; Ge, Zhiqiang; Yuan, Lvjiang; Yang, Xiaolan; Liao, Fei

    2014-07-01

    A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized, and characterized by (1)H NMR, (13)C NMR and MS spectral data. Their inhibition against chitin synthase (CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j, 5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency among these compounds, had a half-inhibition concentration (IC50) of 0.08mmol/L, while polyoxin B as positive drug had IC50 of 0.18mmol/L. These IC50 values of compounds 5i, 5m, 5n, and 5s were greater than 0.75mmol/L, which revealed that those compounds had weak inhibition activity against CHS. Moreover, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the minimum-inhibition-concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to their IC50 values against CHS. Furthermore, these compounds had negligible actions to bacteria. Therefore, these compounds were promising selective antifungal agents. PMID:24856180

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

    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.

  10. Effects of aminoguanidine, a potent nitric oxide synthase inhibitor, on myocardial and organ structure in a rat model of hemorrhagic shock

    Mona M Soliman

    2014-01-01

    Full Text Available Background: Nitric oxide (NO has been shown to increase following hemorrhagic shock (HS. Peroxynitrite is produced by the reaction of NO with reactive oxygen species, leads to nitrosative stress mediated organ injury. We examined the protective effects of a potent inhibitor of NO synthase, aminoguanidine (AG, on myocardial and multiple organ structure in a rat model of HS. Materials and Methods: Male Sprague Dawley rats (300-350 g were assigned to 3 experimental groups (n = 6 per group: (1 Normotensive rats (N, (2 HS rats and (3 HS rats treated with AG (HS-AG. Rats were hemorrhaged over 60 min to reach a mean arterial blood pressure of 40 mmHg. Rats were treated with 1 ml of 60 mg/kg AG intra-arterially after 60 min HS. Resuscitation was performed in vivo by the reinfusion of the shed blood for 30 min to restore normo-tension. Biopsy samples were taken for light and electron microscopy. Results: Histological examination of hemorrhagic shocked untreated rats revealed structural damage. Less histological damage was observed in multiple organs in AG-treated rats. AG-treatment decreased the number of inflammatory cells and mitochondrial swollen in myocardial cells. Conclusion: AG treatment reduced microscopic damage and injury in multiple organs in a HS model in rats.

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

    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

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

    Cho Yu

    2010-08-01

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

  13. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

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

    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.

  15. Spermine synthase

    Pegg, Anthony E; Michael, Anthony J.

    2009-01-01

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

  16. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis

    Lo, Hui-Chen; Hung, Ching-Yi; Huang, Fu-Huan; Su, Tzu-Cheng; Lee, Chien-Hsing

    2016-01-01

    The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats. PMID:27007815

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

    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.

  18. 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

    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), or 50...

  19. Pulmonary hypertension triggered by lipopolysaccharide in ascites-susceptible and -resistant broilers is not amplified by aminoguanidine, a specific inhibitor of inducible nitric oxide synthase.

    Bowen, O T; Erf, G F; Anthony, N B; Wideman, R F

    2006-03-01

    Nitric oxide (NO) is a potent pulmonary vasodilator that modulates the pulmonary vasoconstriction and pulmonary hypertension (PH) triggered by bacterial lipopolysaccharide (LPS) in broilers. The amplitude and duration of the LPS-induced PH are markedly enhanced following pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME), which inhibits NO synthesis by both the constitutive (endothelial) and inducible (inflammatory) forms of nitric oxide synthase (eNOS and iNOS, respectively). In the present study L-NAME and the selective iNOS inhibitor aminoguanidine (AG) were administered to differentiate between iNOS and eNOS as the primary source of NO that attenuates the pulmonary vascular response to LPS. Clinically healthy male progeny from ascites-susceptible and ascites-resistant lines were anesthetized, and their pulmonary artery was cannulated. The initial pulmonary arterial pressure (PAP) was recorded, then the broilers either remained untreated (control group) or were injected i.v. with AG. Ten minutes later all birds received an i.v. injection of LPS, followed 40 min later by an i.v. injection of L-NAME. When compared with untreated controls, AG neither increased the baseline PAP nor did it increase or prolong the PH response to LPS. The ascites-susceptible broilers maintained a higher PAP than the ascites-resistant broilers throughout the experiment, and the ascites-resistant broilers exhibited greater relative increases in PAP in response to LPS than did the ascites-susceptible broilers. Within 40 min after the LPS injection, PAP subsided to a level that did not differ from the respective preinjection value for each line. Injecting L-NAME reversed the decline in PAP, and within 5 min PAP returned to hypertensive levels approaching the maximum peak PH response to LPS. The absence of any impact of AG coupled with the profound response to L-NAME indicates that NO synthesized by eNOS rather than iNOS likely modulated the acute (within 1 h) PH elicited by LPS. Evidently eNOS is activated by the increased shear stress exerted on the endothelium during the PH response to LPS, whereas LPS-mediated up-regulation of iNOS expression may take longer than 1 h before biologically effective quantities of NO are produced. PMID:16553285

  20. Spermine synthase

    Michael, Anthony J.

    2010-01-01

    Spermine is present in many organisms including animals, plants, some fungi, some archaea, and some bacteria. It is synthesized by spermine synthase, a highly specific aminopropyltransferase. This review describes spermine synthase structure, genetics, and function. Structural and biochemical studies reveal that human spermine synthase is an obligate dimer. Each monomer contains a C-terminal domain where the active site is located, a central linking domain that also forms the lid of the catalytic domain, and an N-terminal domain that is structurally very similar to S-adenosylmethionine decarboxylase. Gyro mice, which have an X-chromosomal deletion including the spermine synthase (SMS) gene, lack all spermine and have a greatly reduced size, sterility, deafness, neurological abnormalities, and a tendency to sudden death. Mutations in the human SMS lead to a rise in spermidine and reduction of spermine causing Snyder-Robinson syndrome, an X-linked recessive condition characterized by mental retardation, skeletal defects, hypotonia, and movement disorders. PMID:19859664

  1. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    2010-04-01

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National... preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173.115 Food and Drugs FOOD AND DRUG... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may...

  2. IL-1beta-Induced iNOS Expression, NO Release and Loss in Metabolic Cell Viability Are Resistant to Inhibitors of Ceramide Synthase and Sphingomyelinase in INS 832/13 Cells

    Rajakrishnan Veluthakal

    2006-11-01

    Full Text Available Context Emerging evidence indicates regulatory roles for ceramide in the metabolic dysfunction of the islet beta cell. Recently, potential similarities between IL-1beta and ceramide on their effects on islet beta cell have been reported, including reduction in mitochondrial membrane potential and loss in metabolic cell viability.Objective Herein, we investigated whether IL-1beta-induced nitric oxide synthetase (iNOS expression, nitric oxide (NO release and loss in metabolic cell viability require ceramide biosynthesis either via the activation of sphingomyelinase or ceramide synthase.Setting Insulin-secreting INS 832/13 cells.Results We found that two structurally-distinct inhibitors of sphingomyelinase activation (e.g., 3-O-methylsphingomyelin or desipramine or ceramide biosynthesis inhib-itor (e.g., fumonisin failed to exert clear effects on IL-1beta-induced iNOS expression, NO release and loss in cell viability.Conclusions Taken together, our findings indicate that neither the sphingomyelinase nor the ceramide synthase activation is required for IL-1beta-induced metabolic abnormalities in insulin-secreting INS 832/13 cells.

  3. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, interacts with gastric oxidative metabolism and enhances stress-induced gastric lesions.

    Kwiecien, S; Ptak-Belowska, A; Krzysiek-Maczka, G; Targosz, A; Jasnos, K; Magierowski, M; Szczyrk, U; Brzozowski, B; Konturek, S J; Konturek, P C; Brzozowski, T

    2012-10-01

    Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase known to exert vasoconstriction of vascular bed. The elevation of ADMA has been considered as the cardiovascular risk factor associated with hyperlipidemia, hypercholesterolemia and metabolic syndrome. ADMA is produced by the action of dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Previous studies have shown that endogenous NO plays an important role in the mechanism of gastric mucosal defense, but the role of ADMA in the pathogenesis of serious clinical entity, such as the acute gastric mucosal injury induced by stress has been little studied. In present study, we determined the effect of intragastric (i.g.) pretreatment with ADMA applied in graded doses ranging from 0.1 up to 20 mg/kg on gastric mucosal lesions induced by 3.5 h of water immersion and restraint stress (WRS). The number of gastric lesions was determined by planimetry and the gastric blood flow (GBF) was assessed by laser Doppler technique. The malondialdehyde and 4-hydroxynonenal (MDA+4-HNE) concentration, as an index of oxygen radical-lipid peroxidation was assessed in the gastric mucosa in rats exposed to WRS with or without ADMA administration. Proinflammatory cytokines IL-1β, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1β and TNF-α were analyzed by RT-PCR and ELISA, respectively. The exposure of rats to WRS for 3.5 h produced acute gastric lesions accompanied by a significant rise in the plasma ADMA levels and a significant fall in the GBF, an increase in MDA+4-HNE concentrations and the significant increase in the expression and release of IL-1β and TNF-α. The pretreatment with ADMA, applied i.g. 30 min before WRS dose-dependently, aggravated WRS damage and this effect was accompanied by a further significant fall in the GBF. The ADMA induced exacerbation of WRS lesions and the accompanying rise in the plasma ADMA levels and the fall in GBF were significantly attenuated by concurrent treatment with glyceryl trinitrate (GTN) (10 mg/kg i.g.) in the presence of ADMA. Administration of ADMA resulted in a significant decrease in the expression of SOD and GPx mRNAs and the up-regulation of mRNA for IL-1β and TNF-α followed by an increase in these plasma cytokine levels as compared to respective values observed in vehicle-pretreated animals. We conclude that 1) ADMA could be implicated in the mechanism of WRS-induced ulcerogenesis, 2) ADMA exacerbates WRS-induced gastric lesions due to enhancement in neutrophil dependent lipid peroxidation and overexpression and release of proinflammatory cytokines IL-1β and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity. PMID:23211305

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

    M.A. Rizzardi; R.A. VIDAL; N.G. Fleck; D. Agostinetto

    2002-01-01

    A resistncia de plantas aos herbicidas conseqncia, na maioria das vezes, de mutao ou da preexistncia de genes que conferem resistncia populao. No caso dos herbicidas inibidores da acetolactato sintase (ALS) ocorreram casos de resistncia tanto em plantas daninhas quanto em culturas. Essa reviso foi realizada com o objetivo de discutir aspectos bioqumicos, genticos e moleculares da resistncia de plantas aos herbicidas inibidores da ALS, sendo destacados tambm os efeitos na ec...

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

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

    2013-10-18

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

  6. Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

    Thotala, D K; Hallahan, D E; Yazlovitskaya, E M

    2011-01-01

    Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulat...

  7. Activation of β-catenin by inhibitors of glycogen synthase kinase-3 ameliorates cisplatin-induced cytotoxicity and pro-inflammatory cytokine expression in HEI-OC1 cells

    Graphical abstract: - Abstract: Cisplatin is used in the treatment of a wide variety of solid tumors, but its use is limited by its serious adverse effects, including ototoxicity. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions by phosphorylating its substrates. However, the otoprotective effect of GSK-3 inhibitors is poorly understood. Here, we investigated whether GSK-3 is involved in cisplatin-induced ototoxicity in HEI-OC1 cells and organs of Corti (OCs). GSK-3 inhibitors suppressed cisplatin-induced apoptosis determined by decreased p53 activity, and also decreased expression of PARP and p53 target genes such as p21 and PUMA. The effect of GSK-3 inhibitors was mediated by markedly increased nuclear β-catenin that in turn blocked nuclear translocation of NF-κB. siRNA-mediated β-catenin knockdown markedly increased the expression of NF-κB target genes, such as TNF-α and IL-6. Our data suggest that the GSK-3/β-catenin pathway may play a central role in cisplatin-mediated cytotoxicity in HEI-OC1 cells and hair cells of OCs in vitro

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

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

    2015-11-25

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

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

    Brožíčková, Carole; Otáhal, Jakub

    2013-01-01

    Roč. 62, Suppl.1 (2013), S57-S66. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/10/0999; GA ČR(CZ) GPP304/11/P386; GA ČR(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

  10. New Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Inhibitors, Nalidixic Acid Linked to Isatin Schiff Bases via Certain l-Amino Acid Bridges.

    Naglah, Ahmed M; Ahmed, Atallah F; Wen, Zhi-Hong; Al-Omar, Mohamed A; Amr, Abd El-Galil E; Kalmouch, Atef

    2016-01-01

    A series of new Schiff bases were synthesized by condensation of isatins with the nalidixic acid-l-amino acid hydrazides. Prior to hydrazide formation, a peptide linkage has been prepared via coupling of nalidixic acid with appropriate l-amino acid methyl esters to yield 3a-c. The chemical structures of the new Schiff bases (5b and 5d-h) were confirmed by means of IR, NMR, mass spectroscopic, and elemental analyses. The anti-inflammatory activity of these Schiff bases was evaluated via measurement of the expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells model. The Schiff bases exhibited significant dual inhibitory effect against the induction of the pro-inflammatory iNOS and COX-2 proteins with variable potencies. However, they strongly down-regulated the iNOS expression to the level of 16.5% ± 7.4%-42.2% ± 19.6% compared to the effect on COX-2 expression (acid, isatins (4a-c), and l-amino acid moieties against iNOS expression. These synthesized nalidixic acid-l-amino acid-isatin conjugates can be regarded as a novel class of anti-inflammatory antibacterial agents. PMID:27092477

  11. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values. PMID:26586251

  12. Imidazolinones and Acetohydroxyacid Synthase from Higher Plants

    Muhitch, Michael J.; Shaner, Dale L.; Stidham, Mark A.

    1987-01-01

    Acetohydroxyacid synthase has been purified from maize (Zea mays, var Black Mexican Sweet) suspension culture cells 49-fold by a combination of ion exchange chromatography, gel filtration, and hydroxyapatite chromatography. Use of the nondenaturing, zwitterionic detergent 3-([3-cholamidopropyl]dimethyl-ammonio)-1-propanesulfonate was necessary to dissociate the enzyme from the heterogeneous, high molecular weight aggregates in which it appears to reside in vitro. The solubilized maize acetohydroxyacid synthase had a relative molecular mass of 440,000. The purified enzyme was highly unstable. Acetohydroxyacid synthase activities in crude extracts of excised maize leaves and suspension cultured cells were reduced 85 and 58%, respectively, by incubation of the tissue with 100 micromolar (excised leaves) and 5 micromolar (suspension cultures) of the imidazolinone imazapyr prior to enzyme extraction, suggesting that the inhibitor binds tightly to the enzyme in vivo. Binding of imazapyr to maize acetohydroxyacid synthase could also be demonstrated in vitro. Evidence is presented which suggests that the interaction between imazapyr and the enzyme is reversible. Imazapyr also exhibited slow-binding properties when incubated with maize cell acetohydroxyacid synthase in extended time course experiments. Initial and final Ki values for the inhibition were 15 and 0.9 micromolar, respectively. The results suggest that imazapyr is a slow, tight-binding inhibitor of acetohydroxyacid synthase. PMID:16665267

  13. Experimental study on the effects of 7-nitroindazol, selective inhibitor of neuronal nitric oxide synthase (nNOS, on some brain and hepatic biochemical parameters

    Vessela Vitcheva

    2010-10-01

    Full Text Available 7-nitroindazole (7-NI is a selective nNOS inhibitor, which has been proved to have a beneficial effect on different physical processes and behaviors, related to drug abuse, such as tolerance, withdrawal, neurotoxicity and reward. However there are no information available for its toxicity and hepatic biotransformation. The objective of the following study was to investigate the effects of 7-NI on rat brain and liver, after multiple alone administartion. Male Wistar rats were treated with 7-NI (25 mg/kg i.p. for 5 days. 24 hours after the last administration brains and livers were taken for biochemical assay. Being an inhibitor of nNOS, 7-NI significantly decreased the enzyme activity by 41%. For tracing the possible toxic effect of the compound, the quantity of malondialdehyde (MDA and the level of reduced glutathion (GSH were measured both in the brain and in the liver. Multiple administration of 7-NI did not affect the MDA quantity, neither in the brain, nor in the liver that corresponds with the literature data about the positive effect of 7-NI on lipid peroxidation. On the other hand, the GSH level was depleated by 43% in the brain and by 27% in the liver, which suggest a toxic effect of 7-NI. The chemical structure of 7-NI, benzpyrazole, which is a part of the structure of such substrates of cytochrome P 450 as antichelmintes, suggests hepatic metabolism of the compound. The cytochrome P 450 quantity and the activity of ethylmorphine-N-demethylase (EMND and anilinehydroxilase (AH, were also assessed. 7-NI decreased P 450 quantity by 30% and AH activity by 26%. At the same time EMND activity remained unchanged. On the basis of these results we proved a hepatic metabolism of 7-NI that might be responsible for the detected GSH depletion in the liver and could be regarded as a precondition for hepatic drug interactions.

  14. 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

    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 N...

  15. Nitric Oxide Synthase Inhibitors as Antidepressants

    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......, 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...

  16. Nitric Oxide Synthase Inhibitors as Antidepressants

    Vallo Volke

    2010-01-01

    Full Text Available 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. Higher plant cellulose synthases

    Richmond, Todd

    2000-01-01

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

  18. An Arabidopsis callose synthase

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

    2002-01-01

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

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

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

  20. Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase; Sintese e modificacoes de derivados heterociclicos de d-arabinose: potenciais inibidores de glicose-6-fosfato isomerase e de glicosamina-6-fosfato sintase

    Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Fac. de Farmacia. Dept. de Produtos Farmaceuticos]. E-mail: ricardodylan@farmacia.ufmg.br

    2008-07-01

    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. Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

    Muramatsu, Masayoshi; Ohto, Chikara; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

    2008-09-01

    Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol. PMID:18636253

  2. Geranyl diphosphate synthase from mint

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

    1999-03-02

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

  3. Geranyl diphosphate synthase from mint

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

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

  4. A rapid, radiometric assay for sucrose synthase

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

  5. Hybrid polyketide synthases

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

    2016-05-10

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

  6. An Arabidopsis callose synthase

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

    2002-01-01

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

  7. Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin*

    Machutta, Carl A.; Bommineni, Gopal R.; Luckner, Sylvia R.; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J.

    2009-01-01

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in...

  8. Fatty acid synthase: a novel target for antiglioma therapy

    zhao, w; Kridel, S; Thorburn, A; Kooshki, M; Little, J; Hebbar, S; Robbins, M.

    2006-01-01

    High levels of fatty acid synthase (FAS) expression have been observed in several cancers, including breast, prostate, colon and lung carcinoma, compared with their respective normal tissue. We present data that show high levels of FAS protein in human and rat glioma cell lines and human glioma tissue samples, as compared to normal rat astrocytes and normal human brain. Incubating glioma cells with the FAS inhibitor cerulenin decreased endogenous fatty acid synthesis by approximately 50%. Cel...

  9. Properties of prostacyclin synthase.

    Ullrich, V; Brugger, R; Lottspeich, F; Siegle, I

    1997-01-01

    Prostacyclin synthase (PGIS) was isolated from bovine aortic microsomes after detergent solubilisation following purification by DEAE-Sephacel, immobilized metal affinity, and hydroxy apatite chromatography. The homogenous protein exhibited spectral characteristics of a heme-thiolate protein (P450) like the enzyme purified earlier from porcine microsomes and had an apparent mass of 52 kDa on SDS/PAGE. Three peptides from an endoproteinase Lys-C digest were isolated and sequenced. An antiserum was prepared from rabbits and purified by affinity chromatography. This allowed Western blots of microsomes from cultured endothelial cells. After treatment with IL-1 the activity of the cells in producing 6-keto-PGF1 alpha increased about threefold over 27 h which was accompanied by an increase in PGIS mass. A monoclonal antibody was used to set up an ELISA which served for the quantitation of PGIS in bovine tissues. PMID:9547545

  10. Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region.

    Kulmacz, R J

    1989-08-25

    Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2503512

  11. Structure of a cephalosporin synthase

    Valegård, Karin; Terwisscha Van Scheltinga, Anke C; Lloyd, Matthew D.; Hara, Takane; Ramaswamy, S; Perrakis, Anastassis; Thompson, Andy; Lee, Hwei-Jen; Baldwin, Jack E.; Schofield, Christopher J.; Hajdu, Janos; Andersson, Inger

    1998-01-01

    Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the ...

  12. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

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

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

    2001-01-01

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

  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

    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. Bone marrow-derived versus parenchymal sources of inducible nitric oxide synthase in experimental autoimmune encephalomyelitis

    Zehntner, Simone P; Bourbonniere, Lyne; Hassan-Zahraee, Mina; Tran, Elise; Owens, Trevor

    The role of nitric oxide (NO) in central nervous system (CNS) inflammation is uncertain. Whereas experimental autoimmune encephalomyelitis (EAE) is exacerbated in mice deficient in inducible nitric oxide synthase (iNOS), inhibitor studies have suggested a pro-inflammatory role for NO. These discr...

  16. Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine

    Še; #269; kut; #279; , Jolita; McCloskey, Diane E.; Thomas, H. Jeanette; Secrist III, John A.; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Southern Research); (UPENN-MED)

    2011-11-17

    Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 {angstrom} resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K{sub d} of 1.1 {+-} 0.3 {mu}M in the absence of putrescine and 3.2 {+-} 0.1 {mu}M in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

  17. Extramitochondrial citrate synthase activity in bakers' yeast.

    Rickey, T M; Lewin, A S

    1986-01-01

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

  18. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs. PMID:26976449

  19. Sucrose Synthase: Expanding Protein Function

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  20. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    MarinaKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  1. Proteasome inhibitors.

    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

  2. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

    Grundy, Daniel J; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J; Christianson, David W; Allemann, Rudolf K

    2016-04-12

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent. PMID:26998816

  3. Mechanism of germacradien-4-ol synthase controlled water capture

    Grundy, Daniel J.; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J.; Christianson, David W.; Allemann, Rudolf K.

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class-1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (~50% germacrene A) but no direct evidence was obtained about the mechanism of water incorporation in the active site. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent non-competitive inhibitors of GdolS. 12,13-diF-FDP generated 12,13-(E)-β-farnesene when incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with 1-2H2-FDP and (R)-1-2H-FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined and the deuteron in the final product was scrambled. Since no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent. PMID:26998816

  4. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides.

    Laughlin, Thomas F; Ahmad, Zulfiqar

    2010-04-01

    Previously melittin, the alpha-helical basic honey bee venom peptide, was shown to inhibit F(1)-ATPase by binding at the beta-subunit DELSEED motif of F(1)F(o)-ATP synthase. Herein, we present the inhibitory effects of the basic alpha-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F(1) and membrane bound F(1)F(0)Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (approximately 96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of approximately 13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC(50) approximately 3.25 microM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase ( approximately 20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F(1)F(0) membrane preparations as well as in isolated purified F(1). Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F(1)-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase. PMID:20100509

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

    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.

  6. 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

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

    2006-01-01

    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. Plant...

  7. A functional cellulose synthase from ascidian epidermis

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

    2004-01-01

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

  8. Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

    Niamsiri, Nuttawee; Delamarre, Soazig C.; Kim, Young-Rok; Carl A. Batt

    2004-01-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on co...

  9. Efficient heterocyclisation by (di)terpene synthases.

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

    2015-09-11

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

  10. Producing biofuels using polyketide synthases

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

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  11. Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron 1

    Saxena, Praveen K.; King, John

    1990-01-01

    Two cell lines of Datura innoxia resistant to two imidazolinone herbicides, imazapyr and imazaquin, were isolated from mutagenized, predominantly haploid cell suspension cultures. Both of the resistant variants were >1000-fold more resistant than the wild-type to the two imidazolinones. The variant resistant to imazapyr showed cross-resistance to imazaquin and vice versa, but no cross-resistance to a structurally different inhibitor, chlorsulfuron, a sulfonylurea herbicide, was observed. The target enzyme, acetolactate synthase, extracted from imidazolinone-resistant cell lines was not inhibited by imazapyr or imazaquin but was sensitive to chlorsulfuron indicating separable sites of action for these inhibitors. The variation in resistance and cross-resistance of chlorsulfuron-resistant (PK Saxena, J King [1988] Plant Physiol 86: 863-867) and imidazolinone-resistant cell lines of Datura innoxia demonstrates the possibility of separate mutations of acetolactate synthase gene resulting in specific phenotypes. PMID:16667804

  12. Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum.

    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

  13. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  14. Properties of phosphorylated thymidylate synthase

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Pawel; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat......, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous m......RNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and Fd...

  15. Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

    Wilson, Nedra F.; Lefebvre, Paul A.

    2004-01-01

    Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins ...

  16. Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

    Kasten, T P; Collin-Osdoby, P; Patel, N.; Osdoby, P; Krukowski, M; Misko, T. P.; Settle, S L; Currie, M G; Nickols, G. A.

    1994-01-01

    We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the pr...

  17. Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

    Ketabchi Farzaneh

    2012-01-01

    Full Text Available Abstract Background Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV optimizes gas exchange during local acute (0-30 min, as well as sustained (> 30 min hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. Method We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate, and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min and endothelial permeability. Results In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA, a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS, decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc. This increase disappeared after administration of 1400 W. Conclusion Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.

  18. Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

    Wang, Dan; Strandgaard, Svend; Iversen, Jens; Wilcox, Christopher S

    2008-01-01

    We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadien...

  19. ACE inhibitors

    ... including anything you bought without a prescription, diuretics (water pills), potassium pills, or herbal or dietary supplements. Do not take ACE inhibitors if you are planning to become pregnant, pregnant, ...

  20. Angiogenesis Inhibitors

    ... treatment with angiogenesis inhibitors can include problems with bleeding, clots in the arteries (with resultant stroke or heart attack), hypertension, and protein in the urine ( 3 – 5 ). Gastrointestinal perforation and fistulas also appear to be rare ...

  1. Purification and characterization of prostaglandin F synthase from bovine liver.

    Chen, L Y; Watanabe, K; Hayaishi, O

    1992-07-01

    Prostaglandin D2 11-ketoreductase activity of bovine liver was purified 340-fold to apparent homogeneity. The purified enzyme was a monomeric protein with a molecular weight of about 36 kDa, and had a broad substrate specificity for porstaglandins D1, D2, D3, and H2, and various carbonyl compounds (e.g., phenanthrenequinone and nitrobenzaldehyde, etc.). Prostaglandin D2 was reduced to 9 alpha,11 beta-prostaglandin F2 and prostaglandin H2 to prostaglandin F2 alpha with NADPH as a cofactor. Phenanthrenequinone competitively inhibited the reduction of prostaglandin D2, while it did not inhibit that of prostaglandin H2. Moreover, chloride ion stimulated the reduction of prostaglandin D2 and carbonyl compounds, while it had no effect on that of prostaglandin H2. Besides, the enzyme was inhibited by flavonoids (e.g., quercetin) that inhibit carbonyl reductase, but was not inhibited by barbital and sorbinil, which are the inhibitors of aldehyde and aldose reductases, respectively. These results indicate that the bovine liver enzyme has two different active sites, i.e., one for prostaglandin D2 and carbonyl compounds and the other for prostaglandin H2, and appears to be a kind of carbonyl reductase like bovine lung prostaglandin F synthase (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O., 1985, J. Biol. Chem. 260, 7035-7041). However, the bovine liver enzyme was different from prostaglandin F synthase of bovine lung with regard to the Km value for prostaglandin D2 (10 microM for the liver enzyme and 120 microM for the lung enzyme), the sensitivity to chloride ion (threefold greater activation for the liver enzyme) and the inhibition by CuSO4 and HgCl2 (two orders of magnitude more resistant in the case of the liver enzyme). These results suggest that the bovine liver enzyme is a subtype of bovine lung prostaglandin F synthase. PMID:1605628

  2. Corrosion inhibitors

    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

  3. Arabidopsis CDS blastp result: AK242817 [KOME

    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 ...

  4. Arabidopsis CDS blastp result: AK058963 [KOME

    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 ...

  5. Arabidopsis CDS blastp result: AK109628 [KOME

    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 ...

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

    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-3β. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3β 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

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

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

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

  8. Genetics Home Reference: GM3 synthase deficiency

    ... Some affected individuals have changes in skin coloring (pigmentation), including dark freckle-like spots on the arms ... shortage of GM3 synthase and changes in skin pigmentation is also unknown. Read more about the ST3GAL5 ...

  9. Fungal type III polyketide synthases.

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs. PMID:25182423

  10. Identification of avian wax synthases

    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.

  11. The tomato terpene synthase gene family.

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

    2011-10-01

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

  12. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa.

    Ninnemann, H; Maier, J

    1996-08-01

    Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitic oxide donor, sodium nitroprusside, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives NG -nitro-L-arginine (L-NA) and NG-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness nad in the light, whereas the stereoisomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photo-physiology. PMID:8760579

  13. Molecular evolution of dihydrouridine synthases

    Kasprzak Joanna M

    2012-06-01

    Full Text Available Abstract Background Dihydrouridine (D is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS. DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family. Results To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model. Conclusions We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.

  14. Properties of phosphorylated thymidylate synthase.

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  15. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed. PMID:26123522

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

    Marcelo Carnier Dornelas

    2007-01-01

    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 airborne 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.

  17. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  18. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

    Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å. Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP-354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents

  19. Glycogen synthase kinase 3, circadian rhythms, and bipolar disorder: a molecular link in the therapeutic action of lithium

    Kaladchibachi, Sevag A; Doble, Brad; Anthopoulos, Norman; Woodgett, James R; Manoukian, Armen S.

    2007-01-01

    Background Bipolar disorder (BPD) is a widespread condition characterized by recurring states of mania and depression. Lithium, a direct inhibitor of glycogen synthase kinase 3 (GSK3) activity, and a mainstay in BPD therapeutics, has been proposed to target GSK3 as a mechanism of mood stabilization. In addition to mood imbalances, patients with BPD often suffer from circadian disturbances. GSK3, an essential kinase with widespread roles in development, cell survival, and metabolism has been d...

  20. Loop residues and catalysis in OMP synthase

    Wang, Gary P.; Hansen, Michael Riis; Grubmeyer, Charles

    2012-01-01

    Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100?109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 104-fold decrease...

  1. Producing dicarboxylic acids using polyketide synthases

    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.

  2. Nuclear genetic defects of mitochondrial ATP synthase

    Houštěk, Josef; Kmoch, S.; Mayr, J. A.; Sperl, W.; Zeman, J.

    Bari : University of Bari, 2008. L5.3-L5.3. [IUBMB Symposium S1. 22.06.2008-26.06.2008, Bari] R&D Projects: GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50110509 Keywords : spr2 * mitochondrial disease * ATP synthase defects * nuclear mutation Subject RIV: EB - Genetics ; Molecular Biology

  3. Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase

    Bmmel, Heike M.; Reif, Andreas; Frhlich, Lothar G.; Frey, Armin; Hofmann, Heinrich; Marecak, Dale M.; Groehn, Viola; Kotsonis, Peter; La, Mylinh; Kster, Sandra; Meinecke, Matthias; Bernhardt, Manfred; Weeger, Monika; Ghisla, Sandro; Prestwich, Glenn D.

    1998-01-01

    Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors...

  4. 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

    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.

  5. Competition effects with mixed stands of wheat and kochia (Kochia scoparia biotypes resistant and susceptible to acetolactase synthase inhibitor herbicides Efeitos competitivos da mistura de stands de trigo e biotipos de kochia (Kochia scoparia resistentes e susceptveis aos herbicidas inibidores da acetolactase sintase

    P.J. Christoffoleti

    1994-08-01

    Full Text Available Greenhouse experiments were conducted to compare the competitive ability of sulfonylurea resistant and susceptible kochia (Kochia scoparia L. Schard compared to wheat. The results of several replacement series experiments indicate that wheat was the dominant competitor, and an average of one wheat plant reduced resistant kochia yield per plant equal to the effect of 4.8 resistant kochia or 5.4 susceptible kochia plants. Intraspeciflc competition was more important than interspecific competition for wheat, whereas the reverse was true for the resistant and susceptible kochia. The results of the niche differentiation index (NDI indicate that wheat and either resistant or susceptible kochia are only partly limited by the same resources. The resistant and susceptible kochia, however, are limited by the same resources.Experimentos foram instalados em condies de casa-de-vegetao com o objetivo de comparar a capacidade competitiva de biotipos resistentes e suscetveis aos herbicidas inibidores da enzima acetolactase synthase da planta daninha kochia (Kochia scoparia L. Schard comparada com trigo. Os resultados de diversos experimentos, utilizando a metodologia chamada de substitutiva, indicaram que o trigo foi o competidor dominante, e em mdia uma planta de trigo reduziu o crescimento da planta de kochia resistente igual ao efeito de 4,8 plantas de kochia resistente ou 5,4 plantas de kochia suscetvel. A competio chamada de intraespecfca foi mais importante que a competio interespecfica para o trigo, porm o inverso foi verdadeiro para os biotpos resistentes e susceptveis de kochia. Os resultados do ndice de diferenciao ecolgica indicaram que trigo e qualquer um dos dois biotpos de kochia estudados foram limitados apenas parcialmente pelos mesmos recursos de crescimento. No entanto, o crescimento dos biotpos resistentes e susceptveis de kochia foram limitados pelos mesmos fatores de crescimento.

  6. Mitochondrial β-Cyanoalanine Synthase Is Essential for Root Hair Formation in Arabidopsis thaliana[W

    García, Irene; Castellano, José María; Vioque, Blanca; Solano, Roberto; Gotor, Cecilia; Romero, Luis C.

    2010-01-01

    Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development. PMID:20935247

  7. Mitochondrial beta-cyanoalanine synthase is essential for root hair formation in Arabidopsis thaliana.

    García, Irene; Castellano, José María; Vioque, Blanca; Solano, Roberto; Gotor, Cecilia; Romero, Luis C

    2010-10-01

    Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development. PMID:20935247

  8. Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus.

    Braden, B C; Velikovsky, C A; Cauerhff, A A; Polikarpov, I; Goldbaum, F A

    2000-04-14

    We have determined the three-dimensional structure of 6, 7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals. This enzyme catalyses the formation of 6, 7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. The three-dimensional X-ray crystal structure of the enzyme from B. abortus has been solved and refined at 2.7 A resolution to a final R-value of 0.18 (R(free)=0.23). The macromolecular assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known. While the protein from B. subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B. abortus is pentameric in its crystalline form. Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range. Furthermore, we compare the structures of the enzyme from B. subtilis and B. abortus and describe the C teminus structure which accounts for the differences in quaternary structure. PMID:10764570

  9. Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

    Hiltunen, M. (Maarit); Söderhäll, K

    1992-01-01

    The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.

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

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger; Harholt, Jesper; Sørensen, Susanne Oxenbøll; Moller, Isabel Eva; Blaukopf, Claudia; Johansen, Bo Buchholt; Delotto, Robert; Pauly, Markus; Scheller, Henrik Vibe; Willats, William George Tycho

    2007-01-01

    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...

  11. Caffeine synthase and related methyltransferases in plants.

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region. PMID:14977590

  12. Nitric oxide synthase in the pineal gland

    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 ...

  13. (R)-Citramalate Synthase in Methanogenic Archaea

    Howell, David M.; Xu, Huimin; White, Robert H.

    1999-01-01

    The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate. Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine.

  14. The Eucalyptus terpene synthase gene family

    Külheim, Carsten; Padovan, Amanda; Hefer, Charles; Krause, Sandra T; Köllner, Tobias G; Myburg, Alexander A.; Degenhardt, Jörg; Foley, William J.

    2015-01-01

    Background Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. Results The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissu...

  15. Tertiary model of a plant cellulose synthase

    Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

    2013-01-01

    A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a ...

  16. Allosteric Inhibition of Human Porphobilinogen Synthase*

    Lawrence, Sarah H.; Ramirez, Ursula D.; Selwood, Trevor; Stith, Linda; Jaffe, Eileen K.

    2009-01-01

    Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states ...

  17. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

  18. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  19. Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to α-Difluoromethylornithine*♦

    Wang, Xiaojing; Levic, Snezana; Gratton, Michael Anne; Doyle, Karen Jo; Yamoah, Ebenezer N.; Pegg, Anthony E.

    2009-01-01

    Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear. PMID:19001365

  20. Biochemical and Structural Basis for Inhibition of Enterococcus faecalis Hydroxymethylglutaryl-CoA Synthase, mvaS, by Hymeglusin

    Skaff, D. Andrew; Ramyar, Kasra X.; McWhorter, William J.; Barta, Michael L.; Geisbrecht, Brian V.; Miziorko, Henry M. (UMKC)

    2012-07-25

    Hymeglusin (1233A, F244, L-659-699) is established as a specific {beta}-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 {angstrom}) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

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

    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.

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

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

    1997-01-01

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

  3. Cellulose synthase interacting protein: A new factor in cellulose synthesis

    Gu, Ying; Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the re...

  4. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  5. In Vivo Enzyme Immobilization by Use of Engineered Polyhydroxyalkanoate Synthase

    Peters, Verena; Rehm, Bernd H A

    2006-01-01

    This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The ?-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase...

  6. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    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. 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. 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. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

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

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

    2001-12-01

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

  8. Interleukin 1 beta induces diabetes and fever in normal rats by nitric oxide via induction of different nitric oxide synthases

    Reimers, J I; Bjerre, U; Mandrup-Poulsen, T; Nerup, J

    1994-01-01

    Substantial in vitro evidence suggests that nitric oxide may be a major mediator of interleukin 1 (IL-1) induced pancreatic beta-cell inhibition and destruction in the initial events leading to insulin-dependent diabetes mellitus. Using NG-nitro-L-arginine methyl ester, an inhibitor of both the......, glucagon, corticosterone and leukocyte- and differential-counts in normal rats injected once daily for 5 days with interleukin 1 beta (IL-1 beta) (0.8 microgram/rat = 4.0 micrograms/kg). Inhibition of both the constitutive and the inducible forms of nitric oxide synthase prevented IL-1 beta-induced fever......, hyperglycaemia, hypoinsulinemia, and hyperglucagonemia, and partially prevented lymphopenia and neutrophilia, but had no effect on IL-1 beta-induced anorexia and changes in plasma corticosterone. Preferential inhibition of the inducible form of nitric oxide synthase using two daily injections of 5 mg/rat of...

  9. Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice

    eyler Nurcan

    2010-03-01

    Full Text Available Abstract Background Although it has been largely demonstrated that nitric oxide synthase (NOS, a key enzyme for nitric oxide (NO production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results Intraperitoneal (i.p. pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor, aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor, L-N(G-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor, but not L-N(5-(1-iminoethyl-ornithine (L-NIO, a selective endothelial NOS inhibitor, significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl. injection of complete Freund's adjuvant (CFA. Real-time reverse transcription-polymerase chain reaction (RT-PCR revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF, interleukin-1 beta (IL-1?, and interleukin-10 (IL-10 gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1?. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO mice had lower gene expression of TNF, IL-1?, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

  10. Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage

    The use of radiation therapy has been linked to an increased risk of cardiovascular disease. To understand the mechanisms underlying radiation-induced vascular dysfunction, we employed two models. First, we examined the effect of X-ray irradiation on vasodilation in rabbit carotid arteries. Carotid arterial rings were irradiated with 8 or 16 Gy using in vivo and ex vivo methods. We measured the effect of acetylcholine-induced relaxation after phenylephrine-induced contraction on the rings. In irradiated carotid arteries, vasodilation was significantly attenuated by both irradiation methods. The relaxation response was completely blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a potent inhibitor of soluble guanylate cyclase. Residual relaxation persisted after treatment with L-Nω-nitroarginine (L-NA), a non-specific inhibitor of nitric oxide synthase (NOS), but disappeared following the addition of aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS). The relaxation response was also affected by tetraethylammonium, an inhibitor of endothelium-derived hyperpolarizing factor activity. In the second model, we investigated the biochemical events of nitrosative stress in human umbilical-vein endothelial cells (HUVECs). We measured iNOS and nitrotyrosine expression in HUVECs exposed to a dose of 4 Gy. The expression of iNOS and nitrotyrosine was greater in irradiated HUVECs than in untreated controls. Pretreatment with AG, L-N6-(1-iminoethyl) lysine hydrochloride (a selective inhibitor of iNOS), and L-NA attenuated nitrosative stress. While a selective target of radiation-induced vascular endothelial damage was not definitely determined, these results suggest that NO generated from iNOS could contribute to vasorelaxation. These studies highlight a potential role of iNOS inhibitors in ameliorating radiation-induced vascular endothelial damage. (author)

  11. Engineering of chimeric class II polyhydroxyalkanoate synthases.

    Niamsiri, Nuttawee; Delamarre, Soazig C; Kim, Young-Rok; Batt, Carl A

    2004-11-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes. PMID:15528546

  12. Sphingomyelin Synthases Regulate Protein Trafficking and Secretion

    Subathra, Marimuthu; Qureshi, Asfia; Luberto, Chiara

    2011-01-01

    Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 r...

  13. Topographic studies of microsomal and pure prostaglandin H synthase.

    Kulmacz, R J; Wu, K K

    1989-02-01

    Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2492419

  14. Proton pump inhibitors

    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 ...

  15. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: The human pharmacology of a selective inhibitor of COX-2

    McAdam, B.F.; Catella-Lawson, F.; Mardini, I. A.; S. Kapoor; Lawson, J. A.; Fitzgerald, G A

    1999-01-01

    Prostaglandins (PG) are synthesized by two isoforms of the enzyme PG G/H synthase [cyclooxygenase (COX)]. To examine selectivity of tolerated doses of an inhibitor of the inducible COX-2 in humans, we examined the effects of celecoxib on indices of COX-1-dependent platelet thromboxane (Tx) A2 and on systemic biosynthesis of prostacyclin in vivo. Volunteers received doses of 100, 400, or 800 mg of celecoxib or 800 mg of a nonselective inhibitor, ibuprofen. Ibuprofen, but not celecoxib, signifi...

  16. Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

    Epelbaum, S.; Chipman, D M; Barak, Z

    1996-01-01

    The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The...

  17. Localization of nitric oxide synthase in human skeletal muscle

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

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed in...

  18. Loss of LRPPRC causes ATP synthase deficiency.

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Kühlbrandt, Werner; Larsson, Nils-Göran

    2014-05-15

    Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria. PMID:24399447

  19. Organic corrosion inhibitors

    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)

  20. STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE

    T. Femlin Blessia

    2012-02-01

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

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

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

    1995-01-01

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

  2. Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum

    Bitencourt, Tamires Aparecida; Komoto, Tatiana Takahasi; Massaroto, Bruna Gabriele; MIRANDA, Carlos Eduardo Saraiva; Beleboni, Rene Oliveira; Marins, Mozart; Fachin, Ana Lúcia

    2013-01-01

    Background Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis. Methods The antifungal activity of th...

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

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

    2014-01-01

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

  4. Synthesis of 1,2[3H]-1,2-epoxy analogue of fructose-6P, an affinity label of Escherichia coli glucosamine-6P synthase

    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)

  5. Pseudouridines and pseudouridine synthases of the ribosome.

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

    2001-01-01

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

  6. Expression in Arabidopsis of a strawberry linalool synthase gene under the control of the inducible potato P12 promoter

    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...

  7. Inhibition of nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects.

    Handy, R. L.; Harb, H. L.; Wallace, P.; Gaffen, Z.; Whitehead, K J; Moore, P K

    1996-01-01

    1. The ability of a range of substituted imidazole compounds to inhibit mouse cerebellar neuronal nitric oxide synthase (nNOS), bovine aortic endothelial NOS (eNOS) and inducible NOS (iNOS) from lungs of endotoxin-pretreated rats was investigated. In each case the substrate (L-arginine) concentration employed was 120 nM. 2. 1-(2-Trifluoromethylphenyl) imidazole (TRIM) was a relatively potent inhibitor of nNOS and iNOS (IC50S of 28.2 microM and 27.0 microM respectively) but was a relatively we...

  8. Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: effect of cold acclimation.

    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

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

    Rossi Adriano G

    2006-09-01

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

  10. Glycogen synthase kinase-3: A promising therapeutic target for Fragile X Syndrome

    Richard Scott Jope

    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.

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

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

  12. Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes

    Li, Shundai; LEI, LEI; Somerville, Chris R; Gu, Ying

    2011-01-01

    Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtu...

  13. Biosynthetic potential of sesquiterpene synthases: Alternative products of tobacco 5-epi-aristolochene synthase

    O’Maille, Paul E.; Chappell, Joe; Noel, Joseph P.

    2006-01-01

    Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC–MS assay. The combined use of chiral and non...

  14. Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

    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 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

  15. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly. PMID:26734760

  16. Pyridoxal-phosphate dependent mycobacterial cysteine synthases: Structure, mechanism and potential as drug targets.

    Schnell, Robert; Sriram, Dharmarajan; Schneider, Gunter

    2015-09-01

    The alarming increase of drug resistance in Mycobacterium tuberculosis strains poses a severe threat to human health. Chemotherapy is particularly challenging because M. tuberculosis can persist in the lungs of infected individuals; estimates of the WHO indicate that about 1/3 of the world population is infected with latent tuberculosis providing a large reservoir for relapse and subsequent spread of the disease. Persistent M. tuberculosis shows considerable tolerance towards conventional antibiotics making treatment particularly difficult. In this phase the bacilli are exposed to oxygen and nitrogen radicals generated as part of the host response and redox-defense mechanisms are thus vital for the survival of the pathogen. Sulfur metabolism and de novo cysteine biosynthesis have been shown to be important for the redox homeostasis in persistent M. tuberculosis and these pathways could provide promising targets for novel antibiotics for the treatment of the latent form of the disease. Recent research has provided evidence for three de novo metabolic routes of cysteine biosynthesis in M. tuberculosis, each with a specific PLP dependent cysteine synthase with distinct substrate specificities. In this review we summarize our present understanding of these pathways, with a focus on the advances on functional and mechanistic characterization of mycobacterial PLP dependent cysteine synthases, their role in the various pathways to cysteine, and first attempts to develop specific inhibitors of mycobacterial cysteine biosynthesis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. PMID:25484279

  17. Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

    Suzuki, Motoshi; Cao, Ke; Kato, Seiichi; Komizu, Yuji; Mizutani, Naoki; Tanaka, Kouji; Arima, Chinatsu; Tai, Mei Chee; Yanagisawa, Kiyoshi; Togawa, Norie; Shiraishi, Takahiro; Usami, Noriyasu; Taniguchi, Tetsuo; Fukui, Takayuki; Yokoi, Kohei; Wakahara, Keiko; Hasegawa, Yoshinori; Mizutani, Yukiko; Igarashi, Yasuyuki; Inokuchi, Jin-Ichi; Iwaki, Soichiro; Fujii, Satoshi; Satou, Akira; Matsumoto, Yoko; Ueoka, Ryuichi; Tamiya-Koizumi, Keiko; Murate, Takashi; Nakamura, Mitsuhiro; Kyogashima, Mamoru; Takahashi, Takashi

    2016-01-01

    Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC. PMID:26650179

  18. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    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.

  19. Bacterial Na+-ATP synthase has an undecameric rotor

    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 α-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 atomic ...

  20. Pseudoguaianolides isolated from Inula britannica var. chinenis as inhibitory constituents against inducible nitric oxide synthase.

    Lee, Hyun-Tai; Yang, Seung-Won; Kim, Kyeong Ho; Seo, Eun-Kyoung; Mar, Woongchon

    2002-04-01

    Three pseudoguaianolide type sesquiterpenes, bigelovin (1), 2,3-dihydroaromaticin (2), and ergolide (3) were isolated as inhibitory constituents against inducible nitric oxide synthase (iNOS) from the flowers of Inula britannica var. chinensis. Bigelovin (1) exhibited a highly potent inhibitory activity on lipopolysaccharide (LPS)-induced iNOS in murine macrophage RAW 264.7 cells with an IC50 value of 0.46 mM, which is about 8 times more potent than the known selective inhibitor of iNOS, L-N6-(1-iminoethyl)lysine (IC50 3.49 microM). 2,3-Dihydroaromaticin (2) and ergolide (3) also exhibited potent inhibitory activities on LPS-induced iNOS with IC50 values of 1.05 and 0.69 microM, respectively. PMID:12009027

  1. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower. PMID:23123550

  2. INHIBITOR IN HEMOPHILIA

    Veny K Yantie

    2013-03-01

    Full Text Available Hemophilia is an X-linked recessive disorder which is believed to affect approximately one in 5000-10.000 male birth. An inhibitor is a type of antibody. In hemophilia patients type A, B, and C are directly destroy factor VII, IX, and XI. The incidence of antibody development in hemophilia A is between 20% and 40%, hemophilia B inhibitors only 1 to 6 %. The presence of an inhibitor is usually confirmed using a specific blood test called the Bethesda inhibitor assay. The treatment of hemophilic bleeding in a person with an inhibitor can be a challenging experience. Patients hemophilia with inhibitor have poor prognostic. (MEDICINA 2012;43:31-36.

  3. Seed dormancy is modulated in recently evolved chlorsulfuron-resistant Turkish biotypes of wild mustard (Sinapis arvensis)

    Topuz, Muhamet; Nemli, Yildiz; Fatima, Tahira; Mattoo, Autar K.

    2015-01-01

    Biotypes of the broad-leaved wild mustard (Sinapis arvensis L.) found in wheat fields of Aegean and Marmara region of Turkey were characterized and shown to have developed resistance to sulfonylurea (chlorsulfuron), an inhibitor of acetolactate synthase (ALS). DNA sequence analysis of the ALS genes from two such resistant (“R”) biotypes, KNF-R1 and KNF-R2, revealed point mutations, CCT (Pro 197) to TCT (Ser 197) in KNF-R1 and CCT (Pro 197) to ACT (Thr 197) in KNF-R2; these substitutions are c...

  4. A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    Schulz, Sarah; Iglesias-Cans, Marina; Krah, Alexander; Yildiz, zkan; Leone, Vanessa; Matthies, Doreen; Cook, Gregory M.

    2013-01-01

    The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. PMID:23824040

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

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

    1987-05-01

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

  6. Comparative toxicity of 20 herbicides to 5 periphytic algae and the relationship with mode of action.

    Nagai, Takashi; Taya, Kiyoshi; Yoda, Ikuko

    2016-02-01

    The authors used 5 species of periphytic algae to conduct toxicity assays of 20 herbicides. The 5 tested species represent riverine primary producers most likely to be affected by herbicides. A fluorescence microplate toxicity assay was used as an efficient and economical high-throughput assay. Toxicity characteristics were analyzed, focusing on their relationship to herbicide mode of action. The relative differences between 50% and 10% effect concentrations depended on herbicide mode of action, rather than tested species. Moreover, a clear relationship between sensitive species and herbicide mode of action was also observed. Green alga was most sensitive to herbicides of 2 mode of action groups: inhibitors of protoporphyrinogen oxidase and very long-chain fatty acid synthesis. Diatoms were most sensitive to herbicides of 1 mode of action group: 4-hydroxyphenyl-pyruvate-dioxygenase inhibitors. Cyanobacterium was most sensitive to herbicides of 1 mode of action group: inhibitors of acetolactate synthase. The species sensitivity distribution based on obtained data was also analyzed. The slopes of the species sensitivity distribution significantly differed among modes of action, suggesting that difference in species sensitivity is specific to the mode of action. In particular, differences in species sensitivity were markedly large for inhibitors of acetolactate synthase, protoporphyrinogen oxidase, and very long-chain fatty acid synthesis. The results clearly showed that a single algal species cannot represent the sensitivity of an algal assemblage. Therefore, multispecies algal toxicity data are essential for substances with specific modes of action. Environ Toxicol Chem 2016;35:368-375. 2015 SETAC. PMID:26174500

  7. INHIBITOR IN HEMOPHILIA

    Veny K Yantie; Ariawati K

    2013-01-01

    Hemophilia is an X-linked recessive disorder which is believed to affect approximately one in 5000-10.000 male birth. An inhibitor  is a type of antibody. In hemophilia patients type A, B, and C are directly destroy factor VII, IX, and XI. The incidence of antibody development in hemophilia A is between 20% and 40%, hemophilia B inhibitors only 1 to 6 %. The presence of an inhibitor is usually confirmed using a specific blood test called the Bethesda inhibitor assay. The treatment of hemophil...

  8. Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum

    Nakai, Tomonori; Tonouchi, Naoto; Konishi, Teruko; Kojima, Yukiko; Tsuchida, Takayasu; Yoshinaga, Fumihiro; Sakai, Fukumi; Hayashi, Takahisa

    1999-01-01

    Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-β-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the...

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

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

    2015-01-01

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

  10. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather......Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase in the...... human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than in the...

  11. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

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

    2015-01-01

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

  12. Inhibition of Fatty Acid Synthase Sensitizes Prostate Cancer Cells to Radiotherapy.

    Rae, Colin; Haberkorn, Uwe; Babich, John W; Mairs, Robert J

    2015-11-01

    Many common human cancers, including colon, prostate and breast cancer, express high levels of fatty acid synthase compared to normal human tissues. This elevated expression is associated with protection against apoptosis, increased metastasis and poor prognosis. Inhibitors of fatty acid synthase, such as the cerulenin synthetic analog C75, decrease prostate cancer cell proliferation, increase apoptosis and decrease tumor growth in experimental models. Although radiotherapy is widely used in the treatment of prostate cancer patients, the risk of damage to neighboring normal organs limits the radiation dose that can be delivered. In this study, we examined the potential of fatty acid synthase inhibition to sensitize prostate cancer cells to radiotherapy. The efficacy of C75 alone or in combination with X irradiation was examined in monolayers and in multicellular tumor spheroids. Treatment with C75 alone decreased clonogenic survival, an effect that was abrogated by the antioxidant. C75 treatment also delayed spheroid growth in a concentration-dependent manner. The radiosensitizing effect of C75 was indicated by combination index values between 0.65 and 0.71 and the reduced surviving fraction of clonogens, in response to 2 Gy X irradiation, from 0.51 to 0.30 and 0.11 in the presence of 25 and 35 μM C75, respectively. This increased sensitivity to radiation was reduced by the presence of the antioxidant. The C75 treatment also enhanced the spheroid growth delay induced by X irradiation in a supra-additive manner. The level of radiation-induced apoptosis in prostate cancer cells was increased further by C75, which induced cell cycle arrest in the G2/M phase, but only at a concentration greater than that required for radiosensitization. Radiation-induced G2/M blockade was not affected by C75 treatment. These results suggest the potential use of fatty acid synthase inhibition to enhance the efficacy of radiotherapy of prostate carcinoma and that C75-dependent cell cycle arrest is not responsible for its radiosensitizing effect. PMID:26484401

  13. Improvement of Dolichol-linked Oligosaccharide Biosynthesis by the Squalene Synthase Inhibitor Zaragozic Acid*

    Haeuptle, Micha A.; Welti, Michael; Troxler, Heinz; Hlsmeier, Andreas J; Imbach, Timo; Hennet, Thierry

    2010-01-01

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG....

  14. Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters

    Brožíčková, Carole; Mikulecká, Anna; Otáhal, Jakub

    2014-01-01

    Roč. 63, č. 5 (2014), s. 637-648. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/10/0999; GA ČR(CZ) GPP304/11/P386; GA ČR(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: 1.293, year: 2014

  15. Regulation of mitochondrial ATP synthase in cardiac pathophysiology

    Long, Qinqiang; Yang, Kevin; Yang., Qinglin

    2015-01-01

    Mitochondrial function is paramount to energy homeostasis, metabolism, signaling, and apoptosis in cells. Mitochondrial complex V (ATP synthase), a molecular motor, is the ultimate ATP generator and a key determinant of mitochondrial function. ATP synthase catalyzes the final coupling step of oxidative phosphorylation to supply energy in the form of ATP. Alterations at this step will crucially impact mitochondrial respiration and hence cardiac performance. It is well established that cardiac ...

  16. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

    Khosla, Chaitan; Herschlag, Daniel; Cane, David E.; Walsh, Christopher T.

    2014-01-01

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase doma...

  17. Complete Reconstitution of a Highly-Reducing Iterative Polyketide Synthase

    Ma, Suzanne M.; Li, Jesse W.-H.; Choi, Jin W; Zhou, Hui; Lee, K. K. Michael; Moorthie, Vijayalakshmi A.; Xie, Xinkai; Kealey, James T.; Da Silva, Nancy A.; Vederas, John C.; Tang, Yi

    2009-01-01

    Highly-reducing iterative polyketide synthases are large multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of LovB (the Lovastatin Nonaketide Synthase) from an engineered strain of Saccharomyces cerevisiae, and complete reconstitution of its catalytic function in the presence and absence of cofactors (NADPH, SAM) and its partner enzyme, the enoyl reductase LovC. The results...

  18. Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

    Wong, H C; Fear, A L; Calhoon, R D; Eichinger, G H; Mayer, R; Amikam, D; Benziman, M; Gelfand, D H; Meade, J H; Emerick, A W

    1990-01-01

    An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the co...

  19. Subcellular Targeting Domains of Sphingomyelin Synthase 1 and 2

    Yeang Calvin; Ding Tingbo; Chirico William J; Jiang Xian-Cheng

    2011-01-01

    Abstract Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS...

  20. Early stages of crystallization of lumazine synthase from Bacillus subtilis

    Neumaier, Nikolaus

    2007-01-01

    The subject of this study was the analysis of solubility, particle interactions and aggregation behavior of lumazine synthase from Bacillus subtilis. Lumazine synthase crystallizes in sodium/potassium phosphate buffer, pH 8.7. Contrary to the decrease in solubility with increasing salt concentration, the osmotic second virial coefficient remains almost constant and reveals the same slight attractive interactions under storage and crystallization conditions. In search of protein clusters that ...

  1. Regulation by glycogen synthase kinase-3 of inflammation and T cells in CNS diseases

    Eleonore Beurel

    2011-08-01

    Full Text Available Elevated markers of neuroinflammation have been found to be associated with many psychiatric and neurodegenerative diseases, such as mood disorders, Alzheimer's disease, and multiple sclerosis. Since neuroinflammation is thought to contribute to the pathophysiology of these diseases and to impair responses to therapeutic interventions and recovery, it is important to identify mechanisms that regulate neuroinflammation and potential targets for controlling neuroinflammation. Recent findings have demonstrated that glycogen synthase kinase-3 (GSK3 is an important regulator of both the innate and adaptive immune systems' contributions to inflammation. Studies of the innate immune system have shown that inhibitors of GSK3 profoundly alter the repertoire of cytokines that are produced both by peripheral and central cells, reducing proinflammatory cytokines and increasing anti-inflammatory cytokines. Furthermore, inhibitors of GSK3 promote tolerance to inflammatory stimuli, reducing inflammatory cytokine production upon repeated exposure. Studies of the adaptive immune system have shown that GSK3 regulates the production of cytokines by T cells and the differentiation of T cells to subtypes, particularly Th17 cells. Regulation of transcription factors by GSK3 appears to play a prominent role in its regulation of immune responses, including of NF-κB, cyclic AMP response element binding protein (CREB, and signal transducer and activator of transcription-3 (STAT3. In vivo studies have shown that GSK3 inhibitors ameliorate clinical symptoms of both peripheral and central inflammatory diseases, particularly experimental autoimmune encephalomyelitis (EAE, the animal model of multiple sclerosis. Therefore, the development and application of GSK3 inhibitors may provide a new therapeutic strategy to reduce neuroinflammation associated with many CNS diseases.

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

    Ting Xu

    2015-03-01

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

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

    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 37.5C 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.

  4. What Is the True Structure of D609, a Widely Used Lipid Related Enzyme Inhibitor?

    Kato, Mikako; Hammam, Mostafa A S; Taniguchi, Tohru; Suga, Yoshiko; Monde, Kenji

    2016-02-19

    D609 (1) has been used as a lipid-related enzyme inhibitor during the past three decades. Although it has eight possible stereoisomers, no systematic research considering its chirality has been performed. In this paper, eight possible chiral alcohols as direct precursors of D609 were synthesized, and their stereochemistries were elucidated by a vibrational circular dichroism (VCD) technique. Phosphatidylcholine-specific phospholipase C and sphingomyelin synthase inhibition assays of these isomers showed considerable differences in their activities. PMID:26854352

  5. Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase

    Danley, Dennis E; Baima, Eric T.; Mansour, Mahmoud; Fennell, Kimberly F.; Chrunyk, Boris A.; Mueller, John P.; Liu, Shenping; Qiu, Xiayang

    2014-01-01

    Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2....

  6. Hypothyroid phenotype is contributed by mitochondrial complex I inactivation due to translocated neuronal nitric-oxide synthase.

    Franco, María C; Antico Arciuch, Valeria G; Peralta, Jorge G; Galli, Soledad; Levisman, Damián; López, Lidia M; Romorini, Leonardo; Poderoso, Juan J; Carreras, María C

    2006-02-24

    Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-alpha (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3',5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. PMID:16361261

  7. Low toxicity corrosion inhibitors

    This paper discusses the design and testing of low toxicity corrosion inhibitors. New chemistries have been investigated with respect to corrosion protection and impact on the marine environment. The resulting chemicals, while they are effective corrosion inhibitors, present significant improvements in terms of environmental properties over current products. The discussion includes results of the corrosion inhibition, toxicity, biodegradability and partitioning studies

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

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

    2014-01-01

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

  9. Inducible nitric oxide synthase in the myocard.

    Buchwalow, I B; Schulze, W; Karczewski, P; Kostic, M M; Wallukat, G; Morwinski, R; Krause, E G; Mller, J; Paul, M; Slezak, J; Luft, F C; Haller, H

    2001-01-01

    Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle. PMID:11269668

  10. Concerted versus stepwise mechanism in thymidylate synthase.

    Islam, Zahidul; Strutzenberg, Timothy S; Gurevic, Ilya; Kohen, Amnon

    2014-07-16

    Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design. PMID:24949852

  11. Homocystinuria due to cystathionine beta synthase deficiency

    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 mol/L (normal levels: 5.90-16mol/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.

  12. An evolutionarily ancient NO synthase (NOS) in shrimp.

    Wu, Chun-Hung; Siva, Vinu S; Song, Yen-Ling

    2013-11-01

    Nitric oxide (NO) is a well known essential molecule that is involved in multiple functions such as neuron transduction, cardiac disease, immune responses, etc.; nitric oxide synthase (NOS) is a critical enzyme that catalyzes the synthesis of it. A very few crustacean NOS molecules were biochemically characterized so far. In the present study, we cloned and characterized a NOS cDNA from haemocytes of tiger shrimp (Penaeus monodon) (PmNOS). The full-length of PmNOS cDNA contained 3997 bp, including a 5'UTR of 249 bp, ORF of 3582 bp and a 3'UTR of 166 bp. The putative peptide was 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. Structurally, PmNOS contained oxygenase and reductase domains at N-terminal and C-terminal, respectively, and connected with a calmodulin binding motif. The deduced amino acid sequence of PmNOS shared 98% identical to the Chinese shrimp (Fenneropenaeus chinensis) NOS. Phylogenetically, PmNOS clustered with invertebrate NOS, but not clustered with iNOS, eNOS or nNOS found in vertebrates. PmNOS mRNA was expressed in many tissues or organs including thoracic and ventral nerves, midgut, gill, eyestalk, haemocytes, subcuticular epithelium and heart, but not found in hepatopancreas, muscle and lymphoid organ. But there was no significant difference in PmNOS mRNA expression after stimulation with LPS either by different concentration or time course or against CpG-ODN 2006. The enzyme activities of rPmNOS or crude homogenates from different tissues were detected, and were shown its highest activity in thoracic and ventral nerves, moderate in midgut and haemocytes but the lowest activity were seen in muscle. The addition of NOS antibody against NADPH binding domain leads to less activity which suggested that NADPH was an essential cofactor for PmNOS catalytic activity. The calcium dependency of PmNOS was ascertained using calmodulin inhibitor, Trifluroperazine. To confirm the population of haemocyte which produce NOS, the florescence test was assayed, and it implicated that the production of NO was catalyzed by subset of granulocytic NOS. Since the MW range, inducible/noninducible transcript, calcium-dependent activity and tissue distribution, we suggest that PmNOS may recognize as an ancient NOS evolutionarily. PMID:23994281

  13. Changes in the profile of NO synthases affect coronary blood flow autoregulation and myocardial contractile activity during restraint stress in rats.

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

    2014-12-01

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

  14. Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

    Jahnke, Wolfgang; Rondeau, Jean-Michel; Cotesta, Simona; Marzinzik, Andreas; Pell, Xavier; Geiser, Martin; Strauss, Andr; Gtte, Marjo; Bitsch, Francis; Hemmig, Ren; Henry, Chrystle; Lehmann, Sylvie; Glickman, J Fraser; Roddy, Thomas P; Stout, Steven J; Green, Jonathan R

    2010-09-01

    Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue. PMID:20711197

  15. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

    Choudhury, Swarup Roy; Singh, Sanjay Kumar; Roy, Sujit; Sengupta, Dibyendu N

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP. PMID:20689184

  16. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine

    Swarup Roy Choudhury; Sanjay Kumar Singh; Sujit Roy; Dibyendu N Sengupta

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of -adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana-ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5′-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.

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

    Romero, Ibeth; Tllez, Jair; Romanha, Alvaro Jos; Steindel, Mario; Grisard, Edmundo Carlos

    2015-08-01

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

  18. The rice ent-KAURENE SYNTHASE LIKE 2 encodes a functional ent-beyerene synthase.

    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

  19. Characterisation of the tryptophan synthase alpha subunit in maize

    Gierl Alfons

    2008-04-01

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

  20. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression.

    Kim, Sun-Hee; Hashimoto, Yuuri; Cho, Sung-Nam; Roszik, Jason; Milton, Denái R; Dal, Fulya; Kim, Sangwon F; Menter, David G; Yang, Peiying; Ekmekcioglu, Suhendan; Grimm, Elizabeth A

    2016-05-01

    COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1-specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy. PMID:26801201

  1. Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells.

    Gas-Pascual, Elisabet; Simonovik, Biljana; Schaller, Hubert; Bach, Thomas J

    2015-08-01

    Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished. PMID:26033687

  2. Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones

    Skouloubris, Stéphane; Djaout, Kamel; Lamarre, Isabelle; Lambry, Jean-Christophe; Anger, Karine; Briffotaux, Julien; Liebl, Ursula; de Reuse, Hilde; Myllykallio, Hannu

    2015-01-01

    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

    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. Characterization of Lipoyl Synthase from Mycobacterium tuberculosis.

    Lanz, Nicholas D; Lee, Kyung-Hoon; Horstmann, Abigail K; Pandelia, Maria-Eirini; Cicchillo, Robert M; Krebs, Carsten; Booker, Squire J

    2016-03-01

    The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine. PMID:26841001

  5. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    Bach, Anders*; Clausen, Bettina H; Møller, Magda; Vestergaard, Bente; Chi, Celestine N; Round, Adam; Sørensen, Pernille Louise; Nissen, Klaus Bertram; Kastrup, Jette Sandholm; Gajhede, Michael; Jemth, Per; Kristensen, Anders Skov; Lundström, Patrik; Lambertsen, Kate Lykke; Strømgaard, Kristian* (*Corresponding)

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are...

  6. Tryptophan synthase from Agrobacterium tumefaciens 8628: isolation and properties.

    Rekoslavskaya, N I; Kuznetsova, E V; Vysotskaya, E F; Salyaev, R K

    1997-04-01

    Tryptophan synthase was isolated from a highly virulent strain of Agrobacterium tumefaciens 8628 (octopine type). Separation of tryptophan synthase from thermolabile protease was accomplished using fractionation with polyethylene glycol-6000 followed by ion-exchange chromatography with a pH gradient. Molecular weights of alpha- and beta-subunits are 33 and 51 kD, respectively. The tryptophan synthase is stable at 60 degrees C because of heat-tolerance beta-subunits. After heating the activity of tryptophan synthase increased up to 20 times while temperature-labile proteases lost their activities. Reaction with antibodies showed the presence of four protein bands, one of which was coeluted with nucleic acids during ion-exchange chromatography. It is suggested that the basic tryptophan synthase is encoded by trp genes in a plasmid and its role is to provide the precursor with the prokaryotic pathway of indole-3-acetic acid biosynthesis, which determines the virulence of A. tumefaciens. There is perhaps a cooperation between iaaM, iaaH, and trp genes in the plasmid during plant cell transformation. PMID:9275281

  7. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    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 Ca2+ ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg2+ 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

  8. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    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

  9. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    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.

  10. Neuronal nitric oxide synthase and N-methyl-D-aspartate neurons in experimental carbon monoxide poisoning

    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 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

  11. Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

    Colpo Anna

    2010-10-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK-3 α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB 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-3α and GSK-3β function in multiple myeloma (MM. Methods GSK-3 α and β 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 α and β isoforms. Survival signaling pathways were studied with WB analysis. Results GSK-3α and GSK-3β 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-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α 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-3α and β i play distinct roles in cell survival and ii modulate the sensitivity to proteasome inhibitors.

  12. Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

    Glycogen Synthase Kinase-3 (GSK-3) α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB 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-3α and GSK-3β function in multiple myeloma (MM). GSK-3 α and β 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 α and β isoforms. Survival signaling pathways were studied with WB analysis. GSK-3α and GSK-3β 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-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α 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. These data suggest that in MM cells GSK-3α and β i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

  13. Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

    Dave, Kashyap; Punekar, Narayan S

    2011-09-10

    Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi. PMID:21723343

  14. Evolutionary history of the chitin synthases of eukaryotes.

    Morozov, Alexey A; Likhoshway, Yelena V

    2016-06-01

    Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary. PMID:26887391

  15. Bacterial Na(+)-ATP synthase has an undecameric rotor.

    Stahlberg, H; Müller, D J; Suda, K; Fotiadis, D; Engel, A; Meier, T; Matthey, U; Dimroth, P

    2001-03-01

    Synthesis of adenosine triphosphate (ATP) by the F(1)F(0) ATP synthase involves a membrane-embedded rotary engine, the F(0) domain, which drives the extra-membranous catalytic F(1) domain. The F(0) domain consists of subunits a(1)b(2) and a cylindrical rotor assembled from 9-14 alpha-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 atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits. PMID:11266365

  16. The selectivity of protein kinase inhibitors: a further update.

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C James; McLauchlan, Hilary; Klevernic, Iva; Arthur, J Simon C; Alessi, Dario R; Cohen, Philip

    2007-12-15

    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

  17. Arabidopsis transcriptional responses differentiating closely related chemicals (herbicides) and cross-species extrapolation to Brassica

    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...

  18. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    Chen, Lisa S; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

    2009-01-01

    8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the sub...

  19. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

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

    2015-01-01

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

  20. Single-Molecule Stochastic Analysis of Channeling Enzyme Tryptophan Synthase.

    Loutchko, Dimitri; Gonze, Didier; Mikhailov, Alexander S

    2016-03-10

    The channeling enzyme tryptophan synthase provides a paradigmatic example of a chemical nanomachine. It possesses two active centers and, as a single molecule, catalyzes 13 different reaction steps with a complex pattern of allosteric regulation and with an intermediate product channeled from one active center to another. Here, the first single-molecule stochastic model of the enzyme is proposed and analyzed. All its transition rate constants were deduced from the experimental data available, and no fitting parameters were thus employed. Numerical simulations reveal strong correlations in the states of the active centers and the emergent synchronization of intramolecular processes in tryptophan synthase. PMID:26863529

  1. Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

    Quelas, J. Ignacio; Mongiardini, Elas J.; Prez-Gimnez, Julieta; Parisi, Gustavo; Lodeiro, Anbal R.

    2013-01-01

    Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic...

  2. Propolis attenuates oxidative injury in brain and lung of nitric oxide synthase inhibited rats

    Zeliha Selamoglu-Talas

    2015-10-01

    Full Text Available Background: The blocking of nitric oxide synthase (NOS activity may reason vasoconstriction with formation of reactive oxygen species. Propolis has biological and pharmacological properties, such as antioxidant. The aim of this study was to examine the antioxidant effects of propolis which natural product on biochemical parameters in brain and lung tissues of acute nitric oxide synthase inhibited rats by Nω-nitro-L-arginine methyl ester (L-NAME.Methods: Rats have been received L-NAME (40 mg/kg, intraperitoneally, NOS inhibitor for 15 days to produce hypertension and propolis (200mg/kg, by gavage the lastest 5 of 15 days.Results: There  were  the  increase  (P<0.001  in  the  malondialdehyde  levels  in  the  L-NAME treatment groups when compared to control rats, but the decrease (P<0.001 in the catalase activities in both brain and lung tissues. There were statistically changes (P<0.001 in these parameters of L-NAME+propolis treated rats as compared with L-NAME-treated group.Conclusion: The application of L-NAME to the Wistar rats resulted in well developed oxidative stress. Also, propolis may influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of hypertensive diseases and oxidative stress.

  3. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-01

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed. PMID:26546087

  4. Novel Nuclear Localization of Fatty Acid Synthase Correlates with Prostate Cancer Aggressiveness

    Madigan, Allison A.; Rycyna, Kevin J.; Parwani, Anil V.; Datiri, Yeipyeng J.; Basudan, Ahmed M.; Sobek, Kathryn M; Cummings, Jessica L; Basse, Per H.; Bacich, Dean J.; O'Keefe, Denise S.

    2014-01-01

    Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is express...

  5. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

    Fliedner, Stephanie MJ; Yang, Chunzhang; Thompson, Eli; ABU-ASAB, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S.; WESLEY, ROBERT; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor ...

  6. Inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression and inducible nitric oxide synthase by 4-[(2′-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate from Moringa oleifera

    Park, Eun-Jung; Cheenpracha, Sarot; Chang, Leng Chee; Tamara P. Kondratyuk; Pezzuto, John M.

    2011-01-01

    Moringa oleifera Lamarack is commonly consumed for nutritional or medicinal properties. We recently reported the isolation and structure elucidation of novel bioactive phenolic glycosides, including 4-[(2′-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate (RBITC), which was found to suppress inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 mouse macrophage cells. Inhibitors of proteins such as cyclooxygenase-2 (COX-2) and i...

  7. SGLT2 inhibitors.

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM. PMID:26362302

  8. Characterization of spermidine synthase and spermine synthase--The polyamine-synthetic enzymes that induce early flowering in Gentiana triflora.

    Imamura, Tomohiro; Fujita, Kohei; Tasaki, Keisuke; Higuchi, Atsumi; Takahashi, Hideyuki

    2015-08-01

    Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS, from the gentian plant, Gentiana triflora. Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants. PMID:26056006

  9. Phosphodiesterase-5 inhibitors.

    Cockrill, Barbara A; Waxman, Aaron B

    2013-01-01

    Nitric oxide (NO) signaling plays a key role in modulating vascular tone and remodeling in the pulmonary circulation. The guanylate cyclase/cyclic guanylate monophosphate-signaling pathway primarily mediates nitric oxide signaling. This pathway is critical in normal regulation of the pulmonary vasculature, and is an important target for therapy in patients with pulmonary hypertension. In the pulmonary vasculature, degradation of cGMP is primarily regulated by PDE-5, and inhibition of this enzyme has important effects on pulmonary vasculature smooth muscle tone. Large randomized placebo-controlled trials of PDE-5 inhibitors demonstrated improved exercise capacity, hemodynamics and quality of life in adult patients with PAH. This chapter will discuss the mechanisms of NO signaling in the vasculature, characteristics of the PDE5-inhibitors approved for treatment of PH, and review available data on the use of phosphodiesterase inhibitors in PH. PMID:24092343

  10. Cholinesterase inhibitors from botanicals

    Faiyaz Ahmed

    2013-01-01

    Full Text Available Alzheimer′s disease (AD is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh, appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE and butyrylcholinesterase (BChE. Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com are also presented and the scope for future research is discussed.

  11. The design and clinical development of inhibitors of glycosphingolipid synthesis: will invention be the mother of necessity?

    Shayman, James A

    2013-01-01

    The treatment of glycosphingolipid storage diseases by synthesis inhibition was first proposed 40 years ago as an alternative approach to enzyme replacement therapy. We have pursued this strategy through the rational design of potent and selective inhibitors of glucosylceramide synthase, the first step in glycosphingolipid synthesis. Eliglustat tartrate was the result of these efforts and is currently the focus of phase 3 trials for type 1 Gaucher disease. Phase 2 studies showed a reduction in splenomegaly and hepatomegaly and improvements of anemia and thrombocytopenia at levels equivalent to or exceeding the historic response to imiglucerase. Structural analogues of eliglustat have also been designed that lack pgp-1 recognition and cross the blood brain barrier. These may have utility for central nervous system- based sphingolipidoses. Because glycosphingolipids are important regulators of receptor tyrosine kinases, glucosylceramide synthase inhibitors may also be beneficial for disorders such as type 2 diabetes mellitus and polycystic kidney disease. PMID:23874009

  12. Cathepsin D inhibitors

    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.

  13. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    Edman, U; Edman, J C; Lundgren, B; Santi, D V

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar t...

  14. Plant polyketide synthases: a fascinating group of enzymes.

    Flores-Sanchez, Isvett J; Verpoorte, Robert

    2009-03-01

    The polyketide synthases (PKSs) are condensing enzymes which form a myriad of polyketide compounds. Several PKSs have been identified and studied in plants. This mini-review summarizes what is known about plant PKSs and some of their aspects such as specificity, reaction mechanisms, structure, as well as their possible evolution are highlighted. PMID:19071029

  15. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    Napolitano, H. B.; Sculaccio, S. A.; Thiemann, O H; G Oliva

    2004-01-01

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa.

  16. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    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.

  17. ATP synthase--a marvellous rotary engine of the cell.

    Yoshida, M; Muneyuki, E; Hisabori, T

    2001-09-01

    ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale. PMID:11533724

  18. JAK Inhibitors AG-490 and WHI-P154 Decrease IFN-γ-Induced iNOS Expression and NO Production in Macrophages

    Eeva Moilanen; Hannu Kankaanranta; Riina Nieminen; Outi Kärpänniemi; Riku Korhonen; Outi Sareila

    2006-01-01

    In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ (IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α (STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also...

  19. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    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

  20. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    Halavaty, Andrei S. [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Kim, Youngchang [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Zhou, Min [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Onopriyenko, Olena; Skarina, Tatiana [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N. [Center for Structural Genomics of Infectious Diseases, (United States); J. Craig Venter Institute, Rockville, MD 20850 (United States); Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Savchenko, Alexei [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Anderson, Wayne F., E-mail: wf-anderson@northwestern.edu [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States)

    2012-10-01

    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 (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} 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 AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  1. Inhibition of Nitric Oxide Synthase Prevents Muscarinic and Purinergic Functional Changes and Development of Cyclophosphamide-Induced Cystitis in the Rat

    Patrik Aronsson; Renata Vesela; Martin Johnsson; Yasin Tayem; Vladimir Wsol; Michael Winder; Gunnar Tobin

    2014-01-01

    Nitric oxide (NO) has pivotal roles in cyclophosphamide- (CYP-) induced cystitis during which mucosal nitric oxide synthase (NOS) and muscarinic M5 receptor expressions are upregulated. In cystitis, urothelial muscarinic NO-linked effects hamper contractility. Therefore we wondered if a blockade of this axis also affects the induction of cystitis in the rat. Rats were pretreated with saline, the muscarinic receptor antagonist 4-DAMP (1 mg/kg ip), or the NOS inhibitor L-NAME (30 mg/kg ip) for ...

  2. The Design and Clinical Development of Inhibitors of Glycosphingolipid Synthesis: Will Invention Be the Mother of Necessity?

    Shayman, James A

    2013-01-01

    The treatment of glycosphingolipid storage diseases by synthesis inhibition was first proposed 40 years ago as an alternative approach to enzyme replacement therapy. We have pursued this strategy through the rational design of potent and selective inhibitors of glucosylceramide synthase, the first step in glycosphingolipid synthesis. Eliglustat tartrate was the result of these efforts and is currently the focus of phase 3 trials for type 1 Gaucher disease. Phase 2 studies showed a reduction i...

  3. Detailed characterization of the substrate specificity of mouse wax synthase.

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity. PMID:23730681

  4. Functional reconstitution of cellulose synthase in Escherichia coli.

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

    2014-11-10

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

  5. Corrosion inhibitor compositions

    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. Inhibitors of Ceramidases.

    Saied, Essa M; Arenz, Christoph

    2016-05-01

    The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. Acid ceramidase inhibition sensitizes certain types of cancer to chemo- and radio-therapy and this is suggestive of a role of acid ceramidase inhibitors as chemo-sensitizers which can act synergistically with chemo-therapeutic drugs. In this review, we summarize the development of ceramide analogues as first-generation ceramidase inhibitors together with data on their activity in cells and disease models. Furthermore, we describe the recent developments that have led to highly potent second-generation ceramidase inhibitors that act at nanomolar concentrations. In the third part, various assays of ceramidases are described and their relevance for accurately measuring ceramidase activities and for the development of novel inhibitors is highlighted. Besides potential clinical implications, the recent improvements in ceramidase inhibition and assaying may help to better understand the mechanisms of ceramide biology. PMID:26220616

  7. Thymidine Phosphorylase Inhibitors

    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 ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors * angiogenesis * cancer chemotherapy Subject RIV: CC - Organic Chemistry

  8. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    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.

  9. Homology modeling and Molecular docking studies of AS1 (Anthranilate synthase component I (TrpE model of Mycobacterium tuberculos

    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.

  10. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions. PMID:26589785

  11. Crystallization and preliminary crystallographic studies of dihydrofolate reductase-thymidylate synthase from Trypanosoma cruzi, the Chagas disease pathogen

    Crystals of complexes of the T. cruzi dihydrofolate reductase-thymidylate synthase enzyme with three antifolates in two space groups have been obtained that diffracted to 2.1–2.8 Å resolution. The antifolates used for cocrystallization were dihydrotriazine-based and quinazoline-based antifolates. Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase (TcDHFR-TS) was crystallized in complexes with the dihydrotriazine-based or quinazoline-based antifolates C-448, cycloguanil (CYC) and Q-8 in order to gain insight into the interactions of this DHFR enzyme with classical and novel inhibitors. The TcDHFR-TS–C-448–NDP–dUMP crystal belonged to space group C2221 with two molecules per asymmetric unit and diffracted to 2.37 Å resolution. The TcDHFR-TS–CYC, TcDHFR-TS–CYC–NDP and TcDHFR-TS–Q-8–NDP crystals belonged to space group P21 with four molecules per asymmetric unit and diffracted to 2.1, 2.6 and 2.8 Å resolution, respectively. Crystals belonging to the two different space groups were suitable for structure determination

  12. 2,3-Dihydrobenzofuran privileged structures as new bioinspired lead compounds for the design of mPGES-1 inhibitors.

    Di Micco, Simone; Spatafora, Carmela; Cardullo, Nunzio; Riccio, Raffaele; Fischer, Katrin; Pergola, Carlo; Koeberle, Andreas; Werz, Oliver; Chalal, Malik; Vervandier-Fasseur, Dominique; Tringali, Corrado; Bifulco, Giuseppe

    2016-02-15

    2,3-Dihydrobenzofurans are proposed as privileged structures and used as chemical platform to design small compound libraries. By combining molecular docking calculations and experimental verification of biochemical interference, we selected some potential inhibitors of microsomal prostaglandin E2 synthase (mPGES)-1. Starting from low affinity natural product 1, by our combined approach we identified the compounds 19 and 20 with biological activity in the low micromolar range. Our data suggest that the 2,3-dihydrobenzofuran derivatives might be suitable bioinspired lead compounds for development of new generation mPGES-1 inhibitors with increased affinity. PMID:26777299

  13. Nitric oxide in rostral ventrolateral medulla regulates cardiac-sympathetic reflexes: role of synthase isoforms.

    Guo, Zhi-Ling; Tjen-A-Looi, Stephanie C; Fu, Liang-Wu; Longhurst, John C

    2009-10-01

    Our previous studies have shown that nitric oxide (NO) synthase (NOS)-containing neurons in the rostral ventrolateral medulla (rVLM) are activated during cardiac sympathoexcitatory reflexes (Refs. 12 and 13). However, the precise function of NO in the rVLM in regulation of these reflexes has not been defined. Three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), are located in the rVLM. We explored the role of NO, derived from different NOS isoforms in the rVLM, in processing cardiac-sympathetic reflexes using whole animal reflex and electrophysiological approaches. We found that, in anesthetized cats, increased mean arterial blood pressure and renal sympathetic nerve activity elicited by epicardial application of bradykinin (BK; 1-10 microg/ml, 50 microl) were significantly attenuated following unilateral rVLM microinjection of the nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (50 nmol/50 nl), or a specific nNOS inhibitor, 7-nitroindazole (7-NI; 5-10 pmol/50 nl; both P < 0.05). In contrast, the responses of mean arterial blood pressure and renal sympathetic nerve activity to cardiac BK stimulation were unchanged by unilateral rVLM microinjection of N(omega)-nitro-D-arginine methyl ester (inactive isomer of N(omega)-nitro-L-arginine methyl ester, 50 nmol/50 nl), 3-6% methanol (7-NI vehicle), N(6)-(1-iminoethyl)-L-lysine (250 pmol/50 nl; iNOS inhibitor), or N(5)-(1-iminoethyl)-L-ornithine (250 nmol/50 nl; eNOS inhibitor). Furthermore, in separate cats, we noted that iontophoresis of 7-NI (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory rVLM neurons in response to cardiac stimulation with BK (P < 0.05). These neurons were characterized by their responses to inputs from baroreceptors, and their cardiac rhythmicity was determined through frequency and time domain analyses, correlating their discharge to arterial blood pressure and cardiac sympathetic efferent nerve activity. These data suggest that NO, specifically nNOS, mediates sympathetic cardiac-cardiovascular responses through its action in the rVLM. PMID:19684188

  14. Arginase inhibitor attenuates pulmonary artery hypertension induced by hypoxia.

    Chu, YanBiao; XiangLi, XiaoYing; Niu, Hu; Wang, HongChao; Jia, PingDong; Gong, WenBin; Wu, DaWei; Qin, WeiDong; Xing, ChunYan

    2016-01-01

    Hypoxia-induced pulmonary arterial hypertension (HPAH) is a refractory disease characterized by increased proliferation of pulmonary vascular smooth cells and progressive pulmonary vascular remodeling. The level of nitric oxide (NO), a potential therapeutic vasodilator, is low in PAH patients. L-arginine can be converted to either beneficial NO by nitric oxide synthases or to harmful urea by arginase. In the present study, we aimed to investigate whether an arginase inhibitor, S-(2-boronoethyl)-L-cysteine ameliorates HPAH in vivo and vitro. In a HPAH mouse model, we assessed right ventricle systolic pressure (RVSP) by an invasive method, and found that RSVP was elevated under hypoxia, but was attenuated upon arginase inhibition. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured under hypoxic conditions, and their proliferative capacity was determined by cell counting and flow cytometry. The levels of cyclin D1, p27, p-Akt, and p-ERK were detected by RT-PCR or Western blot analysis. Compared to hypoxia group, arginase inhibitor inhibited HPASMCs proliferation and reduced the levels of cyclin D1, p-Akt, p-ERK, while increasing p27 level. Moreover, in mouse models, compared to control group, hypoxia increased cyclin D1 expression but reduced p27 expression, while arginase inhibitor reversed the effects of hypoxia. Taken together, these results suggest that arginase plays an important role in increased proliferation of HPASMCs induced by hypoxia and it is a potential therapeutic target for the treatment of pulmonary hypertensive disorders. PMID:26608181

  15. Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

    Dias, Danielle F.; Alves, Ricardo J., E-mail: ricardodylan@farmacia.ufmg.b [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia; Roux, Celine; Durand, Philippe; Iorga, Bogdan; Badet-Denisot, Marie A.; Badet, Bernard [Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette (France). Inst. de Chimie des Substances Naturelles

    2010-07-01

    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 IC{sub 50} in the mmol L{sup -1} range. (author)

  16. Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

    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)

  17. Tirosine Kinase Inhibitors Adverse Events

    Campiotti, Leonardo; Maresca, Andrea; Guasti, Luigina; Grandi, Anna Maria

    2014-01-01

    The authors discuss dasatinib as a tyrosine kinase inhibitor with cardiovascular toxicity, which Lenihan and Kowey did not include in their article “Overview and Management of Cardiac Adverse Events Associated With Tyrosine Kinase Inhibitors.”

  18. Insights into the structure activity relationship of mPGES-1 inhibitors: Hints for better inhibitor design.

    Gupta, Ashish; Aparoy, Polamarasetty

    2016-07-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane protein which plays crucial role in arachidonic acid metabolism, in the catalysis of PGH2 to PGE2. It is a potential drug target involved in variety of human cancers and inflammatory disorders. In the present study we made an attempt to identify crucial amino acid residues involved in the effective binding of its inhibitors at the active site. Molecular docking and Structure Activity Relationship (SAR) studies were performed. In the present study 127 inhibitors having significant variability in parent scaffold were considered. The results clearly indicated that in the GSH and PGH2 binding site Arg70, Arg73, Asn74, Glu77, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 consistently form crucial interactions with inhibitors of different classes/scaffolds. These findings are consistent with that of existing reports on the active site residues pivotal at mPGES-1 active site. Further analysis suggested that out of all important amino acid residues identified; Arg73, Asn74, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 play a crucial role in hydrogen and π-π interactions. The identified amino acid residues can act as target sites for the design and development of drug candidates against mPGES-1. PMID:27012893

  19. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

    Nanson, Jeffrey D; Himiari, Zainab; Swarbrick, Crystall M D; Forwood, Jade K

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  20. Traffic of Chitin Synthase 1 (CHS-1) to the Spitzenkrper and Developing Septa in Hyphae of Neurospora crassa: Actin Dependence and Evidence of Distinct Microvesicle Populations ?

    Snchez-Len, Eddy; Verdn, Jorge; Freitag, Michael; Roberson, Robert W.; Bartnicki-Garcia, Salomon; Riquelme, Meritxell

    2011-01-01

    We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkrper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex. PMID:21296914

  1. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    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.

  2. Nitric oxide synthase inhibition abrogates hydrogen sulfide-induced cardioprotection in mice.

    Sojitra, Bhavesh; Bulani, Yogesh; Putcha, Uday Kumar; Kanwal, Abhinav; Gupta, Prachi; Kuncha, Madhusudana; Banerjee, Sanjay Kumar

    2012-01-01

    The cardioprotective property of hydrogen sulfide (H(2)S) is recently reported. However, cellular signaling cascades mediated by H(2)S are largely unclear. This study was undertaken to explore the molecular mechanism of H(2)S-induced cardioprotection in mouse heart by utilizing in vivo model of cardiac injury. We report here that intraperitoneal administration of sodium hydrogen sulfide (NaHS, 50 μmol kg(-1 )day(-1) for 2 days), a H(2)S donor, significantly (P ≤ 0.05) increased nitric oxide levels in serum as well as myocardium without any sign of myocardial injury. Typical characteristics of myocardial injury induced by isoproterenol (ISO) administration was significantly (P ≤ 0.05) abrogated by NaHS administration as evidenced from reduction in elevated thiobarbituric acid reactive substances (TBARS) and normalization of glutathione (GSH), glutathione peroxidase, superoxide dismutase (SOD), and catalase activity. Further, decrease in TNF-α expression and improvement in myocardial architecture was also observed. However, co-administration of N-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, and Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor along with NaHS and ISO abrogated the beneficial effect of H(2)S differentially. Inhibition of NOS significantly (P ≤ 0.05) increased serum creatine kinase, lactate dehydrogenase, serum glutamic oxaloacetic transaminase activity and myocardial TBARS, along with significant (P ≤ 0.05) reduction of myocardial GSH, SOD, and catalase. This was followed by increase in TNF-α expression and histopathological changes. Our results revealed that H(2)S provides myocardial protection through interaction with NOS and COX-2 pathway and inhibition of NOS completely abrogates the hydrogen sulfide-induced cardioprotection in mice. PMID:21879311

  3. Inhibition of glycogen synthase kinase-3 reduces L-DOPA-induced neurotoxicity

    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 μM 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

  4. Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

    Magwamba, Clement Chedza; Rukseree, Kamolchanok; Palittapongarnpim, Prasit

    2016-05-01

    The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 μM, IC50 = 267.4 μM; Ki = 0.84 μM, IC50 = 9.28 μM; and Ki = 0.592 μM, IC50 = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS. PMID:27156617

  5. Histone deacetylase inhibitors impair antibacterial defenses of macrophages.

    Mombelli, Matteo; Lugrin, Jrme; Rubino, Ivana; Chanson, Anne-Laure; Giddey, Marlyse; Calandra, Thierry; Roger, Thierry

    2011-11-01

    Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages. PMID:21921209

  6. Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase.

    Ott, K H; Kwagh, J G; Stockton, G W; Sidorov, V; Kakefuda, G

    1996-10-25

    Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems. PMID:8913312

  7. Implication of nitric oxide synthase in carcinogenesis: analysis of the human inducible nitric oxide synthase gene.

    Esumi, H; Ogura, T; Kurashima, Y; Adachi, H; Hokari, A; Weisz, A

    1995-01-01

    Nitric oxide (NO) is a newly identified, multifunctional biological mediator. However, it also has deleterious effects on biological materials. For instance, nucleic acids, proteins, and some prosthetic groups of enzymes can be modified by NO or its reaction products with other reactive oxygen species. Endogenous nitrosamine formation through the reaction of NO or its oxidized products with amines might be involved in carcinogenesis. These deleterious effects of NO are often associated with inflammatory processes both in experimental animals and human. We analyzed the molecular mechanism of control of expression of the inducible nitric oxide synthase (NOS) gene in mouse cells by cloning its putative promoter region. This promoter responded to various cytokines and endotoxin similarly to the endogenous NOS gene in mouse cells. No appreciable induction of NOS was observed in human peripheral blood cells, but induction was detected in a human glioblastoma cell line A-172. Therefore, the human inducible NOS cDNA was cloned from A-172 cells and its cDNA-deduced amino acid sequence found to have about 80% similarity to those of both mouse and rat inducible NOSs. The effects of various cytokines on the induction of the gene were somewhat different from those observed in mouse cells, but the mouse promoter responded to these cytokines similarly to the endogenous NOS gene in human cells, indicating functional similarity of cis-elements of the genes encoding both human and mouse inducible NOS. Structural analysis of the human inducible NOS gene by Southern blot analysis revealed putative genetic restriction fragment length polymorphism in intron 5.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7581489

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

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji

    2014-01-01

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

  9. Biosynthesis of non-melanin pigment by a divergent polyketide synthase in Metarhizium robertsii.

    Chen, Yixiong; Feng, Peng; Shang, Yanfang; Xu, Yong-Jiang; Wang, Chengshu

    2015-08-01

    Fungal polyketide synthases (PKSs) and their related gene clusters are highly diversified at both inter- and intra-specific levels. The most well characterized PKS enzymes include those responsible for the biosynthesis of polyketide pigments such as melanins. The genome of the insect pathogenic fungus Metarhizium robertsii contains 20 type I PKSs but none has been functionally characterized. In this study, two PKS genes (designated as MrPks1 and MrPKs2) showing homologies to those counterparts for the biosynthesis of heptaketide pigments and dihydroxynaphthalene (DHN)-melanins, respectively, were deleted in two different strains of M. robertsii. The results indicated that disruption of MrPks1 but not MrPks2 impaired fungal culture pigmentation and cell wall structure. In addition to the negative effect of the DHN-melanin pathway inhibitor, it was postulated that DHN-melanin would not be produced by M. robertsii. Various assays revealed that the stress resistance abilities against ultraviolet radiation, heat shock and oxidants, as well as virulence against insects were not impaired in ΔMrPks1 and ΔMrPks2 isolates when compared with the wild-type strain. Thus, the non-melanin pigment(s) produced by the fungus do not contribute to cell damage protection and pathogenicity in M. robertsii. Physiological differences were evident in the two examined wild-type strains. The results from this study advance the understanding of functional divergence of fungal PKSs. PMID:25445307

  10. Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

    Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.

  11. Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-{kappa}B

    Leung, Kin Chung; Li, Ming-Yue; Leung, Billy C.S.; Hsin, Michael K.Y. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Mok, Tony S.K. [Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Underwood, Malcolm J. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Chen, George G., E-mail: gchen@cuhk.edu.hk [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong)

    2010-12-10

    Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-{kappa}B activity was determined in human lung cancer cells. The roles of ROS and NF-{kappa}B in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-{kappa}B by reducing phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-{kappa}B. Antioxidants can significantly block the inhibitory effect of 1-BI.

  12. Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

    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 3β (GSK3β, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl or specific GSK3β 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, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  13. Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.

    Muscari, Claudio; Capanni, Cristina; Giordano, Emanuele; Stefanelli, Claudio; Bonavita, Francesca; Stanic, Ivana; Bonaf, Francesca; Caldarera, Claudio Marcello; Guarnieri, Carlo

    2010-11-01

    The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species. PMID:20828747

  14. Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate

    Yokoyama, Takeshi; Ostermann, Andreas; Mizuguchi, Mineyuki; Niimura, Nobuo; Schrader, Tobias E.; Tanaka, Ichiro

    2014-01-01

    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 mm3) 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

  15. Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation.

    Jaffe, Eileen K; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J; Myler, Peter J; Roos, David S

    2011-04-29

    Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit ?-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors. PMID:21383008

  16. Human farnesyl pyrophosphate synthase inhibition by nitrogen bisphosphonates: a 3D-QSAR study.

    Fernández, David; Ortega-Castro, Joaquín; Frau, Juan

    2013-08-01

    We report the results of a comparative molecular field analysis and comparative molecular similarity index analysis of the human farnesyl pyrophosphate synthase (FPPS) inhibition by nitrogen bisphosphonates (NBPs) taking into account their time-dependent inhibition efficacies. The 3D-QSAR models obtained provide steric, electrostatic and hydrophobic contour maps consistent with the interactions into the active site of human FPPS observed in available crystallographic structures. Furthermore, the 3D-QSAR models obtained provide accurately IC50 values of the NBPs of the training set. The predictive ability of these 3D-QSAR models was found to rely on the choice of biologically active conformations of the target molecules and on a careful examination of the protonation status of the NBPs in the training set. The best models obtained can be useful to predict biological values of a high number of NBPs that have been used for the treatment of different diseases as potential inhibitors of the activity of the FPPS enzyme. PMID:23979193

  17. Resveratrol- and α-viniferin-induced alterations of acetylcholinesterase and nitric oxide synthase in Raillietina echinobothrida.

    Giri, Bikash Ranjan; Roy, Bishnupada

    2015-10-01

    Phytostilbenes, like resveratrol and α-viniferin, which occur mainly in the plants and belong to the families Cyperaceae, Vitaceae, and Gnetaceae are extensively popular for their medicinal and nutritional properties. In Northeast India, the Jaintia tribes consume these phytochemicals through aqueous extract of the medicinal plant Carex baccans to control helminthiasis. The present study aimed to investigate the inhibitory effect of the phytochemicals on neurotransmitters and its related enzymes in helminth parasite Raillietina echinobothrida. Viability of the parasites exposed to the phytostilbenes and extent of inhibition of cholinergic and nitrergic enzymes were evaluated in comparison to reference anthelmintic drug praziquantel and two known enzyme inhibitors, namely Nω-nitro-L-arginine and pyridostigmine. On exposure to resveratrol, α-viniferin, and reference drug praziquantel, the parasites ceased movement at 9.37, 11.38, and 0.24 h followed by death at 23.65, 34.13, and 1.87 h, respectively. Exposed parasites also showed a significant decrease in the activity of acetylcholinesterase (46.101, 65.935, and 63.645%) and nitric oxide synthase (61.241, 55.046, and 29.618%) in comparison to the controls. In addition, a decreased trend in nitric oxide (NO) level was also detected in the tissue of different phytochemical-exposed parasites compared to control. The present study suggests that anthelmintic potential of both the phytochemicals is mediated through inhibition of two vital enzymes which play diverse role in intracellular communications through neuromuscular system. PMID:26141435

  18. Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury

    The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process

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

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

  20. Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

    Yasumoto, Yuki; Miyazaki, Hirofumi; Vaidyan, Linda Koshy; Kagawa, Yoshiteru; Ebrahimi, Majid; Yamamoto, Yui; Ogata, Masaki; Katsuyama, Yu; Sadahiro, Hirokazu; Suzuki, Michiyasu; Owada, Yuji

    2016-01-01

    Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 ?M cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma. PMID:26808816

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

    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.

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

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

    2010-04-09

    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.

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

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

    1990-01-01

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

  4. Benzoylurea Chitin Synthesis Inhibitors.

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs. PMID:26168369

  5. Acetylcholinesterase inhibitors attenuate angiogenesis

    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...

  6. Complete reconstitution of a highly reducing iterative polyketide synthase.

    Ma, Suzanne M; Li, Jesse W-H; Choi, Jin W; Zhou, Hui; Lee, K K Michael; Moorthie, Vijayalakshmi A; Xie, Xinkai; Kealey, James T; Da Silva, Nancy A; Vederas, John C; Tang, Yi

    2009-10-23

    Highly reducing iterative polyketide synthases are large, multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding of the functions and structures of this family of enzymes. PMID:19900898

  7. Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo

    Antos, Christopher L.; McKinsey, Timothy A.; Frey, Norbert; Kutschke, William; McAnally, John; Shelton, John M.; James A. Richardson; Hill, Joseph A.; Eric N. Olson

    2002-01-01

    The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) ph...

  8. The effect of ketotifen on nitric oxide synthase activity

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

    1997-01-01

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

  9. Catalytic subunit stoichiometry within the cellulose synthase complex

    Gonneau, Martine; Desprez, Thierry; Guillot, Alain; Vernhettes, Samantha; Hfte, Herman

    2014-01-01

    Cellulose synthesis is driven by large plasma membrane-inserted protein complexes, which in plants have 6-fold symmetry. In Arabidopsis (Arabidopsis thaliana), functional cellulose synthesis complexes (CSCs) are composed of at least three different cellulose synthase catalytic subunits (CESAs), but the actual ratio of the CESA isoforms within the CSCs remains unresolved. In this work, the stoichiometry of the CESAs in the primary cell wall CSC was determined, after elimination of CESA redunda...

  10. The cellulose synthase superfamily in fully sequenced plants and algae

    Xu Ying

    2009-07-01

    Full Text Available 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 found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  11. Use of linalool synthase in genetic engineering of scent production

    Pichersky, Eran (Chelsea, MI)

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

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

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

    2008-01-01

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

  13. Piriformospora indica requires kaurene synthase activity for successful plant colonization.

    Li, Liang; Chen, Xi; Ma, Chaoyang; Wu, Hongqing; Qi, Shuting

    2016-05-01

    Ent-kaurene (KS) synthases and ent-kaurene-like (KSL) synthases are involved in the biosynthesis of phytoalexins and/or gibberellins which play a role in plant immunity and development. The relationship between expression of five synthase genes (HvKSL1, HvKS2, HvKS4, HvKS5, HvKSL4) and plant colonization by the endophytic fungus Piriformospora indica was assessed in barley (Hordeum vulgare). The KS gene family is differently up-regulated at 1, 3 and 7 day after P. indica inoculation. By comparison, the HvKSL4 gene expression pattern is more significantly affected by UV irradiation and P. indica colonization. The characterizations of two silencing lines (HvKSL1-RNAi, HvKSL4-RNAi) also were analyzed. HvKSL1-RNAi and HvKSL4-RNAi lines in the first generation lead to less dark green leaves and slower plant development. Further, reduced spikelet fertility in progenies of RNAi plants heterozygous for HvKSL1 were observed, but not for HvKSL4. T2 generation of HvKSL1-RNAi line showed semi-dwarf phenotype while the wild type phenotype could be restored by applying GA3. Silencing of HvKSL4 and HvKSL1 resulted in reduced colonization by P. indica especially in the HvKSL1-RNAi line. These results probably suggest the presence of two ent-KS synthase in barley, one (HvKSL1) that participates in the biosynthesis of GAs and another (HvKSL4) that is involved in the biosynthesis of phytoalexins. PMID:26943021

  14. Polymorphism in Argininosuccinate Synthase Gene in Indian Holstein

    Uma Gaur, Tejaswini G. Sathe, Arpita Roy, Rajesh K. Patel* and P. S. Satish Sunkara

    2012-01-01

    The present study investigated the occurrence of an autosomal recessive genetic disease, Bovine Citrullinaemia caused by mutation in Argininosuccinate Synthase (ASS) gene, in Indian Holstein cattle. The Polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 120 Holstein bulls to identify carrier (heterozygous) animals. Two out of 120 (1.67%) animals were found carrier for Bovine Citrullinaemia. The gene and genotype frequency of rece...

  15. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Roč. 183, č. 2 (2012), s. 189-192. ISSN 0166-6851 R&D Projects: GA ČR(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * Sleeping sickness * Ubiquinone * Solanesyl-diphosphate synthase * Digitonin permeabilization * In situ tagging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112000539

  16. Enhancing Human Spermine Synthase Activity by Engineered Mutations

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

    2013-01-01

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

  17. The impact of spermine synthase (SMS) mutations on brain morphology

    Kesler, Shelli R; Schwartz, Charles; Stevenson, Roger E.; Reiss, Allan L.

    2009-01-01

    Snyder–Robinson syndrome (SRS) is a form of X-linked mental retardation resulting from mutations in spermine synthase (SMS), which impact neurodevelopment and cognitive outcome. We obtained cerebral, cerebellum, hippocampus, and red nucleus volumes from two males with SRS and 24 age- and gender-matched typically developing controls using volumetric neuroimaging analyses. Total brain volume was enlarged in males with SRS while cerebellum, hippocampus, and red nucleus volumes tended to be reduc...

  18. Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

    Escartin, Frédéric; Skouloubris, Stéphane; Liebl, Ursula; Myllykallio, Hannu

    2008-01-01

    We have investigated the hitherto unexplored possibility that differences in the catalytic efficiencies of thymidylate synthases ThyX and ThyA, enzymes that produce the essential DNA precursor dTMP, have influenced prokaryotic genome evolution. We demonstrate that DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10-fold decreased compared with species that contain the catalytically more efficient ThyA. Our statistical studies of >400 genomes ind...

  19. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research. PMID:26638491

  20. Neuronal Nitric Oxide Synthase is Involved in Vascular Hyporeactivity and Multiple Organ Dysfunction Associated with Hemorrhagic Shock.

    Sordi, Regina; Chiazza, Fausto; Collino, Massimo; Assreuy, Jamil; Thiemermann, Christoph

    2016-05-01

    Severe hemorrhage can lead to global ischemia and hemorrhagic shock (HS), resulting in multiple organ failure (MOF) and death. Restoration of blood flow and re-oxygenation is associated with an exacerbation of tissue injury and inflammatory response. The neuronal nitric oxide synthase (nNOS) has been implicated in vascular collapse and systemic inflammation of septic shock; however, the role of nNOS in HS is poorly understood. The aim of this study was to evaluate the role of nNOS in the MOF associated with HS.Rats were subjected to HS under anesthesia. Mean arterial pressure was reduced to 30 mmHg for 90 min, followed by resuscitation with shed blood. Rats were randomly treated with two chemically distinct nNOS inhibitors [ARL 17477 (1 mg/kg) and 7-nitroindazol (5 mg/kg)] or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed.HS was associated with MOF development. Inhibition of nNOS activity at resuscitation protected rats against the MOF and vascular dysfunction. In addition, treatment of HS rats with nNOS inhibitors attenuated neutrophil infiltration into target organs and decreased the activation of NF-κB, iNOS expression, NO production, and nitrosylation of proteins. Furthermore, nNOS inhibition also reduced the levels of pro-inflammatory cytokines TNF-α and IL-6 in HS rats.In conclusion, two distinct inhibitors of nNOS activity reduced the MOF, vascular dysfunction, and the systemic inflammation associated with HS. Thus, nNOS inhibitors may be useful as an adjunct therapy before fluids and blood administration in HS patients to avoid the MOF associated with reperfusion injury during resuscitation. PMID:26863124

  1. Structure of isochorismate synthase DhbC from Bacillus anthracis

    The crystal structure of B. anthracis isochorismate synthase DhbC, which is involved in the biosynthesis of bacillibactin, was determined at 2.4 Å resolution. It was compared with other chorismate-utilizing enzymes and both structural and bioinformatics analyses were performed. The putative active site was pinpointed. The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg2+-dependent catalytic mechanism

  2. From bacterial to human dihydrouridine synthase: automated structure determination

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer

  3. Energy transduction in the F1 motor of ATP synthase

    Wang, Hongyun; Oster, George

    1998-11-01

    ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis, after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central `shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

  4. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases. PMID:26799780

  5. From bacterial to human dihydrouridine synthase: automated structure determination

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  6. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-01

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthes...

  7. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    R. GUTIERREZ; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D W

    2009-01-01

    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide...

  8. Wide Distribution among Halophilic Archaea of a Novel Polyhydroxyalkanoate Synthase Subtype with Homology to Bacterial Type III Synthases?

    Han, Jing; Hou, Jing; Liu, Hailong; Cai, Shuangfeng; Feng, Bo(Zhejiang Institute of Modern Physics, Zhejiang University, 38 Zheda Road, Hangzhou, 310027, P.R China); Zhou, Jian; Xiang, Hua

    2010-01-01

    Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of th...

  9. Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase[S

    Lim, Sing Mei; Chen, Dan; Teo, Hsiangling; Roos, Annette; Jansson, Anna Elisabet; Nyman, Tomas; Trésaugues, Lionel; Pervushin, Konstantin; Nordlund, Pär

    2013-01-01

    Lipocalin prostaglandin D synthase (L-PGDS) regulates synthesis of an important inflammatory and signaling mediator, prostaglandin D2 (PGD2). Here, we used structural, biophysical, and biochemical approaches to address the mechanistic aspects of substrate entry, catalysis, and product exit of this enzyme. Structure of human L-PGDS was solved in a complex with a substrate analog (SA) and in ligand-free form. Its catalytic Cys 65 thiol group was found in two different conformations, each making...

  10. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.).

    Zheng, Desen; Hrazdina, Geza

    2008-02-15

    Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3' region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product. PMID:18068110

  11. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis.

    Lei, Lei; Singh, Abhishek; Bashline, Logan; Li, Shundai; Yingling, Yaroslava G; Gu, Ying

    2015-10-01

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress. PMID:26443667

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

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

    2011-01-01

    The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a ~220 amino acid internal sequence element (now recognized as the ? domain) gave rise to the precursor of modern mono- and sesqui-TPSs found i...

  13. Role of nitric oxide synthase isoforms for ophthalmic artery reactivity in mice.

    Laspas, Panagiotis; Goloborodko, Evgeny; Sniatecki, Jan J; Kordasz, Marcin L; Manicam, Caroline; Wojnowski, Leszek; Li, Huige; Patzak, Andreas; Pfeiffer, Norbert; Gericke, Adrian

    2014-10-01

    Nitric oxide synthases (NOS) are involved in regulation of ocular vascular tone and blood flow. While endothelial NOS (eNOS) has recently been shown to mediate endothelium-dependent vasodilation in mouse retinal arterioles, the contribution of individual NOS isoforms to vascular responses is unknown in the retrobulbar vasculature. Moreover, it is unknown whether the lack of a single NOS isoform affects neuron survival in the retina. Thus, the goal of the present study was to examine the hypothesis that the lack of individual nitric oxide synthase (NOS) isoforms affects the reactivity of mouse ophthalmic arteries and neuron density in the retinal ganglion cell (RGC) layer. Mice deficient in one of the three NOS isoforms (nNOS-/-, iNOS-/- and eNOS-/-) were compared to respective wild type controls. Intraocular pressure (IOP) was measured in conscious mice using rebound tonometry. To examine the role of each NOS isoform for mediating vascular responses, ophthalmic arteries were studied invitro using video microscopy. Neuron density in the RGC layer was calculated from retinal wholemounts stained with cresyl blue. IOP was similar in all NOS-deficient genotypes and respective wild type controls. In ophthalmic arteries, phenylephrine, nitroprusside and acetylcholine evoked concentration-dependent responses that did not differ between individual NOS-deficient genotypes and their respective controls. In all genotypes except eNOS-/- mice, vasodilation to acetylcholine was markedly reduced after incubation with L-NAME, a non-isoform-selective inhibitor of NOS. In contrast, pharmacological inhibition of nNOS and iNOS had no effect on acetylcholine-induced vasodilation in any of the mouse genotypes. Neuron density in the RGC layer was similar in all NOS-deficient genotypes and respective controls. Our findings suggest that eNOS contributes to endothelium-dependent dilation of murine ophthalmic arteries. However, the chronic lack of eNOS is functionally compensated by NOS-independent vasodilator mechanisms. The lack of a single NOS isoform does not appear to affect IOP or neuron density in the RGC layer. PMID:25017185

  14. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    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.

  15. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    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.

  16. Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.

    Tian, Dan; Zhu, Min; Chen, Wen-shu; Li, Jian-Sha; Wu, Ren-Liang; Wang, Xi

    2006-09-01

    Epidemiological evidence suggests that cigarette smoke induces squamous metaplasia in human tracheobronchial epithelium that can progress to lung squamous carcinoma. But it is not well understood how tracheobronchial epithelial cells transduce the signals that mediate cigarette smoke-induced squamous differentiation or squamous metaplasia. In the present study, we found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) and induced the expression of involucrin, a marker of squamous differentiation. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine tracheobronchial epithelial cells (PTBECs). Moreover, we demonstrated that cigarette smoke components significantly promoted activator protein-1 (AP-1) binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components. Taken together, we conclude that GSK3 is involved in involucrin expression induced by cigarette smoke in PTBEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke. PMID:16750592

  17. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    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.

  18. Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei.

    Vega, Claudia; Moreno-Carranza, Bibiana; Zamorano, Miriam; Quintanar-Stéphano, Andrés; Méndez, Isabel; Thebault, Stéphanie; Martínez de la Escalera, Gonzalo; Clapp, Carmen

    2010-12-01

    Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction. PMID:20943859

  19. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells.

    Fliedner, Stephanie Mj; Yang, Chunzhang; Thompson, Eli; Abu-Asab, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S; Wesley, Robert; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the ? and ? subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase ? (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs. PMID:26101719

  20. ACE INHIBITORS: A COMPREHENSIVE REVIEW

    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, α-blockers, β-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.

  1. Synthesis of Lysine Methyltransferase Inhibitors

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  2. High performance oilfield scale inhibitors

    Duccini, Y.; Dufour, A. [NorsoHaas S.A., Verneuil en Hallatte (France); Hann, W.M.; Sanders, T.W.; Weinstein, B. [Rohm and Haas Co., Spring House, PA (United States)

    1997-08-01

    Sea water often reacts with the formation water in offshore fields to produce barium, calcium and strontium sulfate deposits that hinder oil production. Newer fields often have more difficult to control scale problems than older ones, and current technology scale inhibitors are not able to control the deposits as well as needed. In addition, ever more stringent regulations designed to minimize the impact of inhibitors on the environment are being enacted. Three new inhibitors are presented that overcome many of the problems of older technology scale inhibitors.

  3. Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents.

    Piano, Valentina; Benjamin, Daniel I; Valente, Sergio; Nenci, Simone; Marrocco, Biagina; Mai, Antonello; Aliverti, Alessandro; Nomura, Daniel K; Mattevi, Andrea

    2015-11-20

    Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy. PMID:26322624

  4. Assembly line polyketide synthases: mechanistic insights and unsolved problems.

    Khosla, Chaitan; Herschlag, Daniel; Cane, David E; Walsh, Christopher T

    2014-05-13

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology. PMID:24779441

  5. Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses.

    Schwerdt, Julian G; MacKenzie, Katrin; Wright, Frank; Oehme, Daniel; Wagner, John M; Harvey, Andrew J; Shirley, Neil J; Burton, Rachel A; Schreiber, Miriam; Halpin, Claire; Zimmer, Jochen; Marshall, David F; Waugh, Robbie; Fincher, Geoffrey B

    2015-07-01

    Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-?-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-?-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels. PMID:25999407

  6. Squalene synthase-deficient mutant of Chinese hamster ovary cells.

    Bradfute, D L; Silva, C J; Simoni, R D

    1992-09-15

    Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation. PMID:1526971

  7. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

    Kim, Sung Soo; Grienenberger, Etienne; Lallemand, Benjamin; Colpitts, Che C; Kim, Sun Young; Souza, Clarice de Azevedo; Geoffroy, Pierrette; Heintz, Dimitri; Krahn, Daniel; Kaiser, Markus; Kombrink, Erich; Heitz, Thierry; Suh, Dae-Yeon; Legrand, Michel; Douglas, Carl J

    2010-12-01

    Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors. PMID:21193570

  8. Role of interferon regulatory factor-1 and mitogen-activated protein kinase pathways in the induction of nitric oxide synthase-2 in retinal pigmented epithelial cells.

    Faure, V; Hecquet, C; Courtois, Y; Goureau, O

    1999-02-19

    Bovine retinal pigmented epithelial cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Experiments were performed to investigate the involvement of interferon regulatory factor-1 (IRF-1) on NOS-2 induction and its regulation by NOS-2 inhibitors such as pyrrolidine dithiocarbamate (PDTC), an antioxidant, or protein kinase inhibitors. Analysis by transitory transfections showed that LPS, alone or with IFN-gamma, stimulated activity of the murine NOS-2 promoter fragment linked upstream of luciferase and its suppression by PDTC and by the different protein kinase inhibitors, genistein (tyrosine kinase inhibitor), PD98059 (mitogen-actived protein (MAP) kinase kinase inhibitor), and SB 203580 (p38 MAP inhibitor). Using specific antibodies, we have confirmed that extracellular signal-regulated kinases and p38 MAP kinase were activated by LPS and IFN-gamma in retinal pigmented epithelial cells. Analysis by reverse transcriptase-polymerase chain reaction, Western blot, and electrophoretic mobility shift assay demonstrated that IFN-gamma alone or combined with LPS induced an accumulation of IRF-1 mRNA and protein and IRF-1 DNA binding. Transfections assays with the IRF-1 promoter showed an induction of this promoter with IFN-gamma, potentiated by LPS. The decrease of LPS/IFN-gamma-induced IRF-1 promoter activity, IRF-1 synthesis, and IRF-1 activation, by PDTC, genistein, PD98059, and SB 203580, could explained in part the inhibition of the NOS-2 induction by these compounds. Our results demonstrate that IRF-1 is necessary for NOS-2 induction by LPS and IFN-gamma and that its synthesis requires the involvement of a redox-sensitive step, the activation of tyrosine kinases, and extracellular signal-regulated kinases 1/2 and p38 MAP kinases. PMID:9988718

  9. Adhesion Development and the Expression of Endothelial Nitric Oxide Synthase

    Michael P. Diamond

    2001-01-01

    Full Text Available Objective: This study was conducted to determine whether nitric oxide (NO, a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions.

  10. Inducible nitric oxide synthase immunoreactivity in healthy rat pancreas.

    Nurullah Keklikoglu

    2008-01-01

    Nitric oxide (NO) is produced by NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). It is believed that, while nNOS and eNOS are effective in regulation of normal physiological processes, iNOS is expressed at an increasing rate especially in inflammatory process. The aim of this study was to determine the presence of iNOS immunoreactivity (iNOS-IR) and, to compare the iNOS-IR in islet of Langerhans cells (LC), acinar cells (AC), centroacinar cell...

  11. Activity of endothelial nitric oxide synthase: substrates, modulators and products

    Bevers, L.M.

    2006-01-01

    In this thesis, the activity of endothelial nitric oxide synthase (eNOS) and the effects of its substrates, modulators and products are described. eNOS produces nitric oxide (NO), which is involved in vascular biology. L-arginine and BH4 are both essential factors for adequate eNOS function. Reduced levels of the substrate or cofactor lead to the formation of superoxide by eNOS, a process referred to as eNOS uncoupling. In all studies described here, a microvascular endothelial cell line (bEn...

  12. Isoflavone synthase genes in legumes and non-leguminous plants

    Pičmanová, Martina; Koblovská, R.; Lapčík, O.; Honys, David

    Washington, D.C : IEEE Computer Society, 2012 - (Sloan, K.), s. 344-347 ISBN 978-0-7695-4706-0. [International Conference on Biomedical Engineering and Biotechnology /2012/. Macau (CN), 28.05.2012-30.05.2012] R&D Projects: GA ČR GA525/09/0994; GA ČR(CZ) GAP501/11/1462; GA MŠk(CZ) OC10054 Institutional support: RVO:61389030 Keywords : legumes * non-leguminous plants * isoflavone synthase Subject RIV: EF - Botanics

  13. CTP Limitation Increases Expression of CTP Synthase in Lactococcus lactis

    Jrgensen, Casper Mller; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which ?-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced le...

  14. Inter-domain Communication of Human Cystathionine β-Synthase

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

    2014-01-01

    Cystathionine β-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at t...

  15. Regulation of endothelial nitric oxide synthase by phosphorylation

    Mohamed, Annisuddin

    2007-01-01

    Since its recognition as an endothelium-derived relaxing factor, the control and consequences of nitric oxide (NO) production have been investigated intensely. We know now that NO is not simply a vasodilator or regulator of smooth muscle tone but is a potent anti-platelet agent, neuromodulator and regulator of gene expression. NO is synthesized from the amino acid Larginine by a family of enzymes termed NO synthases (NOS). The endothelial (eNOS or NOS III) and neuronal (nNOS, NOS I or bNO...

  16. Producing a trimethylpentanoic acid using hybrid polyketide synthases

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

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  17. Inhibitors of the Hydrolytic Enzyme Dimethylarginine Dimethylaminohydrolase (DDAH): Discovery, Synthesis and Development.

    Murphy, Rhys B; Tommasi, Sara; Lewis, Benjamin C; Mangoni, Arduino A

    2016-01-01

    Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (l-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed. PMID:27187323

  18. Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway.

    Park, Sung Ryeol; Tripathi, Ashootosh; Wu, Jianfeng; Schultz, Pamela J; Yim, Isaiah; McQuade, Thomas J; Yu, Fengan; Arevang, Carl-Johan; Mensah, Abraham Y; Tamayo-Castillo, Giselle; Xi, Chuanwu; Sherman, David H

    2016-01-01

    Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A-C), with cahuitamycin C being the most effective inhibitor (IC50=14.5??M). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5??M). PMID:26880271

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

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

    2016-02-01

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

  20. Corrosion inhibitors. Manufacture and technology

    Detailed information is presented relating to corrosion inhibitors. Areas covered include: cooling water, boilers and water supply plants; oil well and refinery operations; fuel and lubricant additives for automotive use; hydraulic fluids and machine tool lubes; grease compositions; metal surface treatments and coatings; and general processes for corrosion inhibitors

  1. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

    Culbertson, Justin E; Chung, Dong hee; Ziebart, Kristin T; Espiritu, Eduardo; Toney, Michael D

    2015-04-14

    The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS. PMID:25710100

  2. Proteinase inhibitors in Brazilian leguminosae

    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.

  3. Selection criteria for corrosion inhibitors

    Webb, L.; Boivin, J. [Cormetrics, Calgary, AB (Canada)

    2008-07-01

    The use of a corrosion inhibitor is the primary method to control internal corrosion of pipelines and to reduce costly failures. This presentation discussed the selection criteria for corrosion inhibitors. The selection process requires a detailed analysis of system chemistry; modeling flow regime; and laboratory testing protocols that challenge the inhibitor under conditions analogous to the field. The nature of corrosion inhibitors and inhibitor requirements were described. Physical factors were also presented. These included viscosity and pour point; stability; density; effect on elastomers and other materials; emulsion tendency; foaming tendency; gunking; polymerization/sludging; and reaction with gases and liquids. Other topics that were discussed included compatibility; solubility; partitioning; environmental effects; and selection requirements. Film tenacity was described in terms of corrosivity; water chemistry; and flow. The presentation concluded with a discussion of performance testing and wheel testing. figs.

  4. Proteinaceous alpha-araylase inhibitors

    Svensson, Birte; Fukuda, Kenji; Nielsen, P.K.; Bønsager, Birgit Christine

    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...

  5. Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase

    Chen, Shaolin; Ehrhardt, David W.; Somerville, Chris R

    2010-01-01

    The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in aniso...

  6. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose

    Huan Huang; McIntosh, Avery L.; Martin, Gregory G.; Petrescu, Anca D.; Landrock, Kerstin K.; Danilo Landrock; Kier, Ann B.; Friedhelm Schroeder

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while bind...

  7. MOLECULAR CLONING AND CHARACTERIZATION OF CHROMOSOME-ENCODED CITRATE SYNTHASE GENE FROM SINORHIZOBIUM FREDII USDA257

    Citrate synthase, a key metabolic enzyme that condenses acetyl-CoA and oxaloacetate to citrate, plays an important role in nodulation and nitrogen fixation. We have isolated a citrate synthase gene by screening a Sinorhizobium fredii USDA257 cosmid library with a heterologous probe from S. meliloti....

  8. GENOTYPIC VARIATION IN THE PROMOTER REGION OF SUCROSE SYNTHASE-2 IN THE GENUS SACCHARUM

    Sucrose synthase (EC 2.4.1.13) is an important enzyme of sucrose metabolism in sugarcane (Saccharum sp. hybrids). One of the genes for sucrose synthase (Sus2) is more highly expressed in sucrose-storing genotypes than low-sucrose genotypes. We designed primers to amplify the 5' end of the Sus2 gene...

  9. Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

    In cereal seeds mutations in one or more starch synthases lead to decreased amylopectin and increased amylose content. Here, the impact of starch synthase IIa (SSIIa or SGP-1) mutations upon durum starch was investigated. A screen of durum accessions identified two lines lacking SGP-A1, the A geno...

  10. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    Napolitano, H. B.; Sculaccio, S. A.; Thiemann, O. H.; Oliva, G., E-mail: oliva@if.sc.usp.br [Instituto de Fsica de So Carlos, Universidade de So Paulo, Caixa Postal 369, 13560-970 So Carlos, SP (Brazil)

    2005-01-01

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa. Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P2{sub 1}2{sub 1}2 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 . The crystals diffract to a maximum resolution of 3.3 and a complete data set to 3.5 resolution was collected and analysed.

  11. Polyketide synthases from poison hemlock (Conium maculatum L.).

    Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

    2015-11-01

    Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. PMID:26260860

  12. Diagnosis of cystathionine beta-synthase deficiency by genetic analysis.

    Suri, Fatemeh; Narooie-Nejad, Mehrnaz; Safari, Iman; Moazzeni, Hamidreza; Rohani, Mohammad-Reza; Khajeh, Ali; Klotzle, Brandy; Fan, Jian-Bing; Elahi, Elahe

    2014-12-15

    Intellectual disability like other common diseases is often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. We present diagnosis of cystathionine beta-synthase (CBS) deficiency in a multiply affected Iranian family with obvious intellectual disability based on whole genome SNP homozygosity mapping. Diagnosis based on clinical presentations had not been made because of unavailability of appropriate medical services. Genetic analysis led to identification of homozygous c.346G>A in CBS that causes p.Gly116Arg in the encoded protein, cystathionine beta-synthase. CBS is the most common causative gene of homocystinurea. Later, the same mutation was found in three other apparently unrelated Iranian homocystinuria patients. p.Gly116Arg was reported once before in a Turkish patient, suggesting it may be a common CBS deficiency causing mutation in the Middle East. Clinical features of the patients are reported that evidence to variable presentations caused by the same mutation. Finally, observations in heterozygous carriers of the mutation suggest data that a single allele of the p.Gly116Arg causing mutation may have phenotypic consequences, including cardiac related phenotypes. Our study attests to the powers of genetic analysis for diagnosis especially for some forms of intellectual disability, with known genetic causing agents. PMID:25455305

  13. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency

    GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methane sulfonate, and three clones deficient in thymidylate synthase 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'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS-c1 and TSminusc2 but decreased levels in TS-c3. Clone TSminusc3 was 32-, 750-, and >100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo

  14. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa. Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P21212 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed

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

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

    2015-09-23

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

  16. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity

  17. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    Serer, María I.; Bonomi, Hernán R. [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina); Guimarães, Beatriz G. [Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin BP 48, 91192 Gif-sur-Yvette CEDEX (France); Rossi, Rolando C. [Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires (Argentina); Goldbaum, Fernando A.; Klinke, Sebastián, E-mail: sklinke@leloir.org.ar [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina)

    2014-05-01

    This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

  18. Methylation and Gene Expression Responses to Ethanol Feeding and Betaine Supplementation in the Cystathionine Beta Synthase-Deficient Mouse

    Medici, Valentina; Schroeder, Diane I.; Woods, Rima; LaSalle, Janine M.; Geng, Yongzhi; Shibata, Noreene M.; Peerson, Janet; Hodzic, Emir; Dayal, Sanjana; Tsukamoto, Hidekazu; Kharbanda, Kusum K.; Tillman, Brittany; French, Samuel W.; Halsted, Charles H.

    2014-01-01

    Background Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol on hepatic methionine metabolism. Methods To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-wk intragastric ethanol feeding with and without the methyl donor betaine in cystathionine beta synthase (C?S) heterozygous C57BL/6J mice. Results The histopathology of early ASH was induced by ethanol feeding and prevented by betaine supplementation, while ethanol feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-Seq genomic sequencing of heterozygous liver samples from each diet group found 24% reduced methylation in gene bodies but not promoter regions of all autosomes of ethanol fed mice, each of which were normalized in samples from mice fed the betaine supplemented diet. The transcript levels of inducible nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-a (Ppar?) were reduced in ethanol fed mice, and each was normalized in mice fed the betaine supplemented diet. DNA pyrosequencing of C?S heterozygous samples found reduced methylation in a gene body of Nos2 by ethanol feeding that was restored by betaine supplementation, and was correlated inversely with its expression and positively with SAM: SAH ratios. Conclusions The present studies have demonstrated relationships among ethanol induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that ethanol-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH is a result of altered methionine metabolism that can be reversed through dietary supplementation of methyl donors. PMID:24730561

  19. Nuclear glycogen synthase kinase-3 {beta} (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    Mentzingen, Leticia; Andrade, Josiana G. de; Logullo, Carlos [Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ (Brazil). Centro de Biociencias e Biotecnologia. Lab. de Quimica e Funcao de Proteinas e Peptideos (LQFPP); Andrade, Caroline P. de; Vaz Junior, Itabajara [Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS (Brazil). Centro de Biotecnologia

    2008-07-01

    Full text: Glycogen synthase kinase-3 (GSK3) is recognized as a key component of a large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. Recent observations point to functions for phosphorylases several transcription factors in the nucleus. Also, GSK3b participate of the canonical W nt signalling pathway, which has been studied intensively in embryonic and cancer cells. Like in many other signaling pathways, most components in W nt signal transduction were highly conserved during the evolution. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Although GSK3 is traditionally considered a cytosolic protein, it is also present in nuclei. Nuclear GSK3 is particularly interesting because of the many transcription factors that it regulates enabling GSK3 to influence many signaling pathways that converge on these transcription factors, thereby regulating the expression of many genes. Our group identified that GSK-3 {beta} could be detected in different stage eggs of R. micro plus. In this work we detected the GSK-3 in isolated nuclear fraction from the egg homogenates of R. micro plus by western-blot analysis, using anti-GSK- 3 {beta} antibodies. The enzyme activity was also detected radiochemically throughout embryogenesis in same fraction. The GSK-3 activity was inhibiting by using SB 216763 (selective molecule inhibitors of GSK-3). Taken together our results suggest that GSK-3 {beta} isoform probably is involved in gene transcription factors during R. micro plus embryo development.

  20. Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide synthase knockout mice.

    Agarwal, Rakhee; Hennings, Leah; Rafferty, Tonya M; Letzig, Lynda G; McCullough, Sandra; James, Laura P; MacMillan-Crow, Lee Ann; Hinson, Jack A

    2012-01-01

    In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity. PMID:22001257