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

Directory of Open Access Journals (Sweden)

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

Fabiano André Petter

2013-09-01

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

Scientific Electronic Library Online (English)

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

Patrícia Andrea, Monqueiro; Pedro Jacob, Christoffoleti.

2001-03-01

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

Scientific Electronic Library Online (English)

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

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

1999-12-01

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

Directory of Open Access Journals (Sweden)

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.

Ribas A. Vidal

1999-12-01

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

Science.gov (United States)

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

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

2015-01-01

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Detailed Structure-Function Correlations of Bacillus subtilis Acetolactate Synthase.  

Science.gov (United States)

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

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

2015-01-01

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

Scientific Electronic Library Online (English)

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

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

2014-06-01

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

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

International Nuclear Information System (INIS)

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

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

OpenAIRE

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

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

2013-01-01

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

Science.gov (United States)

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

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

2014-11-01

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

Directory of Open Access Journals (Sweden)

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

De Cauwer, Benny

2014-02-01

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

Science.gov (United States)

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

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

2013-04-01

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

Science.gov (United States)

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

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

2015-01-01

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

Science.gov (United States)

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

Délye, Christophe; Pernin, Fanny; Scarabel, Laura

2011-02-01

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

Directory of Open Access Journals (Sweden)

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

Patrick W. Geier

2013-04-01

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

Scientific Electronic Library Online (English)

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

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

2006-07-01

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

Science.gov (United States)

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

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

2013-01-16

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

Science.gov (United States)

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

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

2003-11-01

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

Scientific Electronic Library Online (English)

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

Alexandre Magno, Brighenti.

2012-06-01

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

Science.gov (United States)

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

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

2014-11-01

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

Science.gov (United States)

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

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

2014-08-01

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Nitric oxide synthase inhibitors and cerebral vasospasm.  

Science.gov (United States)

L-arginine is a source of nitric oxide (NO) that is cleaved from the terminal guanidino nitrogen atom by nitric oxide synthase (NOS). NO evokes, because of its free radical properties and affinity to heme, ferrous iron and cysteine, a wide spectrum of physiological and pathophysiological effects. For many years, different exogenous NOS inhibitors were used to elucidate the role of NOS and NO in health and disease. Later, endogenous NOS inhibitors, as asymmetric dimethylarginine (ADMA) were discovered. Endogenous inhibitors as ADMA are produced by post-translational methylation of L-arginine which is catalyzed by a family of protein N-methyltransferases (PRMT), using S-adenosylmethionine as a methyl group donor. ADMA is eliminated by dimethylarginine dimethylaminohydrolases (DDAH I or II). ADMA hydrolysis increases NOS activity and NO production. Furthermore, L-citrulline, a by-product of ADMA hydrolysis as well as of NO production by NOS, can in turn inhibit DDAH. Therefore, endogenous inhibition of NOS can be modified via different ways (1) changing the availability of L-arginine and/or of L-citrulline; (2) stimulating or inhibiting DDAH activity; (3) modifying methylation via regulating availability of adenosylmethionine; or (4) modifying PRMT activity. Research elucidating the role of NOS inhibitors in respect of delayed cerebral vasospasm after subarachnoid hemorrhage is summarized. PMID:21116921

Jung, C S

2011-01-01

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Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.  

Science.gov (United States)

There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ?2 ?M, Ki ?300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 ?g/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

2014-07-10

25

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

Directory of Open Access Journals (Sweden)

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.

D.L.P. Gazziero

2006-01-01

26

Characterization of microsomal prostaglandin E synthase 1 inhibitors.  

Science.gov (United States)

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible terminal synthase in PGE2 biosynthesis by inflammatory and cancer cells. Clinical and experimental data emphasize that mPGES-1 might be a valuable target, with improved selectivity and safety compared to traditional NSAIDs or selective COX-2 inhibitors, in the treatment of inflammatory diseases, different types of cancer as well as central symptoms elicited by peripheral inflammation. Since the first characterization of mPGES-1, the numbers of publications on mPGES-1 structure, pathogenic role and inhibitor development have increased exponentially; however, there are currently no selective mPGES-1 inhibitors available for clinical use. In this MiniReview, we focus on recent advances in the development of selective inhibitors of mPGES-1 activity, with the aim to discuss the effects of targeting mPGES-1 in different inflammatory models in vitro and in vivo. PMID:24138533

Korotkova, Marina; Jakobsson, Per-Johan

2014-01-01

27

Novel inhibitors of nitric oxide synthase with antioxidant properties.  

Science.gov (United States)

We previously described a series of imidazole-based inhibitors substituted at N-1 with an arylethanone chain as interesting inhibitors of neuronal nitric oxide synthase (nNOS), endowed with good selectivity vs endothelial nitric oxide synthase (eNOS). As a follow up of these studies, several analogs characterized by the presence of substituted imidazoles or other mono or bicyclic nitrogen-containing heterocycles instead of simple imidazole were synthesized, and their biological evaluation as in vitro inhibitors of both nNOS and eNOS is described herein. Most of these compounds showed improved nNOS and eNOS inhibitory activity with respect to reference inhibitors. Selected compounds were also tested to analyze their antioxidant properties. Some of them displayed good capacity to scavenge free radicals and ability to reduce lipid peroxidation. PMID:22280820

Salerno, Loredana; Modica, Maria N; Romeo, Giuseppe; Pittalà, Valeria; Siracusa, Maria A; Amato, Maria E; Acquaviva, Rosaria; Di Giacomo, Claudia; Sorrenti, Valeria

2012-03-01

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Enhancement of vascular targeting by inhibitors of nitric oxide synthase  

International Nuclear Information System (INIS)

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

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Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.  

Science.gov (United States)

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis. PMID:10620343

Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

2000-01-01

30

Design, development and synthesis of ribitylaminopyrimidinedione derivatives as lumazine synthase mechanistic probes and trifluoromethylated pyrazoles as riboflavin synthase inhibitors  

OpenAIRE

Inhibition of riboflavin synthase and lumazine synthase, the last two enzymes in the riboflavin biosynthetic pathway, provides a strategy for the development of therapeutically useful antibiotics.^ A homologous series of four derivatives of 5-amino-6-ribitylamino-1 H-pyrimidine-2,4-dione were designed and synthesiszed as potential enzyme inhibitors and mechanistic probes. Some of them displayed moderate inhibition activity on Mycobacterium tuberculosis lumazine synthase and riboflavin synth...

Zhao, Yujie

2009-01-01

31

Can P-glycoprotein influence the bioavailability of iminosugar-based glucosylceramide synthase inhibitors?  

OpenAIRE

Recently developed glucosylceramide synthase inhibitors with enhanced hydrophobicity display increased bioavailability in the central nervous system (CNS). Have these improvements come at a potential risk given that the improved glucosylceramide synthase inhibitors bear the hallmarks of P-glycoprotein substrates? This question warrants attention given the potential to induce adverse drug interactions or toxicity, if glucosylceramide synthase inhibitors are administered with other P-glycoprote...

Norris-cervetto, E.; Butters, Td; Martin, C.; Modok, S.; Dwek, Ra; Callaghan, R.

2006-01-01

32

Fatty acid synthase inhibitors isolated from Punica granatum L  

International Nuclear Information System (INIS)

The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC50 value of 10.3 ?mol L-1. (author)

33

Fatty acid synthase inhibitors isolated from Punica granatum L  

Energy Technology Data Exchange (ETDEWEB)

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)

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

34

Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase  

Energy Technology Data Exchange (ETDEWEB)

Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E. (Tennessee-HSC); (SJCH)

2010-01-12

35

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

Science.gov (United States)

Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues-sT-CH2 -CoA (26?a) and sTet-CH2 -CoA (26?b)-as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26?a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26?a and 26?b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

2015-01-01

36

Fatty acid synthase inhibitors isolated from Punica granatum L.  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Este trabalho tem por objetivo o isolamento de inibidores da enzima ácido graxo sintase (FAS) a partir de acetato de etila proveniente de extratos de cascas de frutas da Punica granatum L. A investigação química guiada por bioensaios das cascas das frutas resultou no isolamento de dezessete composto [...] s incluindo principalmente triternóides e compostos fenólicos, dos quais um novo triterpeno do tipo oleanano (punicaone) juntamente com quatorze compostos conhecidos foram isolados pela primeira vez a partir desta planta. Sete dos componentes isolados foram avaliados para atividades inibitórias de FAS e dois deles apresentaram-se ativos. Em particular, o ácido flavogalônico que exibiu forte atividade inibitória de FAS com valor de IC50 de 10,3 µmol L-1. Abstract in english The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic co [...] mpounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC50 value of 10.3 µmol L-1.

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

2012-05-01

37

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

Directory of Open Access Journals (Sweden)

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

Hsueh-Fen Juan

2011-08-01

38

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

OpenAIRE

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

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

1994-01-01

39

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

International Nuclear Information System (INIS)

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

40

Nitric oxide synthase inhibitors containing the carboxamidine group or its isosteres  

International Nuclear Information System (INIS)

The review summarises structures, activities and selectivity of NO-synthase (NOS) inhibitors belonging to various classes of chemical compounds. Linear, cyclic and heterocyclic structures containing guanidine, amidine and/or isothiourea fragments are considered. The structure-activity relationships for these inhibitors were analysed in relation to their action on the inducible NOS isoform. This analysis can provide the basis for the synthesis of new more efficient compounds.

41

Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments  

OpenAIRE

Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, sp...

King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Ele?onore

2013-01-01

42

Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo.  

OpenAIRE

1. Three analogues of L-arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat. 2. NG-monomethyl-L-arginine (L-NMMA), N-iminoethyl-L-ornithine (L-NIO) and NG-nitro-L-arginine methyl ester (L-NAME; all at 0.1-100 microM) caused concentration-dependent inhibition of the Ca2(+)-dependent endothelia...

Rees, D. D.; Palmer, R. M.; Schulz, R.; Hodson, H. F.; Moncada, S

1990-01-01

43

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

OpenAIRE

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

Duhoux, Arnaud; De?lye, Christophe

2013-01-01

44

Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.  

OpenAIRE

1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring ...

Szabo?, C.; Bryk, R.; Zingarelli, B.; Southan, G. J.; Gahman, T. C.; Bhat, V.; Salzman, A. L.; Wolff, D. J.

1996-01-01

45

Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors.  

OpenAIRE

1. Electrophysiological (whole-cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea-pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2. The two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA, 1 mM) and NG-nitro-L-arginine (L-NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D-NMMA (1 mM), the stereoi...

Costamagna, Costanzo; Bosia, Amalia; Alloatti, Giuseppe; Gallo, Maria Pia; Ghigo, Dario Antonio; Levi, Renzo; Penna, Claudia

1998-01-01

46

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

International Nuclear Information System (INIS)

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

47

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

Science.gov (United States)

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

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

2014-07-01

48

Good Response to Pemetrexed after Treatment Failure with Another Thymidylate Synthase Inhibitor in Lung Adenocarcinoma: A Case Report  

OpenAIRE

Pemetrexed is an antimetabolic agent and is well-known as a potent inhibitor of thymidylate synthase (TS). It also inhibits dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). We reported an intriguing case in which a non-small cell lung cancer patient who was refractory to pretreatment with S-1, a strong TS inhibitor, showed...

Fumihiko Hirai; Eiko Inamasu; Gouji Toyokawa; Tsukihisa Yoshida; Kaname Nosaki; Tomoyoshi Takenaka; Masafumi Yamaguchi; Mitsuhiro Takenoyama; Takashi Seto; Yukito Ichinose

2014-01-01

49

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

Science.gov (United States)

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

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

2007-02-01

50

Selective L-Nitroargininylaminopyrrolidine and L-Nitroargininylaminopiperidine Neuronal Nitric Oxide Synthase Inhibitors  

OpenAIRE

Selective inhibition of the localized excess production of NO by neuronal nitric oxide synthase (nNOS) has been targeted as a potential means of treating various neurological disorders. Based on observations from the X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N?-nitroguanidine (L-Arg(NO2)-L-Dbu-NH2 (1) and 4-N-(N?-nitro-L-argininyl)-trans-4-amino-Lproline amide (2), a series of descarboxamide analogues was...

Seo, Jiwon; Marta?sek, Pavel; Roman, Linda J.; Silverman, Richard B.

2007-01-01

51

NO synthase inhibitors attenuate locus coeruleus catecholamine metabolism and behavior induced by morphine withdrawal.  

Science.gov (United States)

The effects of nitric oxide synthase (NOS) inhibitors were examined simultaneously on the behavior and on the catecholaminergic metabolism in the locus coeruleus (LC) during morphine withdrawal using microdialysis in freely moving rats. Morphine withdrawal was precipitated by naltrexone administration to morphine-treated rats. Acute pretreatment of rats with NOmicron-nitro-L-arginine-p-nitroanilide (L-NAPNA) or 7-nitroindazole (7-NI) before naltrexone challenge attenuated the behavioral expression of morphine withdrawal and strongly reduced the withdrawal-induced increase in 3,4-dihydroxyphenylacetic acid (DOPAC) in the LC. The two NOS inhibitors also decreased DOPAC in absence of naltrexone challenge. These results suggest a role for NO in the expression of morphine withdrawal syndrome that may be mediated, at least in part, by LC noradrenergic neurons. PMID:11973478

Javelle, Nathalie; Bérod, Anne; Renaud, Bernard; Lambás-Señas, Laura

2002-04-16

52

A human fatty acid synthase inhibitor binds ?-ketoacyl reductase in the keto-substrate site.  

Science.gov (United States)

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the ?-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor. PMID:25086508

Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

2014-09-01

53

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

OpenAIRE

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

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

1992-01-01

54

Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor.  

Science.gov (United States)

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a (11)C-labeled specific 20-HETE synthase inhibitor, N'(4-dimethylaminohexyloxy)phenyl imidazole ([(11)C]TROA). Autoradiographic study showed that [(11)C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [(11)C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [(11)C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [(11)C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia. PMID:22669478

Kawasaki, Toshiyuki; Marumo, Toshiyuki; Shirakami, Keiko; Mori, Tomoko; Doi, Hisashi; Suzuki, Masaaki; Watanabe, Yasuyoshi; Chaki, Shigeyuki; Nakazato, Atsuro; Ago, Yukio; Hashimoto, Hitoshi; Matsuda, Toshio; Baba, Akemichi; Onoe, Hirotaka

2012-09-01

55

Fatty acid synthase inhibitors from the hulls of Nephelium lappaceum L.  

Science.gov (United States)

Natural products inhibiting fatty acid synthase (FAS) are appearing as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Nephelium lappaceum L. resulted in the isolation of ten compounds (1-10) mainly including flavonoids and oleane-type triterpene oligoglycosides, in which all of the compounds were isolated from this plant for the first time. Additionally, compounds 8 and 9 were new hederagenin derivatives and were elucidated as hederagenin 3-O-(2,3-di-O-acetyl-?-l-arabinofuranosyl)-(1?3)-[?-l-rhamnopyranosyl(1?2)]-?-l-arabinopyranoside and hederagenin 3-O-(3-O-acetyl-?-l-arabinofuranosyl)-(1?3)-[?-l-rhamnopyranosyl-(1?2)]-?-l-arabinopyranoside, respectively. All these isolates were evaluated for inhibitory activities of FAS, which showed these isolates had inhibitory activity against FAS with IC(50) values ranging from 6.69 to 204.40 ?M, comparable to the known FAS inhibitor EGCG (IC(50)=51.97 ?M). The study indicates that the hulls of Nephelium lappaceum L. could be considered as potential sources of promising FAS inhibitors and the oleane-type triterpene oligoglycosides could be considered as another type of natural FAS inhibitors. PMID:21605850

Zhao, You-Xing; Liang, Wen-Juan; Fan, Hui-Jin; Ma, Qing-Yun; Tian, Wei-Xi; Dai, Hao-Fu; Jiang, He-Zhong; Li, Ning; Ma, Xiao-Feng

2011-08-16

56

The glucosylceramide synthase inhibitor PDMP sensitizes pancreatic cancer cells to MEK/ERK inhibitor AZD-6244.  

Science.gov (United States)

Here we show that d,l-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a glycosphingolipid biosynthesis inhibitor, increases the sensitivity of pancreatic cancer cells to the novel MEK-ERK inhibitor AZD-6244. AZD-6244 and PDMP co-administration induced massive pancreatic cancer cell death and apoptosis, more potently than either drug alone. We discovered that AZD-6244 induced ceramide production in pancreatic cancer cells, yet the excess ceramide was metabolically removed in the long-term (24-48h). PDMP facilitated AZD-6244-induced ceramide production, and ceramide level remained elevated up to 48h. Meanwhile, exogenously-added cell-permeable short chain ceramide (C2) similarly sensitized AZD-6244's activity, the two caused substantial pancreatic cancer cell death and apoptosis. At the molecular level, PDMP and AZD-6244 co-treatment inactivated ERK1/2 and AKT-mTOR signalings simultaneously in pancreatic cancer cells, while either agent alone only affected one signaling. In summary, PDMP significantly increased the sensitivity of AZD-6244 in pancreatic cancer cells. This appears to involve a sustained ceramide production as well as concurrent block of ERK and AKT-mTOR signalings. PMID:25498501

Wang, Ting; Wei, Jue; Wang, Na; Ma, Jia-Li; Hui, Ping-Ping

2015-01-16

57

Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.  

Science.gov (United States)

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi. PMID:23891162

Gahungu, Mathias; Arguelles-Arias, Anthony; Fickers, Patrick; Zervosen, Astrid; Joris, Bernard; Damblon, Christian; Luxen, André

2013-09-01

58

Nitric oxide synthase inhibitors protect cholinergic neurons against quinolinic acid toxicity in the rat brain  

Directory of Open Access Journals (Sweden)

Full Text Available The aim of this study was to examine the effects of intrastriatally injected nitric oxide synthase (NOS inhibitors, N?-nitro-l-arginine methyl ester (L-NAME and 7-nitroindazole (7-NI, on quinolinic acid (QA-induced toxicity in selective vulnerable brain regions of adult Wistar rats. QA was administered into the striatum unilaterally, in a single dose of 150 nM/L with a stereotaxic instrument. The other two experimental groups were pretreated with L-NAME and 7-NI, respectively. The control group of animals was treated with 0.154 mM/L saline solution. The animals were decapitated seven days after the treatment. Samples of both striatum and forebrain cortex were prepared for measurement of acetylcholinesterase (AChE activity. QA injection revealed a significant increase in AChE activity in both the ipsi- and contralateral striatum and forebrain cortex compared to the control animals. Treatment with NOS inhibitors, followed by QA, very clearly demonstrated lower levels of AChE bilaterally in these brain structures, compared to the QA-treated group.

Stevanovi? Ivana

2013-01-01

59

Polymorphic human prostaglandin H synthase-2 proteins and their interactions with cyclooxygenase substrates and inhibitors.  

Science.gov (United States)

The cyclooxygenase (COX) activity of prostaglandin H synthase-2 (PGHS-2) is implicated in colorectal cancer and is targeted by nonsteroidal anti-inflammatory drugs (NSAIDs) and dietary n-3 fatty acids. We used purified, recombinant proteins to evaluate the functional impacts of the R228H, E488G, V511A and G587R PGHS-2 polymorphisms on COX activity, fatty acid selectivity and NSAID actions. Compared to wild-type PGHS-2, COX activity with arachidonate was ?20% lower in 488G and ?20% higher in 511A. All variants showed time-dependent inhibition by the COX-2-specific inhibitor (coxib) nimesulide, but 488G and 511A had 30-60% higher residual COX activity; 511A also showed up to 70% higher residual activity with other time-dependent inhibitors. In addition, 488G and 511A differed significantly from wild type in Vmax values with the two fatty acids: 488G showed ?20% less and 511A showed ?20% more discrimination against eicosapentaenoic acid. The Vmax value for eicosapentaenoate was not affected in 228H or 587R, nor were the Km values or the COX activation efficiency (with arachidonate) significantly altered in any variant. Thus, the E488G and V511A PGHS-2 polymorphisms may predict who will most likely benefit from interventions with some NSAIDs or n-3 fatty acids. PMID:20548327

Liu, W; Poole, E M; Ulrich, C M; Kulmacz, R J

2011-10-01

60

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

Directory of Open Access Journals (Sweden)

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

Wilson Abrao Saad

2006-01-01

61

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

Science.gov (United States)

...2010-04-01 2009-04-01 true Alpha-acetolactate decarboxylase (Î...Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (?-ALDC...Bacillus subtilis. The food additive alpha-acetolactate decarboxylase...

2010-04-01

62

In vivo study of radioprotective effect of NO-synthase inhibitors and acetyl-L-carnitine.  

Science.gov (United States)

This study investigated the protective effect of two nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg i.p.) and aminoguanidine (AG, 400 mg/kg i.p.), and an antioxidant acetyl-L-carnitine (ALC, 250 mg/kg i.p., once daily for five days) against radiation-induced damage in Wistar rats. Blood samples were collected 6 h after whole-body irradiation with 8 Gy. Plasma concentrations of nitrite+nitrate (NO(x)) and malondialdehyde (MDA) were measured by high-performance liquid chromatography. A single injection of L-NAME one hour before exposure effectively prevented the radiation-induced elevation of plasma NO(x) and it reduced 2.6-fold the risk for death during the subsequent 30-day period. Pretreatment with ALC prevented the radiation-induced increase in plasma MDA and it had similar effect on mortality as L-NAME did. Presumably due to its short half-life, the partially iNOS-selective inhibitor and antioxidant AG given in a single dose before exposure did not attenuate MDA and NO(x) and it failed to significantly improve the 30-day survival. In conclusion, pretreatment with both the nonspecific NOS inhibitor L-NAME and the antioxidant ALC markedly reduce mortality to radiation sickness in rats. The radioprotective effect may be directly related to effective attenuation of the radiation-induced elevation of NO production by L-NAME and of oxidative stress by ALC. PMID:23869893

Babicová, A; Havlínová, Z; Hroch, M; Rezá?ová, M; Pejchal, J; Vávrová, J; Chládek, J

2013-12-20

63

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase.  

Science.gov (United States)

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 ?M, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 ?M, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 ?M, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 ?g/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 ?. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors. PMID:23523771

Cho, June-Haeng; Lee, Mi-Young; Baig, Irshad Ahmed; Ha, Na-Reum; Kim, Joungmok; Yoon, Moon-Young

2013-07-01

64

Pharmacological and toxicological evaluation of a new series of thymidylate synthase inhibitors as anticancer agents.  

Science.gov (United States)

Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3-bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR35) and 6-bromo-3,3-bis(3-chloro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [3H]-thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [3H]-thymidine test was the best in predicting toxicity against melanoma cells. PMID:17094473

Benassi, Luisa; Magnoni, Cristina; Giudice, Stefania; Bertazzoni, Giorgia; Costi, Maria Paola; Rinaldi, Marcella; Venturelli, Alberto; Coppi, Andrea; Rossi, Tiziana

2006-01-01

65

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target.  

Science.gov (United States)

N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

2014-01-01

66

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target  

Science.gov (United States)

N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

2014-01-01

67

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

Science.gov (United States)

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

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

1993-11-25

68

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

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

Irene Mavelli

2012-02-01

69

Effects of nitric oxide synthase inhibitor ?-Nitro-L-Arginine Methyl Ester, on silica-induced inflammatory reaction and apoptosis  

OpenAIRE

Abstract Background Although nitric oxide is overproduced by macrophages and neutrophils after exposure to silica, its role in silica-induced inflammatory reaction and apoptosis needs further clarification. In this study, rats were intratracheally instilled with either silica suspension or saline to examine inflammatory reactions and intraperitoneally injected with ?-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthases, or saline to examine the poss...

Leigh James; Wang He

2006-01-01

70

Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation  

OpenAIRE

Background and purpose: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimers disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. Experimental approach: Bra...

Selenica, Maj-linda; Jensen, Henning S.; Larsen, Anna Kirstine; Pedersen, M. L.; Helboe, Lone; Leist, Marcel; Lotharius, Julie

2007-01-01

71

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

OpenAIRE

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

Gao, Su; Lane, M. Daniel

2003-01-01

72

A phase I study of the lipophilic thymidylate synthase inhibitor Thymitaq™ (nolatrexed dihydrochloride) given by 10-day oral administration  

OpenAIRE

2-Amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (nolatrexed dihydrochloride, Thymitaq, AG337), a specific inhibitor of thymidylate synthase, was developed using protein structure-based drug design. Intravenously administered nolatrexed is active clinically. As oral bioavailability is high (70–100%), nolatrexed was administered orally, 6 hourly for 10 days, at 3-week intervals, and dose escalated from 80 to 572 mg m?2day?1in 23 patients. Common toxicity c...

Jodrell, D. I.; Bowman, A.; Rye, R.; Byrne, B.; Boddy, A.; Rafi, I.; Taylor, G. A.; Johnston, A; Clendeninn, N. J.

1999-01-01

73

Thiazolopyrimidine inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum.  

Science.gov (United States)

A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay. PMID:20480490

Geist, Julie G; Lauw, Susan; Illarionova, Victoria; Illarionov, Boris; Fischer, Markus; Gräwert, Tobias; Rohdich, Felix; Eisenreich, Wolfgang; Kaiser, Johannes; Groll, Michael; Scheurer, Christian; Wittlin, Sergio; Alonso-Gómez, José L; Schweizer, W Bernd; Bacher, Adelbert; Diederich, François

2010-07-01

74

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

75

Identification of a glycogen synthase kinase-3? inhibitor that attenuates hyperactivity in CLOCK mutant mice.  

Science.gov (United States)

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? (GSK-3?) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3?. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC?? values in the range of 4 to 680?nM against human GSK-3?. 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?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?¹) and the antipsychotic haloperidol (1?mg?kg?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? in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3? as a relevant therapeutic target in the identification of new therapies for bipolar patients. PMID:21732538

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

2011-09-01

76

Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis.  

Science.gov (United States)

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor alpha (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested. PMID:12444550

Shchepina, Liarisa A; Pletjushkina, Olga Y; Avetisyan, Armine V; Bakeeva, Liora E; Fetisova, Elena K; Izyumov, Denis S; Saprunova, Valeria B; Vyssokikh, Mikhail Y; Chernyak, Boris V; Skulachev, Vladimir P

2002-11-21

77

Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.  

Science.gov (United States)

Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS. PMID:17928650

Soszynski, D; Chelminiak, M

2007-09-01

78

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

Science.gov (United States)

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

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

2012-12-14

79

Repositioning proton pump inhibitors as anticancer drugs by targeting the thioesterase domain of human Fatty Acid synthase.  

Science.gov (United States)

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 FDA-approved drug database, we identified proton pump inhibitors (PPIs) as effective inhibitors of the thioesterase activity of human FASN. Further investigation showed that PPIs inhibited proliferation and induced apoptosis of cancer cells. Supplementation of palmitate, the end product of FASN catalysis, rescued cancer cells from PPI-induced cell death. These findings provide new evidence for the mechanism by which this FDA-approved class of compounds may be acting on cancer cells. PMID:25513712

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

2015-01-22

80

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

Science.gov (United States)

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

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

2012-06-19

81

Diacetyl and ?-Acetolactate Overproduction by Lactococcus lactis subsp. lactis Biovar Diacetylactis Mutants That Are Deficient in ?-Acetolactate Decarboxylase and Have a Low Lactate Dehydrogenase Activity  

OpenAIRE

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor ?-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in ?-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of ?-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was suppleme...

Monnet, Christophe; Aymes, Fre?de?ric; Corrieu, Georges

2000-01-01

82

The Effects of C75, an Inhibitor of Fatty Acid Synthase, on Sleep and Metabolism in Mice  

OpenAIRE

Sleep is greatly affected by changes in metabolic state. A possible mechanism where energy-sensing and sleep-regulatory functions overlap is related to lipid metabolism. Fatty acid synthase (FAS) plays a central role in lipid metabolism as a key enzyme in the formation of long-chain fatty acids. We studied the effects of systemic administration of C75, an inhibitor of FAS, on sleep, behavioral activity and metabolic parameters in mice. Since the effects of C75 on feeding and metabolism are th...

Pellinen, Jacob; Szentirmai, E?va

2012-01-01

83

Time-dependent enhancement or inhibition of endotoxin-induced vascular injury in rat intestine by nitric oxide synthase inhibitors.  

OpenAIRE

1. The effects of the nitric oxide (NO) synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), on the vascular damage induced by the endotoxin, E. coli lipopolysaccharide (LPS), in the ileum and colon were investigated in the conscious rat over a 5 h period. 2. Administration of LPS (3 mg kg-1, i.v.) increased ileal and colonic vascular injury after a lag period of 2 h, as determined by the leakage of radiolabelled albumin. 3. Administration of L...

Laszlo, F.; Whittle, B. J.; Moncada, S

1994-01-01

84

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

OpenAIRE

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

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

2009-01-01

85

Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.  

Science.gov (United States)

Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families. PMID:23627736

Capasso, Clemente; Supuran, Claudiu T

2014-06-01

86

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

OpenAIRE

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

Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

2013-01-01

87

Effects of various nitric oxide synthase inhibitors on AlCl3-induced neuronal injury in rats  

Directory of Open Access Journals (Sweden)

Full Text Available The present study was aimed at determining the effectiveness of nitric oxide synthase (NOS inhibitors: N-nitro-L-arginine methyl ester, 7-nitroindazole and aminoguanidine in modulating the toxicity of AlCl3 on superoxide production and the malondialdehyde concentration of Wistar rats. The animals were sacrificed 10 min and 3 days after the treatment and the forebrain cortex was removed. The results show that AlCl3 exposure promotes oxidative stress in different neural areas. The biochemical changes observed in the neuronal tissues show that aluminum acts as pro-oxidant, while NOS inhibitors exert an anti-oxidant action in AlCl3-treated animals.

IVANA STEVANOVI?

2009-05-01

88

Use of structure-based drug design approaches to obtain novel anthranilic acid acyl carrier protein synthase inhibitors.  

Science.gov (United States)

Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail. PMID:16335920

Joseph-McCarthy, Diane; Parris, Kevin; Huang, Adrian; Failli, Amedeo; Quagliato, Dominick; Dushin, Elizabeth Glasfeld; Novikova, Elena; Severina, Elena; Tuckman, Margareta; Petersen, Peter J; Dean, Charles; Fritz, Christian C; Meshulam, Tova; DeCenzo, Maureen; Dick, Larry; McFadyen, Iain J; Somers, William S; Lovering, Frank; Gilbert, Adam M

2005-12-15

89

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

International Nuclear Information System (INIS)

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

90

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

2013-11-15

91

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1986-05-01

92

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

International Nuclear Information System (INIS)

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

93

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

Science.gov (United States)

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

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

2012-03-01

94

Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX.  

Science.gov (United States)

Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activity has been found to begin with Intermediate II, which contains a tyrosyl radical. The structure of this radical is altered by cyclooxygenase inhibitors, such as indomethacin and flurbiprofen, and by replacement of heme by manganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation in inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow spectroscopic techniques and by chromatographic and mass spectrometric analysis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-)1 in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(-)1); as with PGHS-1 itself, the peroxidase inactivation rates were independent of peroxide concentration and structure, consistent with an inactivation process beginning with Intermediate II. The changes in metalloporphyrin absorbance spectra during inactivation of inhibitor-treated PGHS-1 were similar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next species were comparable to the inactivation kinetics in each case. In contrast to the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron chlorin and heme-protein adduct species. Structural perturbations at the peroxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PGHS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation. PMID:11279106

Wu, G; Vuletich, J L; Kulmacz, R J; Osawa, Y; Tsai, A L

2001-06-01

95

Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful?  

Directory of Open Access Journals (Sweden)

Full Text Available The glycogen synthase kinase 3 (GSK-3 is a serine/threonine kinase widely expressed in mammalian tissues. Ini-tially identified by its ability to modulate glycogen synthesis, GSK-3 turned out to be a multifunctional enzyme, able to phosphorylate many proteins, including members of the steroid receptor superfamily. Although GSK-3 was shown to phosphorylate the androgen receptor (AR, its effects on AR transcriptional activity remain controversial. Analysis of short hairpin RNA (shRNA–mediated downmodulation of GSK-3 proteins in prostate cancer cells showed a reduction in AR transcriptional activity and AR protein levels. Pharmacological GSK-3 inhibitors such as the maleimide SB216763 or the aminopyrazole GSK inhibitor XIII inhibited AR-dependent reporter gene activity and AR expression in vitro. Analysis of androgen-induced nuclear translocation of the AR was performed in PC3 cells transfected with pAR-t1EosFP coding for EosAR, a green fluorescent AR fusion protein. When grown in pres-ence of androgens, EosAR was predominantly nuclear. Incubation with SB216763 before and after androgen treat-ment almost completely reduced nuclear EosAR. In contrast, the thiazole-containing urea compound AR-A014418 increased rather than decreased AR–expression/function. Although not all GSK inhibitors affected AR–stability/ function, our observations suggest a potential new therapeutic application for some of these compounds in pros-tate cancer.

Ludwig Rinnab

2008-06-01

96

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

Science.gov (United States)

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

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

2013-03-01

97

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

Science.gov (United States)

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

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

2010-12-01

98

Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor.  

Science.gov (United States)

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P. PMID:11371194

Asojo, O; Friedman, J; Adir, N; Belakhov, V; Shoham, Y; Baasov, T

2001-05-29

99

Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.  

Science.gov (United States)

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (??m) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition. PMID:24964211

Rossato, Franco A; Zecchin, Karina G; La Guardia, Paolo G; Ortega, Rose M; Alberici, Luciane C; Costa, Rute A P; Catharino, Rodrigo R; Graner, Edgard; Castilho, Roger F; Vercesi, Aníbal E

2014-01-01

100

The design and synthesis of potent and selective inhibitors of Trypanosoma brucei glycogen synthase kinase 3 for the treatment of human african trypanosomiasis.  

Science.gov (United States)

Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3? enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

Urich, Robert; Grimaldi, Raffaella; Luksch, Torsten; Frearson, Julie A; Brenk, Ruth; Wyatt, Paul G

2014-09-25

101

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

OpenAIRE

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...

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

2014-01-01

102

Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity.  

OpenAIRE

1. The induction of a calcium-independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S-substituted isothioureas, S-methylisothiourea (SMT), S-(2-aminoethyl)isothiourea (aminoethyl-TU), S-ethylisothiourea (ethyl-TU) and S-isopropylisothiourea (isopropyl-TU) potently inhibit iNOS activity in J774.2 macrophages ...

Southan, G. J.; Szabo?, C.; Thiemermann, C.

1995-01-01

103

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

Directory of Open Access Journals (Sweden)

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.

Jesus Olivero-Verbel

2012-07-01

104

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

Science.gov (United States)

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

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

2011-01-01

105

Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors (III).  

Science.gov (United States)

We have previously described a series of 4,5-dihydro-1H-pyrazole as moderately potent nNOS inhibitors. As a follow up of these studies, we report here the preparation and the preliminary evaluation of a series of 1-alkyl-3-benzoyl-4,5-dihydro-1H-pyrazole and 1-alkyl-3-benzoyl-1H-pyrazole as potential inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS). None of the reported compounds exhibited significant iNOS or nNOS inhibition, although the 1-benzyl-3-(2-amino-5-chlorobenzoyl)-1H-pyrazole-5-carboxylic acid ethyl ester derivative (10l), which shows an inhibition of 50% versus iNOS at a 1mM final concentration and no activity against nNOS, is potentially amenable of further optimization. The reasons for the inactivity of the reported series are discussed on the basis of docking studies. PMID:18325637

Carrión, M Dora; López Cara, Luisa C; Camacho, M Encarnación; Tapias, Víctor; Escames, Germaine; Acuña-Castroviejo, Darío; Espinosa, Antonio; Gallo, Miguel A; Entrena, Antonio

2008-11-01

106

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

Science.gov (United States)

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 active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach led to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by Surface Plasmon Resonance (SPR) experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo. PMID:25437621

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

2015-01-27

107

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

Science.gov (United States)

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

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

2003-01-01

108

Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor.  

Science.gov (United States)

R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types. PMID:20038615

Huitric, E; Verhasselt, P; Koul, A; Andries, K; Hoffner, S; Andersson, D I

2010-03-01

109

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

Science.gov (United States)

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

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

2012-12-21

110

Inhibition of stimulated ascorbic acid and luteinizing hormone-releasing hormone release by nitric oxide synthase or guanyl cyclase inhibitors.  

Science.gov (United States)

Ascorbic acid (AA), an antioxidant, is present in high concentrations in the hypothalamus. Previously, we have shown that AA inhibited stimulated release of luteinizing hormone-releasing hormone (LHRH) from medial basal hypothalami in vitro. We have also demonstrated that cell membrane depolarization by high [K(+)] media-induced AA release that is blocked by N(G)-mono-methyl-L-arginine, a competitive inhibitor of nitric oxide synthase (NOS), indicating that the release process is mediated by NO. The release of LHRH is also mediated by NO. We hypothesized that AA is a co-transmitter released with classical transmitters from synaptic vesicles that acts to reduce chemically the NO formed, thereby providing feed-forward inhibitory control over LHRH release. Because NO acts by activating guanylyl cyclase (GC) resulting in production of cGMP, in the present investigation we studied the effects of an NOS inhibitor LY 83583 and GC inhibitor, O.D.Q. to further characterize the role of NO in high [K(+)]-induced AA and LHRH release. Medial basal hypothalami were incubated in 0.5 ml of Krebs-Ringer Bicarbonate buffer or medium containing increased potassium [K(+) = 56 mM] for 1 hr or combinations of high [K(+)] + LY 83583 or O.D.Q. for 1 hr. AA and LHRH released into the incubation medium were measured by high-pressure liquid chromatography and radioimmunoassay, respectively. Cell membrane depolarization with high [K(+)] produced a significant increase in both AA and LHRH release. A combination of high [K(+)] + LY 83583 or high [K(+)] + O.D.Q. decreased basal AA and completely blocked high [K(+)]-induced AA and LHRH release. As in the case of high [K(+)], LHRH release induced by the excitatory amino acid N-methyl-D-aspartic acid (NMDA) was blocked by both the inhibitors. NMDA alone failed to alter AA release, but the combined presence of NMDA and the inhibitors totally blocked AA release. Because LY 83583 and O.D.Q. were shown to inhibit NOS and soluble GC, respectively, the data demonstrate that basal and high [K(+)]-induced AA and high [K(+)] and NMDA-stimulated LHRH release were mediated by NO by its activation of GC and consequent generation of cGMP. PMID:14709779

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

2004-01-01

111

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

International Nuclear Information System (INIS)

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 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

112

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

OpenAIRE

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

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

2013-01-01

113

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

Science.gov (United States)

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 effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

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

2014-10-15

114

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

OpenAIRE

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...

Li, Yi-ching; Chiang, Chia-wang; Yeh, Hui-chun; Hsu, Pei-yung; Whitby, Frank G.; Wang, Lee-ho; Chan, Nei-li

2008-01-01

115

N omega-amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance but increases mortality rates in awake canines challenged with endotoxin  

OpenAIRE

Inhibitors of nitric oxide synthase (NOS) have been reported to increase mean arterial pressure in animal models of sepsis and recently have been given to patients in septic shock. However, controlled studies to determine the effects of these agents on cardiovascular function and survival in awake animal models of sepsis have not been reported. To examine the therapeutic potential of NOS inhibition in septic shock, we challenged canines with endotoxin (2 or 4 mg/kg i.v.) and treated them with...

1992-01-01

116

Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase.  

OpenAIRE

Enhanced formation of nitric oxide (NO) by both the constitutive and the inducible isoforms of NO synthase (NOS) has been implicated in the pathophysiology of a variety of diseases, including circulatory shock. Non-isoform-selective inhibition of NO formation, however, may lead to side effects by inhibiting the constitutive isoform of NOS and, thus, the various physiological actions of NO. S-Methylisothiourea sulfate (SMT) is at least 10- to 30-fold more potent as an inhibitor of inducible NO...

Szabo?, C.; Southan, G. J.; Thiemermann, C.

1994-01-01

117

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.  

Science.gov (United States)

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

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

2014-04-15

118

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.  

Energy Technology Data Exchange (ETDEWEB)

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Angstroms resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal {beta}-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Korolev, S.; Ikeguchi, Y.; Skarina, T.; Beasley, S.; Arrowsmith, C.; Edwards, A.; Joachimiak, A.; Pegg, A. E.; Savchenko, A.; Pennsylvania State Univ. Coll. of Medicine; Milton S. Hershey Medical Center; Banting and Best Department of Medical Research; Univ. of Health Network

2002-01-01

119

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase  

Science.gov (United States)

Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

Snoeberger, Ning-Shiuan Nicole

120

Discovery of thienopyrimidine-based inhibitors of the human farnesyl pyrophosphate synthase--parallel synthesis of analogs via a trimethylsilyl ylidene intermediate.  

Science.gov (United States)

Thienopyrimidine-based bisphosphonates were identified as a new class of nitrogen-containing bisphosphonate (N-BP) inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Analogs were prepared via cyclization of 2-(1-(trimethylsilyl)ethylidene)malononitrile to 2-amino-4-(trimethylsilyl)thiophene-3-carbonitrile in the presence of elemental sulfur. Direct ipso-iododesilylation of this intermediate led to selective iodination at C? of the sulfur atom in high efficiency. The synthetic protocols developed were used in the parallel synthesis of structurally diverse thieno[2,3-d]pyrimidin-4-amine-based bisphosphonate inhibitors of hFPPS. PMID:23477945

Leung, Chun-Yuen; Langille, Adrienne M; Mancuso, John; Tsantrizos, Youla S

2013-04-15

121

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

Science.gov (United States)

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-?. 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

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

2013-01-01

122

Internalization and stability of a thymidylate synthase Peptide inhibitor in ovarian cancer cells.  

Science.gov (United States)

Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability. PMID:25353379

Cannazza, Giuseppe; Cazzato, Addolorata Stefania; Marraccini, Chiara; Pavesi, Giorgia; Pirondi, Silvia; Guerrini, Remo; Pelà, Michela; Frassineti, Chiara; Ferrari, Stefania; Marverti, Gaetano; Ponterini, Glauco; Costi, Maria Paola

2014-12-26

123

Design and syntheses of novel phthalazin-1(2H)-one derivatives as acetohydroxyacid synthase inhibitors.  

Science.gov (United States)

A series of 2-substituted-8-(4,6-dimethoxypyrimidin-2-yloxy)-4-methylphthalazin-1-one derivatives, 7a-7w, were designed via an ortho-substituent cyclization strategy to discover a new herbicidal lead structure. These compounds were synthesized by a seven-step route using 3-hydroxy-acetophenone as a starting material. Determination of the Ki values against wild-type A. thaliana acetohydroxyacid synthase (AHAS) (EC 4.1.3.18) indicated that some of the compounds displayed good enzyme inhibition activity comparable to that of KIH-6127. The further preliminary bioassay data on weeds showed that the synthesized compounds exhibited typical injury symptoms of AHAS-inhibiting herbicides, and some of them showed broad-spectrum and high herbicidal activities in postemergence treatments against Echinochloa crusgalli, Digitaria sanguinalis, Setaria viridis, Brassica juncea, Amaranthus retroflexus, and Chenopodium album at an application rate of 150 g ai/ha. To our knowledge, this is the first report of methylphthalazin-1-one derivatives as AHAS inhibitors. PMID:17117801

Li, Yuan-Xiang; Luo, Yan-Ping; Xi, Zhen; Niu, Congwei; He, Yan-Zhen; Yang, Guang-Fu

2006-11-29

124

Effects of nitric oxide synthase inhibitor ?-Nitro-L-Arginine Methyl Ester, on silica-induced inflammatory reaction and apoptosis  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Although nitric oxide is overproduced by macrophages and neutrophils after exposure to silica, its role in silica-induced inflammatory reaction and apoptosis needs further clarification. In this study, rats were intratracheally instilled with either silica suspension or saline to examine inflammatory reactions and intraperitoneally injected with ?-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthases, or saline to examine the possible role of nitric oxide production in the reaction. Results Results showed that silica instillation induced a strong inflammatory reaction indicated by increased total cell number, number of neutrophils, protein concentration and lactate dehydrogenase (LDH activity in bronchoalveolar lavage fluid (BALF. There were no significant differences in these indices between silica-instilled groups with and without L-NAME injection (p > 0.05 except LDH level. The results also showed that apoptotic leucocytes were identified in BALF cells of silica-instilled groups whereas no significant difference was found between silica-instilled groups with and without L-NAME injection in the apoptotic reaction (p > 0.05. Silica instillation significantly increased the level of BALF nitrite/nitrate and L-NAME injection reduced this increase. Conclusion Intratracheal instillation of silica caused an obvious inflammatory reaction and leucocyte apoptosis, but these reactions were not influenced by intraperitoneal injection of L-NAME and reduced production of NO. This supports the possibility that silica-induced lung inflammation and BALF cell apoptosis are via NO-independent mechanisms.

Leigh James

2006-11-01

125

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

Science.gov (United States)

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

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

2004-07-01

126

Protective effects of poly (ADP-ribose) synthase inhibitors on digoxin-induced cardiotoxicity in guinea-pig isolated hearts.  

Science.gov (United States)

Reactive oxygen species, generated and released during digoxin-induced cardiotoxicity, can produce an activation of poly (ADP-ribose) synthase (PARS). Our objective was to examine the effects of PARS inhibitors, 3-aminobenzamide (3-AB ) and nicotinamide, on digoxin-induced arrhythmias in guinea-pig isolated hearts. 3-AB (0.1-0.3 mM) and nicotinamide (0.3 mM) were added to the perfusion solution starting 10 min before digoxin infusion (8 microg x ml (-1)min (-1)reaching the heart) and maintained throughout the experiments. Electrocardiograms and coronary perfusion pressure were recorded continuously, and digoxin-induced arrhythmias were determined. Nicotinamide markedly inhibited ventricular tachycardia (VT) incidence (from 100%, n= 7, to 29%, n= 7), and abolished ventricular fibrillation (VF) incidence. 3-AB (0.1 mM, n= 9) significantly decreased VT incidence from 100% ( n= 7) to 22% ( n= 9) and VF incidence from 86% ( n= 7) to 11% ( n= 9). Both nicotinamide and 3-AB (0.1 mM) markedly decreased number of ventricular ectopic beats (VEBs) and arrhythmia score. 3-AB at 0.3 mM ( n= 8) appeared to decrease the VT (to 63%) and VF incidence (to 38%), but these reductions did not reach statistically significance levels. Moreover, 3-AB at high concentration (0.3 mM) did not significantly modify the number of VEBs and arrhythmia score. There were no significant changes in coronary perfusion pressure, heart rate or pressure rate index measured at certain time points throughout the experiment in all groups. Our results suggest that PARS activation plays a role in the digitalis-induced cardiotoxicity in guinea-pig isolated hearts. PMID:11884214

Demiryürek, A Tuncay; Yildiz, Gülüzar; E?iyok, Sibel; Altu?, Sedat

2002-03-01

127

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

OpenAIRE

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

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

1991-01-01

128

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.  

Science.gov (United States)

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

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

129

Reduction of carrageenin oedema and the associated c-Fos expression in the rat lumbar spinal cord by nitric oxide synthase inhibitor.  

OpenAIRE

1. Three hours after intraplantar carrageenin (6 mg/150 microliters of saline) Fos-like immunoreactivity (Fos-LI) was mainly observed in L4 and L5 segments of the dorsal horn. Both superficial (I-II) and deep laminae (V-VI) neurones were labelled. 2. We have studied the effect of systemic administration of a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on carrageenin evoked c-Fos expression and thus the contribution of nitric oxide to this expression. 3. Pre-admi...

Honore?, P.; Chapman, V.; Buritova, J.; Besson, J. M.

1995-01-01

130

Structure-Based Design and Synthesis of N?-Nitro-L-Arginine-Containing Peptidomimetics as Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural Water  

OpenAIRE

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N?-nitroguanidine (1) and 4-N-(N?-nitro-L-argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structura...

Seo, Jiwon; Igarashi, Jotato; Li, Huiying; Marta?sek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2007-01-01

131

Effects of estrous synchronization on response to nitric oxide donors, nitric oxide synthase inhibitors, and endothelin-1 in vitro.  

Science.gov (United States)

Two experiments were conducted to determine the effects of nitric oxide (NO) donors, endothelin-(ET-1), and NO synthase (NOS) inhibitors on bovine luteal function in vitro. In experiment 1, estrus in Brahman cows was synchronized with Synchro-Mate-B (SMB) and day-13-14 corpora luteal slices were weighed, diced and incubated in vitro. Treatments (100 ng/ml) were: vehicle, N[see symbol in text]-nitro-L-arginine-L-methyl ester (L-NAME), N(G)-monomethyl-L-arginine acetate (L-NMMA), diethylenetriamine (DETA), DETA-NONOate, sodium nitroprusside (SNP), or ET-1. In experiment 2, estrus was synchronized with Lutalyse, a Controlled Intravaginal Progesterone Releasing Device (CIDR), or cows were not synchronized. Corpora lutea were collected, weighed, and luteal slices were weighed, diced and incubated in vitro with treatments. Treatments (100ng/ml) were: vehicle, L- NAME, L-NMMA, DETA, DETA-NONOate, sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or endothelin-1. Tissues were incubated in M- 199 for 1 h without treatments and for 4 and 8 h in both experiments with treatments in both experiments. Media were analyzed for progesterone, prostaglandins E2 and F2alpha (PGE2, PGF2alpha) by radioimmunoassay (RIA). Hormone data in experiments 1 and 2 were analyzed by 2 x 7 and 3 x 2 x 8 factorial design for analysis of variance (ANOVA), respectively. Luteal weights in experiment 2 were analyzed by a one-way ANOVA. Concentrations of progesterone in media were similar (P > or = 0.05) among treatments within experiments. Concentrations of PGE2 in media in experiment 1 were undetectable in 90 and 57% of the samples at 4 and 8 h, respectively. PGF2alpha increased (P or = 0.05) among treatments. Secretion of PGF2alpha was not affected by treatments (P > or = 0.05). In experiment 2, luteal weights of the induced estrous cycle were decreased (P or = 0.05). No treatment increased (P > or = 0.05) PGF2alpha in any synchronization regimen. It is concluded that either SMB containing norgestomet or a CIDR containing progesterone alters luteal secretion of PGE2, Lutalyse lowers luteal weights in the induced estrous cycle, and NO or ET-1 given alone are not luteolytic agents. It is suggested that NO and ET-1 could have indirect antiluteolytic/luteotropic effects via increasing PGE2 secretion by luteal tissue rather than being luteolytic. PMID:15560115

Weems, Y S; Randel, R D; Tatman, S; Lewis, A W; Neuendorff, D A; Weems, C W

2004-10-01

132

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

International Nuclear Information System (INIS)

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 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.)

133

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

Energy Technology Data Exchange (ETDEWEB)

We recently showed an increase in tumour uptake of 2-[{sup 11}C]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 [{sup 3}H]thymidine after a 1-h pulse was determined at different times after drug treatment. In both cell lines, [{sup 3}H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [{sup 3}H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [{sup 3}H]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 [{sup 3}H]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-[{sup 11}C]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.)

Yau, Kawai; Price, Patricia; Pillai, Radhakrishma G.; Aboagye, Eric [Imperial College, Imaging Sciences, London (United Kingdom)

2006-09-15

134

From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3? Inhibitors that Suppress Proliferation and Survival of Pancreatic Cancer Cells  

OpenAIRE

Recent studies have demonstrated that Glycogen Synthase Kinase 3? (GSK-3?) is overexpressed in human colon and pancreatic carcinomas contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3? inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3? and an enhanced selectivity against Cyclin-dependent Kinase 2 (CDK-2). Selected GSK-3? inhibitor...

Gaisina, Irina N.; Gallier, Franck; Ougolkov, Andrei V.; Kim, Ki H.; Kurome, Toru; Guo, Songpo; Holzle, Denise; Luchini, Doris N.; Blond, Sylvie Y.; Billadeau, Daniel D.; Kozikowski, Alan P.

2009-01-01

135

Transcriptional and Translational Regulation of ?-Acetolactate Decarboxylase of Lactococcus lactis subsp. lactis  

OpenAIRE

The ?-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of ?-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or indepe...

Goupil-feuillerat, Nathalie; Corthier, Ge?rard; Godon, Jean-jacques; Ehrlich, S. Dusko; Renault, Pierre

2000-01-01

136

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

Science.gov (United States)

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

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

2014-10-01

137

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

Science.gov (United States)

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 ?-catenin that in turn blocked nuclear translocation of NF-?B. siRNA-mediated ?-catenin knockdown markedly increased the expression of NF-?B target genes, such as TNF-? and IL-6. Our data suggest that the GSK-3/?-catenin pathway may play a central role in cisplatin-mediated cytotoxicity in HEI-OC1 cells and hair cells of OCs in vitro. PMID:24560772

Kim, Se-Jin; Lim, Jae-Young; Lee, Joon No; Choe, Seong-Kyu; Kim, Yong-Il; Song, Seung Ryel; Cho, Meyoung; So, Hong-Seob; Park, Raekil

2014-06-01

138

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

Science.gov (United States)

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

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

2014-08-01

139

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

OpenAIRE

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

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

2010-01-01

140

Glycogen synthase kinase 3 beta inhibitors induce apoptosis in ovarian cancer cells and inhibit in vivo tumor growth  

OpenAIRE

Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3? (GSK3?), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3? inhi...

Hilliard, Tyvette S.; Gaisina, Irina N.; Muehlbauer, Amanda G.; Gaisin, Arsen M.; Gallier, Franck; Burdette, Joanna E.

2011-01-01

141

Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful?12  

OpenAIRE

The glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase widely expressed in mammalian tissues. Initially identified by its ability to modulate glycogen synthesis, GSK-3 turned out to be a multifunctional enzyme, able to phosphorylate many proteins, including members of the steroid receptor superfamily. Although GSK-3 was shown to phosphorylate the androgen receptor (AR), its effects on AR transcriptional activity remain controversial. Analysis of short hairpin RNA (shRNA)-mediated...

Rinnab, Ludwig; Schu?tz, Stefanie V.; Diesch, Jeannine; Schmid, Evi; Ku?fer, Rainer; Hautmann, Richard E.; Spindler, Klaus-dieter; Cronauer, Marcus V.

2008-01-01

142

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

OpenAIRE

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 3? (GSK-3?) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumul...

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

2011-01-01

143

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

International Nuclear Information System (INIS)

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

144

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor.  

Science.gov (United States)

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E(2) (PGE(2)) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE(2) synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC(50)=16.5+/-3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE(2) synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE(2) synthesis (IC(50) in the range of 0.5-5 microM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF(1alpha) (PGF(1alpha)) and PGF(2alpha). In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE(2), PGF(1alpha) and PGF(2alpha) synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention. PMID:20067770

Mbalaviele, Gabriel; Pauley, Adele M; Shaffer, Alexander F; Zweifel, Ben S; Mathialagan, Sumathy; Mnich, Stephen J; Nemirovskiy, Olga V; Carter, Jeff; Gierse, James K; Wang, Jane L; Vazquez, Michael L; Moore, William M; Masferrer, Jaime L

2010-05-15

145

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

OpenAIRE

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

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

1996-01-01

146

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

2012-05-29

147

Design, synthesis and antibacterial activities of vanillic acylhydrazone derivatives as potential ?-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors.  

Science.gov (United States)

Fatty acid biosynthesis is essential for bacterial survival. FabH, ?-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of acylhydrazone derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong broad-spectrum antibacterial activity. Compounds with potent antibacterial activities were tested for their Escherichia coli FabH inhibitory activity. Especially, compound E9 showed the most potent antibacterial activity with MIC values of 0.39-1.56 ?g/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC(50) of 2.5 ?M. Docking simulation was performed to position compound E9 into the E. coli FabH active site to determine the probable binding conformation. PMID:23124163

Wang, Xiao-Liang; Zhang, Yan-Bin; Tang, Jian-Feng; Yang, Yu-Shun; Chen, Ruo-Qi; Zhang, Fei; Zhu, Hai-Liang

2012-11-01

148

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

Directory of Open Access Journals (Sweden)

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.

Weiss Ingrid M

2007-11-01

149

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

Science.gov (United States)

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling ?-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. Copyright © 2014 John Wiley & Sons, Ltd. PMID:24965182

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

2015-01-01

150

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

Energy Technology Data Exchange (ETDEWEB)

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

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

151

1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols: a new class of potent and selective aldosterone synthase inhibitors.  

Science.gov (United States)

1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols and related compounds were synthesized and evaluated for inhibition of aldosterone synthase (CYP11B2), a potential target for cardiovascular diseases associated with elevated plasma aldosterone levels like congestive heart failure and myocardial fibrosis. Introduction of substituents at the phenylsulfinyl moiety and changes of the substitution pattern at the naphthalene core were examined. Potent compounds were further examined for selectivity versus other important steroidogenic CYP enzymes, i.e. the highly homologous 11?-hydroxylase (CYP11B1), CYP17 and CYP19. The most potent compound (IC50 = 14 nM) discovered was the meta-trifluoromethoxy derivative 11, which also exhibited excellent selectivity toward CYP11B1 (SF = 415), and showed no inhibition of CYP17 and CYP19. PMID:25462268

Grombein, Cornelia M; Hu, Qingzhong; Heim, Ralf; Rau, Sabrina; Zimmer, Christina; Hartmann, Rolf W

2015-01-01

152

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

Directory of Open Access Journals (Sweden)

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 

Henry JL

2011-03-01

153

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

OpenAIRE

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

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

2005-01-01

154

Synthesis and SAR study of imidazoquinolines as a novel structural class of microsomal prostaglandin E? synthase-1 inhibitors.  

Science.gov (United States)

The imidazoquinoline derivative 1 was found as a novel mPGES-1 inhibitor. Optimization of 1 led to the identification of the 2-chlorophenyl group at the C(2)-position and the quinolone structure at the C(4)-position. Compound 33, the most potent synthesized compound, showed excellent mPGES-1 inhibition (IC(50)=9.1nM) with high selectivity (>1000-fold) over both COX-1 and COX-2. PMID:22137787

Shiro, Tomoya; Takahashi, Hirotada; Kakiguchi, Keisuke; Inoue, Yoshifumi; Masuda, Keiki; Nagata, Hidetaka; Tobe, Masanori

2012-01-01

155

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

International Nuclear Information System (INIS)

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

156

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

Energy Technology Data Exchange (ETDEWEB)

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

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

157

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan  

OpenAIRE

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 ?, and with its bound feedback inhibitor, tryptophan, at 2.4 ?. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric or...

Spraggon, Glen; Kim, Choel; Nguyen-huu, Xuong; Yee, Muh-ching; Yanofsky, Charles; Mills, Stanley E.

2001-01-01

158

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

International Nuclear Information System (INIS)

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

159

Protection from inorganic mercury effects on the in vivo dopamine release by ionotropic glutamate receptor antagonists and nitric oxide synthase inhibitors.  

Science.gov (United States)

The possible role of ionotropics glutamate receptors on the HgCl(2)-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective NMDA and AMPA/Kainate receptors antagonists dizocilpine (MK-801), D (-)-2-amino-5-phoshonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moreover, we have also studied the effects of nitric oxide synthase (NOS) inhibitors L-nitro-arginine methyl ester (L-NAME) and 7-nitro-indazol (7-NI) on HgCl(2)-induced DA release. Intraestriatal infusion of 1mM HgCl(2) increased striatal DA to 1717.2+/-375.4% respect to basal levels. Infusion of 1mM HgCl(2) in 400 microM MK-801 pre-treated animals produced an increase on striatal DA levels 61% smaller than that induced in non-pre-treated animals. In the case of AP5, this treatment reduced 92% the increase produced by HgCl(2) as compared to non-pre-treated rats. Nevertheless, the administration of CNQX did not produce any effect on HgCl(2)-induced dopamine release. Intrastriatal infusion of 1mM HgCl(2) in 100 microM L-NAME pre-treated animals produced an increase on extracellular DA levels 82% smaller than produced by HgCl(2) alone. In addition, the pre-treatment with 7-NI reduced 90% the increase produced by infusion of HgCl(2) alone in rats. Thus, HgCl(2)-induced DA release could be produced at last in part, by overstimulation of NMDA receptors with NO production, since administration of NMDA receptor antagonists and NOS inhibitors protected against HgCl(2) effects on DA release. PMID:17624650

Vidal, Lucía; Durán, Rafael; Faro, Lilian F; Campos, Francisco; Cervantes, Rosa C; Alfonso, Miguel

2007-09-01

160

Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3? inhibitors through ligand and structure based drug design.  

Science.gov (United States)

Glycogen synthase kinase-3? elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3? over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3? inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3? and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3?. GSK-3? and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3?. The molecules showed IC50 values ranging from 0.825?M to 1.116?M and were 23- to 57-fold selective for GSK-3?. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825?M. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and ?-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule. PMID:25064440

Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

2014-09-01

161

Interaction of nitric oxide synthase inhibitors and their D-enantiomers with rat neutrophil luminol dependent chemiluminescence response.  

OpenAIRE

1. Formyl-methionyl-leucyl-phenylalanine (FMLP) or arachidonic acid (AA) induced luminol dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was found to be inhibited by nitric oxide synthease inhibitors and their D-enantiomers. 2. Rat PMNLs LCL response was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), D-NAME, NG-monomethyl-L-arginine (L-NMMA) or D-NMMA, in a concentration- and time-dependent manner. 3. It was observed that both L- and D-enantiome...

Dikshit, M.; Chari, S. S.; Seth, P.; Kumari, R.

1996-01-01

162

Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.  

OpenAIRE

1. The synthesis of nitric oxide (NO) from L-arginine by rat peritoneal neutrophils (PMN) and the murine macrophage cell-line J774 and the inhibition of this synthesis by N-iminoethyl-L-ornithine (L-NIO), NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) were investigated. 2. L-NIO was the most potent inhibitor in both types of cells while L-NMMA was less active. L-NNA and L-NAME had no significant effect in PMN and L-NNA produced only approximately ...

Mccall, T. B.; Feelisch, M.; Palmer, R. M.; Moncada, S.

1991-01-01

163

A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.  

Science.gov (United States)

QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1chi(v) (Kier's first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction. PMID:24791414

Bharti, Vishwa Deepak; Gupta, Satya P; Kumar, Harish

2014-02-01

164

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

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

Cho Yu

2010-08-01

165

Heteroatom insertion into 3,4-dihydro-1H-quinolin-2-ones leads to potent and selective inhibitors of human and rat aldosterone synthase.  

Science.gov (United States)

Aldosterone synthase (CYP11B2) catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone. CYP11B2 is regarded as a new target for several cardiovascular diseases which are associated with chronically elevated aldosterone levels such as hypertension, congestive heart failure and myocardial fibrosis. In this paper, we optimized heterocycle substituted 3,4-dihydropyridin-2(1H)-ones as CYP11B inhibitors by systematic introduction of heteroatoms and by bioisosteric exchange of the lactame moiety by a sultame moiety. The most promising compounds regarding inhibition of human CYP11B2 and selectivity versus human enzymes CYP11B1, CYP17, and CYP19 were tested for inhibition of rat CYP11B2. Thus, we discovered compounds 4 and 9 which show potent inhibition of hCYP11B2 (IC50 < 1 nM) and the corresponding rat enzyme (4: 64%, 9: 51% inhibition, at 2 ?M). PMID:25528333

Grombein, Cornelia M; Hu, Qingzhong; Rau, Sabrina; Zimmer, Christina; Hartmann, Rolf W

2015-01-27

166

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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

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

167

Trehalose-6-phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening.  

Science.gov (United States)

Trehalose phosphate synthase (EC 2.4.1.15; TPS) is the crucial enzyme for the biosynthesis of trehalose, the main haemolymph sugar of insects, and therefore a potential insecticidal molecular target. In this study, we report the functional heterologous expression of Drosophila melanogaster TPS, the gene identification, full length cDNA cloning and functional expression of cat flea (Ctenocephalides felis) TPS, and the Michaelis-Menten constants for their specific substrates glucose-6-phosphate and uridinediphosphate-glucose. A novel high throughput screening-compatible TPS assay and its use for the identification of the first potent insect TPS inhibitors from a large synthetic compound collection (>115 000 compounds) is described. One compound class that emerged in this screening, the 4-substituted 2,6-diamino-3,5-dicyano-4H-thiopyrans, was further investigated by analysing preliminary structure-activity relationships. Here, compounds were identified that show low µM to high nM half maximal inhibitory concentrations on insect TPS and that may serve as lead compounds for the development of insecticides with a novel mode of action. PMID:22762304

Kern, C; Wolf, C; Bender, F; Berger, M; Noack, S; Schmalz, S; Ilg, T

2012-08-01

168

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

169

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

Scientific Electronic Library Online (English)

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

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

1047-10-01

170

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

International Nuclear Information System (INIS)

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

171

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

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

Jianrong Gao

2013-05-01

172

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

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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.

Suborov Evgeny V

2012-06-01

173

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

Science.gov (United States)

Echinocandins inhibit the synthesis of ?-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 invasive candidiasis against C. albicans, C. glabrata, and C. parapsilosis. The SCY-078 MICs varied 8-fold. Oral doses of 3.125 to 200 mg/kg SCY-078 salt in sterile water produced peak levels of 0.04 to 2.66 ?g/ml, elimination half-lives of 5.8 to 8.5 h, areas under the concentration-time curve from 0 to 24 h (AUC0-24 h) of 0.61 to 41.10 ?g · h/ml, and AUC from 0 to infinity (AUC0-?) values of 0.68 to 40.31 ?g · h/ml. The pharmacokinetics (PK) were approximately linear over the dose range studied. Maximum response (Emax) and PK/PD target identification studies were performed with 4 C. albicans, 4 C. glabrata, and 3 C. parapsilosis isolates. The PD index AUC/MIC was explored by using total (tAUC) and free (fAUC) drug concentrations. The maximum responses were 4.0, 4.0, and 4.3 log10 CFU/kidney reductions for C. albicans, C. glabrata, and C. parapsilosis, respectively. The AUC/MIC was a robust predictor of efficacy (R(2), 0.53 to 0.91). The 24-h PD targets were a static dose of 63.5 mg/kg, a tAUC/MIC of 500, and an fAUC/MIC of 1.0 for C. albicans; a static dose of 58.4 mg/kg, a tAUC/MIC of 315, and an fAUC/MIC of 0.63 for C. glabrata; and a static dose of 84.4 mg/kg, a tAUC/MIC of 198, and an fAUC/MIC of 0.40 for C. parapsilosis. The mean fAUC/MIC values associated with a 1-log kill endpoint against these species were 1.42, 1.26, and 0.91 for C. albicans, C. glabrata, and C. parapsilosis, respectively. The static and 1-log kill endpoints were measured relative to the burden at the start of therapy. The static and 1-log kill doses, as well as the total and free drug AUC/MIC PD targets, were not statistically different between species but were numerically lower than those observed for echinocandins. SCY-078 is a promising novel oral glucan synthase inhibitor against Candida species, and further investigation is warranted. PMID:25512406

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

2015-02-01

174

Development and Use of a Screening Procedure for Production of (alpha)-Acetolactate by Lactococcus lactis subsp. lactis biovar diacetylactis Strains  

OpenAIRE

A method was developed to screen and isolate mutagenized Lactococcus lactis subsp. lactis biovar diacetylactis strains accumulating (alpha)-acetolactate. This compound is accumulated by (alpha)-acetolactate decarboxylase-deficient strains and undergoes spontaneous degradation into diacetyl on agar plates. The diacetyl produced is detected by a colorimetric reaction yielding a red halo around the colonies.

Monnet, C.; Schmitt, P.; Divies, C.

1997-01-01

175

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

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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.

Morteza Piri

2011-01-01

176

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

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Full Text Available

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

L. A. RODRIGUES

2009-01-01

177

Spermine synthase  

OpenAIRE

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

Pegg, Anthony E.; Michael, Anthony J.

2009-01-01

178

Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp. lactis.  

Science.gov (United States)

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures. PMID:10986242

Goupil-Feuillerat, N; Corthier, G; Godon, J J; Ehrlich, S D; Renault, P

2000-10-01

179

Transcriptional and Translational Regulation of ?-Acetolactate Decarboxylase of Lactococcus lactis subsp. lactis  

Science.gov (United States)

The ?-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of ?-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures. PMID:10986242

Goupil-Feuillerat, Nathalie; Corthier, Gérard; Godon, Jean-Jacques; Ehrlich, S. Dusko; Renault, Pierre

2000-01-01

180

Design and Synthesis of 2-Amino-4-methylpyridine Analogues as Inhibitors for Inducible Nitric Oxide Synthase and in vivo Evaluation of [18F]6-(2-Fluoropropyl)-4-methyl-pyridin-2-amine as a Potential PET Tracer for Inducible Nitric Oxide Synthase  

OpenAIRE

A series of position-6 substituted 2-amino-4-methylpyridine analogues was synthesized and compounds 9, 18, and 20 were identified as the inhibitors with the greatest potential to serve as PET tracers for imaging inducible nitric oxide synthase (iNOS). [18F]9 was synthesized and evaluated in a mouse model of lipopolysaccharide (LPS)-induced iNOS activation. In vivo biodistribution studies of [18F]9 indicate higher tracer uptake in the lungs of the LPS-treated mice when compared to control mic...

Zhou, Dong; Lee, Hsiaoju; Rothfuss, Justin M.; Chen, Delphine L.; Ponde, Datta E.; Welch, Michael J.; Mach, Robert H.

2009-01-01

181

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity  

International Nuclear Information System (INIS)

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N G-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 ± 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 ± 5%, while, SNAP or DETA-NONO increased viability to 66 ± 8 or 71 ± 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAMxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress

182

Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS).  

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UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme. PMID:10413459

Krekel, F; Oecking, C; Amrhein, N; Macheroux, P

1999-07-13

183

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

International Nuclear Information System (INIS)

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

184

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

Science.gov (United States)

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

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

2015-01-27

185

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

OpenAIRE

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

Ferrero, Aldo; Vidotto, Francesco; Busi, Roberto

2006-01-01

186

Substituted 3-imidazo[1,2-a]pyridin-3-yl- 4-(1,2,3,4-tetrahydro-[1,4]diazepino-[6,7,1-hi]indol-7-yl)pyrrole-2,5-diones as highly selective and potent inhibitors of glycogen synthase kinase-3.  

Science.gov (United States)

Glycogen synthase kinase-3 (GSK3) is involved in signaling from the insulin receptor. Inhibitors of GSK3 are expected to effect lowering of plasma glucose similar to insulin, making GSK3 an attractive target for the treatment of type 2 diabetes. Herein we report the discovery of a series of potent and selective GSK3 inhibitors. Compounds 7-12 show oral activity in an in vivo model of type II diabetes, and 9 and 12 have desirable PK properties. PMID:15267232

Engler, Thomas A; Henry, James R; Malhotra, Sushant; Cunningham, Brian; Furness, Kelly; Brozinick, Joseph; Burkholder, Timothy P; Clay, Michael P; Clayton, Joshua; Diefenbacher, Clive; Hawkins, Eric; Iversen, Philip W; Li, Yihong; Lindstrom, Terry D; Marquart, Angela L; McLean, Johnathan; Mendel, David; Misener, Elizabeth; Briere, Daniel; O'Toole, John C; Porter, Warren J; Queener, Steven; Reel, Jon K; Owens, Rebecca A; Brier, Richard A; Eessalu, Thomas E; Wagner, Jill R; Campbell, Robert M; Vaughn, Renee

2004-07-29

187

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

Science.gov (United States)

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

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

188

Structure Determination of Glycogen Synthase Kinase-3 from Leishmania major and Comparative Inhibitor Structure-Activity Relationships with Trypanosoma brucei GSK-3  

OpenAIRE

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 i...

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

2010-01-01

189

Neural Precursor Cells Are Protected from Apoptosis Induced by Trophic Factor Withdrawal or Genotoxic Stress by Inhibitors of Glycogen Synthase Kinase 3*,S  

OpenAIRE

Mechanisms controlling the survival of neural precursor cells (NPCs) are critical during brain development, in adults for neuron replenishment, and after transplantation for neuron replacement. This investigation found that glycogen synthase kinase 3 (GSK3) promotes apoptotic signaling in cultured NPCs derived from embryonic mouse brain subjected to two common apoptotic conditions, trophic factor withdrawal and genotoxic stress. Trophic factor withdrawal activated GSK3 and the key apoptosis m...

Eom, Tae-yeon; Roth, Kevin A.; Jope, Richard S.

2007-01-01

190

Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase  

OpenAIRE

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, ?-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase) was evaluated in a histamine H3 receptor agonist-induced ((R)?-methylhistamine, (R)?-MeHA) endothelium-dependent rat aorta relaxation assay. (R)?-MeHA (0.1 nM – 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 cau...

Djuric, D. M.; Nesic, M. T.; Andjelkovic, I. Z.

1996-01-01

191

Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors  

OpenAIRE

Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic sta...

Ji, Haitao; Stanton, Benjamin Z.; Igarashi, Jotaro; Li, Huiying; Marta?sek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2008-01-01

192

Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme’s active site closure  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Human farnesyl pyrophosphate synthase (FPPS controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme’s C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP. This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme’s active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation. Results We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi, inorganic pyrophosphate (PPi, and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade. Conclusions In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the “off” position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

Park Jaeok

2012-12-01

193

Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.  

Science.gov (United States)

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3-30 ?M), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3?-acetoxy-8,24-dienetirucallic acid (6) and 3?-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 ?M, each. Structure-activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 ?M). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

Verhoff, Moritz; Seitz, Stefanie; Paul, Michael; Noha, Stefan M; Jauch, Johann; Schuster, Daniela; Werz, Oliver

2014-06-27

194

Tetra- and Pentacyclic Triterpene Acids from the Ancient Anti-inflammatory Remedy Frankincense as Inhibitors of Microsomal Prostaglandin E2 Synthase-1  

Science.gov (United States)

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3–30 ?M), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3?-acetoxy-8,24-dienetirucallic acid (6) and 3?-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 ?M, each. Structure–activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 ?M). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

2014-01-01

195

The molecular pharmacology and in vivo activity of 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid (YS121), a dual inhibitor of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase.  

Science.gov (United States)

The microsomal prostaglandin E(2) synthase (mPGES)-1 is one of the terminal isoenzymes of prostaglandin (PG) E(2) biosynthesis. Pharmacological inhibitors of mPGES-1 are proposed as an alternative to nonsteroidal anti-inflammatory drugs. We recently presented the design and synthesis of a series of pirinixic acid derivatives that dually inhibit mPGES-1 and 5-lipoxygenase. Here, we investigated the mechanism of mPGES-1 inhibition, the selectivity profile, and the in vivo activity of alpha-(n-hexyl)-substituted pirinixic acid [YS121; 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid)] as a lead compound. In cell-free assays, YS121 inhibited human mPGES-1 in a reversible and noncompetitive manner (IC(50) = 3.4 muM), and surface plasmon resonance spectroscopy studies using purified in vitro-translated human mPGES-1 indicate direct, reversible, and specific binding to mPGES-1 (K(D) = 10-14 muM). In lipopolysaccharide-stimulated human whole blood, PGE(2) formation was concentration dependently inhibited (IC(50) = 2 muM), whereas concomitant generation of the cyclooxygenase (COX)-2-derived thromboxane B(2) and 6-keto PGF(1alpha) and the COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was not significantly reduced. In carrageenan-induced rat pleurisy, YS121 (1.5 mg/kg i.p.) blocked exudate formation and leukocyte infiltration accompanied by reduced pleural levels of PGE(2) and leukotriene B(4) but also of 6-keto PGF(1alpha). Taken together, these results indicate that YS121 is a promising inhibitor of mPGES-1 with anti-inflammatory efficiency in human whole blood as well as in vivo. PMID:19934399

Koeberle, Andreas; Rossi, Antonietta; Zettl, Heiko; Pergola, Carlo; Dehm, Friederike; Bauer, Julia; Greiner, Christine; Reckel, Sina; Hoernig, Christina; Northoff, Hinnak; Bernhard, Frank; Dötsch, Volker; Sautebin, Lidia; Schubert-Zsilavecz, Manfred; Werz, Oliver

2010-03-01

196

Discovery of bacterial fatty acid synthase inhibitors from a Phoma species as antimicrobial agents using a new antisense-based strategy.  

Science.gov (United States)

Fatty acids are essential for bacterial growth and viability, with the type II fatty acid synthesis (FAS II) pathway being a potential antibacterial target. A new, selective, and highly sensitive whole cell-based antisense strategy has been designed to screen for natural product inhibitors of FabH/F of the FAS II pathway using a high-throughput two-plate agar-based differential sensitivity assay (FabF(2)p). An antisense assay along with the FASII enzyme prepared from Staphylococcus aureus was used for bioactivity-guided fractionation, leading to the isolation of phomallenic acids A-C (1-3) from a leaf litter fungus identified as Phoma sp. Compounds 1-3 exhibited minimum detection concentrations (MDC) of 0.63, 0.31, and 0.15 microg/mL in the FabF(2P) assay, IC(50) values of 22, 3.4, and 0.77 microg/mL in the FASII enzyme assay, and minimum inhibitory concentrations (MIC) of 250, 7.8, and 3.9 microg/mL, respectively, against wild-type S. aureus. Phomallenic acid C (3), the analogue with the longest chain, exhibited the best overall activity within the phomallenic acids obtained and was superior to cerulenin and thiolactomycin, the two most studied and commonly used FabF inhibitors. PMID:16562839

Ondeyka, John G; Zink, Deborah L; Young, Katherine; Painter, Ronald; Kodali, Srinivas; Galgoci, Andrew; Collado, Javier; Tormo, Jose Ruben; Basilio, Angela; Vicente, Francisca; Wang, Jun; Singh, Sheo B

2006-03-01

197

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.  

Science.gov (United States)

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45?M. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300?g/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51?M, mammalian ATPase IC50>100?M, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12?g/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100?g/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5?g/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173?mol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains. PMID:25614114

Singh, Supriya; Roy, Kuldeep K; Khan, Shaheb R; Kashyap, Vivek Kr; Sharma, Abhisheak; Jaiswal, Swati; Sharma, Sandeep K; Krishnan, Manju Yasoda; Chaturvedi, Vineeta; Lal, Jawahar; Sinha, Sudhir; Gupta, Arnab D; Srivastava, Ranjana; Saxena, Anil K

2015-02-15

198

Recent advances in the study of rTS proteins. rTS expression during growth and in response to thymidylate synthase inhibitors in human tumor cells.  

Science.gov (United States)

The rTS proteins have now been shown to be expressed in a variety of cell lines, with expression of rTS beta being found elevated in three cell lines which are resistant to TS inhibitors (3, 4) (Figure 1). In one of these cell lines (K562 B1A), the cells were selected for resistance to MTX, which has a primary site of action on DHFR, but was found to be cross-resistant to FUdR (4). The other two cell lines were selected for resistance to either 5-fluorouracil (H630-1) or a combination of ZD1694 and FU. In each case, elevation of rTS beta appears to be a selected response to thymidylate stress. In HCT-8 and HCT-8/DF2 cells, treatment of cells for a short period of time (2 hr) resulted in the elevation of rTS beta levels, again suggestive that expression of rTS beta is a response to thymidylate stress. rTS beta appears to be regulated with cell growth, its levels increasing at mid-log and at late-log/saturation phase in H630 and H630-1 cells (Fig. 2), and increasing with late-log in several other cell lines as well (Fig. 3). The increase in rTS beta is suggestive of a cellular function associated with a state where growth is no longer desirable, reminiscent of the starvation-sensing protein homolog RSPA in E. coli (22). While this relationship would not explain the spike in rTS beta levels in mid-log H630 and H630-1 cells, it does make sense if the rTS proteins (particularly rTS beta) are involved in down-regulating thymidylate biosynthesis. The potential mechanism of this down-regulation may be speculated to be the catabolism of some precursor for thymidylate biosynthesis or some direct effect upon TS through modulation by some other ligand, either a metabolite or another protein. Studies on the expression of rTS proteins in clinical specimens indicate that rTS beta is expressed at high levels in kidney and kidney tumor (Dolnick, unpublished results). Given the physiologic role of the kidney, high level expression of rTS in this organ is consistent with a role in a catabolic pathway. Since down-regulation of TS activity is expected to increase sensitivity to TS inhibitors, a role for rTS beta in directly down-regulating TS activity in the biochemical sense would seem unlikely. However, the manner of biochemical TS down-regulation may make a difference. In the TS- Cl/Cl cell line, there are two mutations in TS which likely reduce affinity for N-5,10-methylene tetrahydrofolates (23). This cell line is highly resistant to MTX, yet is still tumorigenic in vivo (24), and supplying the cells with high levels of exogenous folate can restore TS function (23). Thus in TS- Cl/Cl cells, the TS phenotype is conditionally dependent upon the presence of high levels of exogenous folate. This suggests that a role of rTS proteins as conditional down-regulators of TS, perhaps through modulating folate binding, may be possible. Two cell lines (K562 B1A and H630-1) that overproduce rTS beta have altered sensitivity to TS inhibitors that differ depending upon the nature of the inhibitor. The K562 B1A cell line was found to be approximately 2000-fold resistant to ZD1694 and BW1843U89 (120 hr exposures), but only three-fold resistant to AG331. The H630-1 cell line is approximately 30-fold resistant to BW1843U89 (120 hr exposure) and 40-fold resistant to ZD1694 (120 hr exposure), but only eight-fold resistant to AG331. Since K562 B1A cells overproduce rTS beta (2), but have no significant alterations in FPGS activity, the possibility that rTS may affect folate binding remains a hypothesis worth examining. The recent discovery that TS is a phosphoprotein and that it is nuclear as well as cytoplasmic (21) raises the possibility that the phosphorylation state of TS may regulate one of its cellular functions, and that the subcellular localization of this enzyme is regulated as well. Since rTS proteins have HSP with proteins that participate in kinase/phosphatase reactions, this also seems to be an avenue worthy of future investigation. (ABSTRACT TRUNCATED PMID:9381988

Dolnick, B J; Lu, K; Yin, M B; Rustum, Y M

1997-01-01

199

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  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese 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, su [...] gerindo 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 (p Abstract in english In 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 i [...] nvolved 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

Claudio Marcio Yudi, Ikino; Roseli Saraiva Moreira, Bittar; Karina Midori, Sato; Newton Macuco, Capella.

2006-04-01

200

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

Directory of Open Access Journals (Sweden)

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.

Claudio Marcio Yudi Ikino

2006-04-01

201

Nitric Oxide Synthase Inhibitors as Antidepressants  

DEFF Research Database (Denmark)

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.

Wegener, Gregers; Volke, Vallo

2010-01-01

202

Nitric Oxide Synthase Inhibitors as Antidepressants  

Directory of Open Access Journals (Sweden)

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.

Vallo Volke

2010-01-01

203

Applying Molecular Dynamics Simulations to Identify Rarely Sampled Ligand-bound Conformational States of Undecaprenyl Pyrophosphate Synthase, an Antibacterial Target  

OpenAIRE

Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ra...

Sinko, William; Oliveira, Ce?sar; Williams, Sarah; Wynsberghe, Adam; Durrant, Jacob D.; Cao, Rong; Oldfield, Eric; Mccammon, J. Andrew

2011-01-01

204

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

OpenAIRE

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

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

1988-01-01

205

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  

OpenAIRE

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 Pa...

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

2006-01-01

206

Higher plant cellulose synthases  

OpenAIRE

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

Richmond, Todd

2000-01-01

207

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

OpenAIRE

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

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

2008-01-01

208

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  

Energy Technology Data Exchange (ETDEWEB)

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)

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

209

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

Scientific Electronic Library Online (English)

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

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

1710-17-01

210

GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS  

OpenAIRE

Inhibiting glycogen synthase kinase-3 (GSK-3) activity via pharmacological intervention has become an important strategy for treating neurodegenerative and psychiatric disorders. The known GSK-3 inhibitors are of diverse chemotypes and mechanisms of action and include compounds isolated from natural sources, cations, synthetic small-molecule ATP-competitive inhibitors, non-ATP-competitive inhibitors, and substrate–competitive inhibitors. Here we describe the variety of GSK-3 inhibitors with...

HagitEldar-Finkelman; AnaMartinez

2011-01-01

211

Geranyl diphosphate synthase from mint  

Energy Technology Data Exchange (ETDEWEB)

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.

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

1999-01-01

212

Geranyl diphosphate synthase from mint  

Energy Technology Data Exchange (ETDEWEB)

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.

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

1999-03-02

213

Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase  

Energy Technology Data Exchange (ETDEWEB)

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg{sup 2+} and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 {angstrom}, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

Wang, Jian-Guo; Lee, Patrick K.-M.; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G.; Li, Zheng-Ming; Guddat, Luke W.; (Queensland); (Nankai); (IHEP-Beijing)

2009-08-17

214

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells 1  

OpenAIRE

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation ...

Zeng, Fu-yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

2009-01-01

215

Imidazole glycerol phosphate synthase: Structural and kinetic studies of a triad glutamine amidotransferase  

OpenAIRE

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase (GAT) that incorporates ammonia derived from glutamine into the unusual nucleotide, PRFAR to form AICAR and IGP. A common feature of all GATs is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisiae IGP synthase shows a 4,900-fold stimulation of glutaminase in the presence of PRFAR. Competitive inhibitors in t...

Myers, Rebecca S.

2005-01-01

216

Slow Onset Inhibition of Bacterial ?-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin*  

OpenAIRE

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the ?-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 i...

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

2010-01-01

217

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

OpenAIRE

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 (dcSA...

S?ec?kute?, Jolita; Mccloskey, Diane E.; Thomas, H. Jeanette; Secrist, John A.; Pegg, Anthony E.; Ealick, Steven E.

2011-01-01

218

Adenosine preconditioning attenuates hepatic reperfusion injury in the rat by preventing the down-regulation of endothelial nitric oxide synthase  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Previous work has suggested that in the liver, adenosine preconditioning is mediated by nitric oxide. Whether the endothelial isoform of nitric oxide synthase plays a part in this mechanism has however not yet been investigated. Methods Wistar rats were used (6 in each group – Groups: (1 sham, (2 ischemia-reperfusion, (3 adenosine + ischemia-reperfusion, (4 endothelial isoform inhibitor + adenosine + ischemia-reperfusion. Results Using immunohistochemistry, this study has revealed a decrease in the expression of endothelial nitric oxide synthase following hepatic ischemia-reperfusion. This was prevented by adenosine pre-treatment. When an inhibitor of endothelial nitric oxide synthase was administered prior to adenosine pre-treatment, pre-conditioning did not occur despite normal expression of endothelial nitric oxide synthase. Conclusions These findings suggest that adenosine attenuates hepatic injury by preventing the downregulation of endothelial nitric oxide synthase that occurs during ischemia-reperfusion.

Williamson Robin CN

2002-09-01

219

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

Science.gov (United States)

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

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

1989-01-23

220

ATP synthase in mycobacteria: special features and implications for a function as drug target.  

Science.gov (United States)

ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. PMID:24513197

Lu, Ping; Lill, Holger; Bald, Dirk

2014-07-01

221

Purification and characterization of dihydrodipicolinate synthase from pea.  

Science.gov (United States)

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-beta-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-beta-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-alpha-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-alpha-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-beta-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-beta-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase. PMID:16668753

Dereppe, C; Bold, G; Ghisalba, O; Ebert, E; Schär, H P

1992-03-01

222

Applying Molecular Dynamics Simulations to Identify Rarely Sampled Ligand-bound Conformational States of Undecaprenyl Pyrophosphate Synthase, an Antibacterial Target  

Energy Technology Data Exchange (ETDEWEB)

Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentally validated undecaprenyl pyrophosphate synthase inhibitors using high- and low-populated conformational states extracted from the molecular dynamics simulations, we show that structurally dissimilar compounds can bind preferentially to different and rarely sampled conformational states. By performing structural analyses on the newly obtained apo-undecaprenyl pyrophosphate synthase and other crystal structures previously published, we show that the changes observed during the molecular dynamics simulation are very similar to those seen in the crystal structures obtained in the presence or absence of ligands. We believe that this is the first time that a rare 'expanded pocket' state, key to drug design and verified by crystallography, has been extracted from a molecular dynamics simulation.

Sinko, William; de Oliveira, César; Williams, Sarah; Van Wynsberghe, Adam; Durrant, Jacob D.; Cao, Rong; Oldfield, Eric; McCammon, J. Andrew (UIUC); (UCSD); (Hamilton)

2012-04-30

223

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

OpenAIRE

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

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

2009-01-01

224

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

Science.gov (United States)

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

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

2001-08-01

225

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.  

OpenAIRE

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounce...

Douglas, C. M.; Foor, F.; Marrinan, J. A.; Morin, N.; Nielsen, J. B.; Dahl, A. M.; Mazur, P.; Baginsky, W.; Li, W.; El-sherbeini, M.

1994-01-01

226

Mechanism of Action and Inhibition of dehydrosqualene Synthase  

Energy Technology Data Exchange (ETDEWEB)

'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.

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

2011-12-31

227

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death  

OpenAIRE

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite forma...

Lam, Philip Y.; Cadenas, Enrique

2008-01-01

228

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

Energy Technology Data Exchange (ETDEWEB)

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.

?e; #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

229

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

Science.gov (United States)

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 Å 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(d) of 1.1 ± 0.3 ?M in the absence of putrescine and 3.2 ± 0.1 ?M in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold. PMID:21898642

Se?kut?, Jolita; McCloskey, Diane E; Thomas, H Jeanette; Secrist, John A; Pegg, Anthony E; Ealick, Steven E

2011-11-01

230

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

OpenAIRE

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

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

1996-01-01

231

Lipoic Acid Synthase (LASY)  

Science.gov (United States)

OBJECTIVE—Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes. RESEARCH DESIGN AND METHODS—We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake. RESULTS—LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with age- and sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-? and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulin-stimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-?–induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response. CONCLUSIONS—Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction. PMID:19074983

Padmalayam, Indira; Hasham, Sumera; Saxena, Uday; Pillarisetti, Sivaram

2009-01-01

232

Cardiolipin synthase from Escherichia coli.  

Science.gov (United States)

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

Tropp, B E

1997-09-01

233

Extramitochondrial citrate synthase activity in bakers' yeast.  

OpenAIRE

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

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

1986-01-01

234

Calcium-Dependent Nitric Oxide Synthase Activity in Rat Thymocytes  

OpenAIRE

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

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

1998-01-01

235

Heterocyclic inhibitors of glycogen synthase kinase GSK-3  

OpenAIRE

Compounds of formula (I) where A, E, G, X, Y, and the bond --- take various meanings are of use in the preparation of a pharmaceutical formulation, for example in the treatment of a disease in which GSK-3 is involved, including Alzheimer's disease or the non-dependent insulin diabetes mellitus, or hyperproliferative disease such as cancer, displasias or metaplasias of tissue, psoriasis, arterosclerosis or restenosis

Marti?nez Garci?a, Ana; Castro Morera, Ana; Pe?rez Marti?n, Mari?a Concepcio?n; Alonso, Mercedes; Dorronsoro Di?az, Isabel; Moreno Mun?oz, Francisco Jose?; Wandosell Jurado, Francisco

2006-01-01

236

Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).  

Science.gov (United States)

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

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

2010-07-01

237

Small-Molecule Inhibitors at the PSD-95/nNOS Interface have Antidepressant-Like Properties in Mice  

OpenAIRE

Previous studies have demonstrated that nitric oxide (NO) synthase inhibitors are as efficacious as tricyclic antidepressants in preclinical antidepressant screening procedures and in attenuating behavioural deficits associated with animal models of depression. The N-methyl--aspartate receptor (NMDA-R) complex gates Ca2+, which interacts with calmodulin to subsequently activate NO synthase. We hypothesised that uncoupling neuronal nitric oxide synthase (nNOS) from the NMDA-R through the s...

Doucet, Mv; Levine, H.; Dev, Kk; Harkin, A.

2013-01-01

238

Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo  

DEFF Research Database (Denmark)

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

Volke, V; Wegener, Gregers

1999-01-01

239

Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates.  

Science.gov (United States)

We have studied the effects of methotrexate (MTX-Glu1) and the polyglutamate derivatives of methotrexate (MTXPGs) with 2, 3, 4, and 5 glutamyl residues on the catalytic activity of thymidylate synthase purified from MCF-7 human breast cancer cells and on the kinetics of the ternary complex formation by 5-fluoro-2'-deoxyuridine 5'-monophosphate, folate cofactor, and thymidylate synthase. MTX-Glu1 exhibited uncompetitive inhibition of thymidylate synthase when reaction kinetics were analyzed by either double reciprocal plots or a computerized mathematical model based on nonlinear least-squares curve fitting. The Ki for MTX-Glu1 inhibition was 13 microM and the I50 was 22 microM, irrespective of the degree of polyglutamation of the folate. In contrast, the polyglutamated derivatives of MTX all acted as noncompetitive inhibitors. The MTXPGs had 75-300-fold greater potency than MTX-Glu1 as inhibitors of thymidylate synthase catalytic activity, with Ki values from 0.17 to 0.047 microM for MTX-Glu2 to MTX-Glu5, respectively. Neither MTX-Glu1 nor MTXPGs promoted the formation of a charcoal-stable ternary complex with thymidylate synthase and 5-fluoro-2'-deoxyuridine 5'-monophosphate. CH2-H4PteGlu5 (where PteGlu represents pteroylglutamic acid) was found to be 40-fold more potent than CH2-H4PteGlu1 in participating in the formation of a ternary complex, and 10 microM MTX-Glu5 significantly inhibited the formation of a ternary complex containing this folate as cofactor. The inhibition was determined to be due to a reduction in the kon. The potency of this inhibition was markedly greater in the presence of CH2-H4PteGlu1 as compared to CH2-H4PteGlu5. This finding suggests that the degree of interference with complex formation in intact cells would depend on the state of polyglutamation of available folate cofactor. Ternary complex formation with H2PteGlu5 as the folate cofactor was also investigated, and a 50% reduction in complex formation was found in the presence of a 2 microM concentration of MTX-Glu5. These findings have significant implications regarding the mechanism of action of MTX-Glu1 and contribute to an understanding of the complex interactions of MTX-Glu1 and 5-fluorouracil. PMID:2410416

Allegra, C J; Chabner, B A; Drake, J C; Lutz, R; Rodbard, D; Jolivet, J

1985-08-15

240

Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.  

Science.gov (United States)

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-?-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. PMID:23550753

Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

2013-04-01

241

Characterization of putative capsaicin synthase promoter activity.  

Science.gov (United States)

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

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

2009-10-31

242

Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides.  

Science.gov (United States)

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

Laughlin, Thomas F; Ahmad, Zulfiqar

2010-04-01

243

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

Scientific Electronic Library Online (English)

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

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

244

SbnG, a Citrate Synthase in Staphylococcus aureus: A NEW FOLD ON AN OLD ENZYME.  

Science.gov (United States)

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

Kobylarz, Marek J; Grigg, Jason C; Sheldon, Jessica R; Heinrichs, David E; Murphy, Michael E P

2014-12-01

245

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

Science.gov (United States)

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

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

1976-04-10

246

Combination therapy targeting ectopic ATP synthase and 26S proteasome induces ER stress in breast cancer cells  

Science.gov (United States)

F1Fo ATP synthase is present in all organisms and is predominantly located on the inner membrane of mitochondria in eukaryotic cells. The present study demonstrated that ATP synthase and electron transport chain complexes were ectopically expressed on the surface of breast cancer cells and could serve as a potent anticancer target. We investigated the anticancer effects of the ATP synthase inhibitor citreoviridin on breast cancer cells through proteomic approaches and revealed that differentially expressed proteins in cell cycle regulation and in the unfolded protein response were functionally enriched. We showed that citreoviridin triggered PERK-mediated eIF2? phosphorylation, which in turn attenuated general protein synthesis and led to cell cycle arrest in the G0/G1 phase. We further showed that the combination of citreoviridin and the 26S proteasome inhibitor bortezomib could improve the anticancer activity by enhancing ER stress, by ameliorating citreoviridin-caused cyclin D3 compensation, and by contributing to CDK1 deactivation and PCNA downregulation. More interestingly, the combined treatment triggered lethality through unusual non-apoptotic caspase- and autophagy-independent cell death with a cytoplasmic vacuolization phenotype. The results imply that by boosting ER stress, the combination of ATP synthase inhibitor citreoviridin and 26S proteasome inhibitor bortezomib could potentially be an effective therapeutic strategy against breast cancer. PMID:25429617

Chang, H-Y; Huang, T-C; Chen, N-N; Huang, H-C; Juan, H-F

2014-01-01

247

A functional cellulose synthase from ascidian epidermis  

OpenAIRE

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...

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

2004-01-01

248

Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase† ?  

OpenAIRE

The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. ...

Li, Fuli; Hagemeier, Christoph H.; Seedorf, Henning; Gottschalk, Gerhard; Thauer, Rudolf K.

2007-01-01

249

Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase  

Energy Technology Data Exchange (ETDEWEB)

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.

Maley, F.; Maley, G.F.

1983-01-01

250

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

Directory of Open Access Journals (Sweden)

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.

Ketabchi Farzaneh

2012-01-01

251

GLYCOGEN SYNTHASE KINASE-3 IS A NEGATIVE REGULATOR OF EXTRACELLULAR SIGNAL-REGULATED KINASE  

OpenAIRE

Glycogen-synthase kinase-3 (GSK-3) and extracellular signal-regulated kinase (ERK) are critical downstream signaling proteins for the PI3-kinase/Akt and Ras/Raf/MEK-1 pathway, respectively, and regulate diverse cellular processes including embryonic development, cell differentiation and apoptosis. Here, we show that inhibition of GSK-3 using GSK-3 inhibitors or RNA interference (RNAi) significantly induced the phosphorylation of ERK1/2 in human colon cancer cell lines HT29 and Caco-2. Pretrea...

Wang, Qingding; Zhou, Yuning; Wang, Xiaofu; Evers, B. Mark

2006-01-01

252

Tissue-Specific Role of Glycogen Synthase Kinase 3? in Glucose Homeostasis and Insulin Action? †  

OpenAIRE

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3? and GSK-3?. Mice engineered to lack GSK-3? die in late embryogenesis from liver apoptosis, whereas mice engineered to lack ...

Patel, Satish; Doble, Bradley W.; Macaulay, Katrina; Sinclair, Elaine M.; Drucker, Daniel J.; Woodgett, James R.

2008-01-01

253

Regulation of Th1 cells and experimental autoimmune encephalomyelitis (EAE) by glycogen synthase kinase-3  

OpenAIRE

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the central nervous system, for which only limited therapeutic interventions are available. Since MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the present study, we tested if inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or ameliorate EAE. G...

Beurel, Ele?onore; Kaidanovich-beilin, Oksana; Yeh, Wen-i; Song, Ling; Palomo, Valle; Michalek, Suzanne M.; Woodgett, James R.; Harrington, Laurie E.; Eldar-finkelman, Hagit; Martinez, Ana; Jope, Richard S.

2013-01-01

254

The Role of Glycogen Synthase Kinase 3 Beta in Neuroinflammation and Pain  

OpenAIRE

Neuroinflammation is a crucial mechanism related to many neurological diseases. Extensive studies in recent years have indicated that dysregulation of Glycogen Synthase Kinase 3 Beta (GSK3?) contributes to the development and progression of these disorders through regulating the neuroinflammation processes. Inhibitors of GSK3? have been shown to be beneficial in many neuroinflammatory disease models including Alzheimer's disease, multiple sclerosis and AIDS dem entia complex. Glial activati...

Maixner, Dylan Warren; Weng, Han-rong

2013-01-01

255

Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3*  

OpenAIRE

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of mini...

Wei, Jing; Liu, Wenhua; Yan, Zhen

2010-01-01

256

Innate and adaptive immune responses regulated by glycogen synthase kinase-3 (GSK3)  

OpenAIRE

In just a few years, glycogen synthase kinase-3 (GSK3) has transformed from an obscure enzyme seldom encountered in the immune literature to one implicated in an improbably large number of roles. GSK3 is a crucial regulator of the balance between pro- and anti-inflammatory cytokine production in both the periphery and the central nervous system, endowing GSK3 inhibitors such as lithium with the capacity to diminish inflammation. T cell proliferation, differentiation, and survival are influenc...

Beurel, Ele?onore; Michalek, Suzanne M.; Jope, Richard S.

2009-01-01

257

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy  

OpenAIRE

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that d...

Wang, Zhong; Smith, Kevin S.; Murphy, Mark; Piloto, Obdulio; Somervaille, Tim C. P.; Cleary, Michael L.

2008-01-01

258

Validation of spermidine synthase as a drug target in African trypanosomes  

OpenAIRE

Abstract The trypanocidal activity of the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomasis, the requirement for additional drug targets is paramount. Here we report the biochemical properties of Trypanosoma brucei spermidine synthase (TbSpSyn), the enzyme immediately down-stream of ODC in this pathway...

Tayor, Martin C.; Kaur, Harparkash; Blessington, Bernard; Kelly, John M.; Wilkinson, Shane R.

2008-01-01

259

Detailed Enzyme Kinetics in Terms of Biochemical Species: Study of Citrate Synthase  

OpenAIRE

The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength,...

Beard, Daniel A.; Vinnakota, Kalyan C.; Wu, Fan

2008-01-01

260

Deoxyribonuclease inhibitors.  

Science.gov (United States)

Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or modify their activities. This review focuses on DNase inhibitors. Some DNase inhibitors have been isolated from various natural sources, such as humans, animals (beef, calf, rabbit and rat), plants (Nicotiana tabacum), and microorganisms (some Streptomyces and Adenovirus species, Micromonospora echinospora and Escherichia coli), while others have been obtained by chemical synthesis. They differ in chemical structure (various proteins, nucleotides, anthracycline and aminoglycoside antibiotics, synthetic organic and inorganic compounds) and mechanism of action (forming complexes with DNases or DNA). Some of the inhibitors are specific toward only one type of DNase, while others are active towards two or more. Physico-chemical properties of DNase inhibitors are calculated using the Molinspiration tool and most of them meet all criteria for good solubility and permeability. DNase inhibitors may be used as pharmaceuticals for preventing, monitoring and treating various diseases. PMID:25042005

Kolarevic, Ana; Yancheva, Denitsa; Kocic, Gordana; Smelcerovic, Andrija

2014-12-17

261

Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis.  

Science.gov (United States)

Systemic administration of the nitric oxide (NO) synthase inhibitor Nomega-nitro--arginine methyl ester (L-NAME) to rabbits bearing a corneal implant blocked vascular endothelial growth factor (VEGF), but not basic fibroblast growth factor (bFGF)-induced angiogenesis. L-NAME completely blocked angiogenesis induced by VEGF-transfected MCF-7 breast carcinoma cells and the cells remained dormant in the cornea. Postcapillary endothelial cell migration and growth induced by VEGF were blocked by both the NO synthase inhibitor Nomega-mono-methyl--arginine and by the guanylate cyclase inhibitor LY 83583. We conclude that NO is a downstream imperative of VEGF-, but not bFGF-induced angiogenesis, and propose that the NO synthase/guanylate cyclase pathway is a potential target for controlling tumor angiogenesis in response to VEGF. Our studies support recent evidence that VEGF and bFGF induce angiogenesis by different mechanistic pathways using the alphavbeta5 and alphavbeta3 integrins, respectively. PMID:9169492

Ziche, M; Morbidelli, L; Choudhuri, R; Zhang, H T; Donnini, S; Granger, H J; Bicknell, R

1997-01-01

262

Analysis of hyaluronan synthase activity.  

Science.gov (United States)

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

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

2015-01-01

263

Producing biofuels using polyketide synthases  

Energy Technology Data Exchange (ETDEWEB)

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.

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

2013-04-16

264

Water stress on the performace of herbicides and biochemical characteristics of Euphorbia heterophylla
Déficit hídrico na eficiência de herbicidas e nas características bioquímicas de Euphorbia heterophylla
 

OpenAIRE

The objective of this work was to evaluate conditions the effectiveness of acetolactate synthase (ALS) and protoporphyrinogen oxidase (PROTOX) inhibitors in the Bidens pilosa control under two water deficit conditions, as well as to determine the action under the content of soluble carbohydrates and protein and free amino acids of weed. The experimental design was randomized completely design, with four replications, with the treatments setup in a factorial scheme 4x2, with four herbicides (f...

Caio Ferraz de Campos; Guilherme Sasso Ferreira Souza; Saulo Ítalo de Almeida Costa; Dagoberto Martins; Hermeson dos Santos Vitorino

2013-01-01

265

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

OpenAIRE

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

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

2011-01-01

266

Threonine phosphorylation of rat liver glycogen synthase  

International Nuclear Information System (INIS)

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine. When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

267

Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi.  

Science.gov (United States)

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

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

2010-02-01

268

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

Science.gov (United States)

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

Paliyath, G.; Poovaiah, B. W.

1988-01-01

269

ACE inhibitors  

Science.gov (United States)

... inhibitors are rare. You may have a dry cough. This may go away after a while. If it does not, tell your doctor. Sometimes reducing your dose helps, but sometimes your doctor will switch you to a different medication. Do not lower ...

270

Crystal structure of riboflavin synthase  

Energy Technology Data Exchange (ETDEWEB)

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

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

2010-03-05

271

Inducible nitric oxide synthase downmodulates contact hypersensitivity by suppressing dendritic cell migration and survival.  

Science.gov (United States)

Nitric oxide (NO) has several important roles in various physiological settings; one of the NO synthases, inducible NO synthase (iNOS), is induced by external stimulation of the skin. A prototypic example of external stimulation is hapten exposure, which induces the T-cell-mediated immune response known as contact hypersensitivity (CHS). We herein report on cutaneous dendritic cell (DC) function in the presence of an iNOS-specific inhibitor during the sensitization phase of CHS. First, we examined epidermal cell (EC) suspensions using flow cytometry with an iNOS antibody and confirmed that iNOS was expressed in the cytoplasm of Langerhans cells (LCs). We then studied the role of iNOS in CHS, and found that responses to DNFB were enhanced by the addition of an iNOS inhibitor during sensitization. Similarly, the iNOS inhibitor augmented FITC-induced migration of cutaneous DCs, including Langerin(+) LCs and Langerin(-) dermal DCs, to draining lymph nodes. Finally, we showed that iNOS inhibitor enhanced LC survival in vitro. We concluded that NO suppresses migration and survival of cutaneous DCs, resulting in a downmodulation of CHS. PMID:19727121

Sugita, Kazunari; Kabashima, Kenji; Yoshiki, Ryutaro; Ikenouchi-Sugita, Atsuko; Tsutsui, Masato; Nakamura, Jun; Yanagihara, Nobuyuki; Tokura, Yoshiki

2010-02-01

272

Genetics Home Reference: GM3 synthase deficiency  

Science.gov (United States)

... Genetic Testing Registry Genetic testing ClinicalTrials.gov Research studies PubMed Recent literature ... synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ...

273

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

Directory of Open Access Journals (Sweden)

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

Ramani Shilpa

2010-08-01

274

Purification and properties of 6-methylsalicylic acid synthase from Penicillium patulum.  

Science.gov (United States)

6-Methylsalicylic acid synthase has been isolated in homogeneous form from Penicillium patulum grown in liquid culture from a spore inoculum. The enzyme is highly susceptible to proteolytic degradation in vivo and in vitro, but may be stabilized during purification by incorporating proteinase inhibitors in the buffers. The enzyme exists as a homotetramer of M(r) 750,000, with a subunit M(r) of 180,000. 6-Methylsalicyclic acid synthase also accepts acetoacetyl-CoA as an alternative starter molecule to acetyl-CoA. The enzyme also catalyses the formation of small amounts of triacetic acid lactone as an oligatory by-product of the reaction. In the absence of NADPH, triacetic acid lactone is the exclusive enzymic product, being formed at 10% of the rate of 6-methylsalicylic acid. The enzyme is inactivated by 1,3-dibromopropan-2-one, leading to the formation of cross-linked dimers similar to that observed with type I fatty acid synthases. Acetyl-CoA protects the enzyme against the inactivation and inhibits dimer formation. An adaptation of the purification method for 6-methylsalicylic acid synthase may be used for the isolation of fatty acid sythase from Penicillium patulum. PMID:1471999

Spencer, J B; Jordan, P M

1992-12-15

275

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

International Nuclear Information System (INIS)

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

276

Nitric Oxide synthases and atrial fibrillation  

OpenAIRE

Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases, which normally produce nitric oxide in the heart. Two nitric oxide synthase isoforms (1 and 3) are normally expressed in the heart. During pathologies such as heart failure, there is induction of nitric oxide syn...

CynthiaAnnCarnes; ArunSridhar; SandorGyorke

2012-01-01

277

Novel Polyketide Synthase from Nectria haematococca  

OpenAIRE

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: ?-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, a...

Graziani, Stephane; Vasnier, Christelle; Daboussi, Marie-josee

2004-01-01

278

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

Science.gov (United States)

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

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

2014-10-13

279

Unique animal prenyltransferase with monoterpene synthase activity.  

Science.gov (United States)

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

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

2009-06-01

280

Malate synthase a membrane protein?  

International Nuclear Information System (INIS)

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

281

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

Directory of Open Access Journals (Sweden)

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.

Marcelo Carnier Dornelas

2007-01-01

282

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

Scientific Electronic Library Online (English)

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

Marcelo Carnier, Dornelas; Paulo, Mazzafera.

283

Terpene synthases are widely distributed in bacteria.  

Science.gov (United States)

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

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

2015-01-20

284

Phosphatidylinositol 3-kinase and glycogen synthase kinase 3 regulate estrogen receptor-mediated transcription in neuronal cells  

OpenAIRE

In addition to 17?-estradiol binding, estrogen receptor (ER) transcriptional activity could be controlled by intracellular kinase signaling pathways activated by growth factors. In this report we present evidence suggesting that glycogen synthase kinase 3 (GSK3), an effector kinase of the phosphatidylinositol 3-kinase (PI3K) pathway, may affect ER? activity in N2a neuroblastoma cells. LiCl, sodium valproate, and SB415286, three inhibitors of GSK3, dose-dependently blocked ER?-mediated tran...

Mendez, P.; Garcia-segura, Luis M.

2006-01-01

285

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

OpenAIRE

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

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

2012-01-01

286

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

OpenAIRE

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

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

2000-01-01

287

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

OpenAIRE

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

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

2002-01-01

288

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

OpenAIRE

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

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

2007-01-01

289

Structural and mechanistic insights into the action of Plasmodium falciparum spermidine synthase.  

Science.gov (United States)

Spermidine synthase is currently considered as a promising drug target in the malaria parasite, Plasmodium falciparum, due to the vital role of spermidine in the activation of the eukaryotic translation initiation factor (eIF5A) and cell proliferation. However, very limited information was available regarding the structure and mechanism of action of the protein at the start of this study. Structural and mechanistic insights of the P. falciparum spermidine synthase (PfSpdSyn) were obtained utilizing molecular dynamics simulations of a homology model based on the crystal structures of the Arabidopsis thaliana and Thermotoga maritima homologues. Our data are supported by in vitro site-directed mutagenesis of essential residues as well as by a crystal structure of the protein that became available recently. We provide, for the first time, dynamic evidence for the mechanism of the aminopropyltransferase action of PfSpdSyn. This characterization of the structural and mechanistic properties of the PfSpdSyn as well as the elucidation of the active site residues involved in substrate, product, and inhibitor interactions paves the way toward inhibitor selection or design of parasite-specific inhibitors. PMID:17196392

Burger, Pieter B; Birkholtz, Lyn-Marie; Joubert, Fourie; Haider, Nashya; Walter, Rolf D; Louw, Abraham I

2007-02-15

290

A Strategy to Discover Inhibitors of Bacillus subtilis Surfactin-type Phosphopantetheinyl Transferase  

OpenAIRE

Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and nonribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need ...

Yasgar, Adam; Foley, Timothy; Jadhav, Ajit; Inglese, James; Burkart, Michael; Simeonov, Anton

2010-01-01

291

Chitin synthase homologs in three ectomycorrhizal truffles.  

Science.gov (United States)

Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii. A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber, and suggested a close relationship between T. magnatum and T. uncinatum. PMID:8593947

Lanfranco, L; Garnero, L; Delpero, M; Bonfante, P

1995-12-01

292

AcEST: DK963523 [AcEST  

Lifescience Database Archive (English)

Full Text Available TST39A01NGRL0016_M07 619 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0016_M07. 5' end seq ... |ILVI_BUCSC Acetolactate synthase large subunit OS=Buch ... 96 1e-19 sp|O08353|ILVB_METAO Probable acetolac ... |ILVI_BUCBP Acetolactate synthase large subunit OS=Buch ... 93 1e-18 sp|O33112|ILVB_MYCLE Acetolactate synt ...

293

AcEST: DK956652 [AcEST  

Lifescience Database Archive (English)

Full Text Available TST39A01NGRL0026_E20 616 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0026_E20. 5' end seq ... |ILVI_BUCSC Acetolactate synthase large subunit OS=Buch ... 98 4e-20 sp|O08353|ILVB_METAO Probable acetolac ... |ILVI_BUCBP Acetolactate synthase large subunit OS=Buch ... 95 3e-19 sp|O33112|ILVB_MYCLE Acetolactate synt ...

294

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens.  

OpenAIRE

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

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

1980-01-01

295

Glycogen Synthase Kinase 3 and h-prune Regulate Cell Migration by Modulating Focal Adhesions†  

OpenAIRE

h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylatio...

Kobayashi, Tsuyoshi; Hino, Shin-ichiro; Oue, Naohide; Asahara, Toshimasa; Zollo, Massimo; Yasui, Wataru; Kikuchi, Akira

2006-01-01

296

Fragment-based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping  

OpenAIRE

Solution state nuclear magnetic resonance (NMR1) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the co-factor binding pocket of hTS. Screening hits were used as search cr...

Begley, Darren W.; Zheng, Suxin; Varani, Gabriele

2010-01-01

297

Gamma-irradiation and exogenous iron induce nitric oxide synthase synthesis in mouse liver in vivo  

International Nuclear Information System (INIS)

The inhibitor of protein biosynthesis, cyclocheximide (CHI) and the exogenous antioxidant, phenazan, attenuated the synthesis of nitric acid oxide (NO) in mouse liver in vivo induced by gamma-irradiation, bacterial lipopolysaccharide (LPS) or LPS+Fe2+-citrate treatment of experimental animals. The latter were formed as a result of NO binding to selective NO traps (DETC complexes with exogenous or endogenous FE2+ ions) and measured by the EPR method. A conclusion is drawn that the activation of NO biosynthesis under the action of gamma-irradiation, LPS or LPS+Fe2+-citrate treatment was due to the induction of NO synthase synthesis inhibited by CHI

298

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

OpenAIRE

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...

Bo?mmel, Heike M.; Reif, Andreas; Fro?hlich, Lothar G.; Frey, Armin; Hofmann, Heinrich; Marecak, Dale M.; Groehn, Viola; Kotsonis, Peter; La, Mylinh; Ko?ster, Sandra; Meinecke, Matthias; Bernhardt, Manfred; Weeger, Monika; Ghisla, Sandro; Prestwich, Glenn D.

1998-01-01

299

Inhibition of Inducible Nitric Oxide Synthase Attenuates Monosodium Urate-induced Inflammation in Mice  

OpenAIRE

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor N6-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependen...

Ju, Tae-jin; Dan, Jin-myoung; Cho, Young-je; Park, So-young

2011-01-01

300

Induction of nitric oxide synthase in cultured vascular smooth muscle cells: the role of cyclic AMP.  

OpenAIRE

1. Interleukin-1 beta (IL-1 beta) is a potent stimulant of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production in vascular smooth muscle (VSM) cells in culture. These studies investigate the role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in this process. 2. Dibutyryl cyclic AMP (db cyclic AMP, 0.1-1 mM), forskolin (1-10 microM) and the phosphodiesterase inhibitor, Ro 20-1724 (1-10 microM), all of which increase intracellular cyclic AMP, had no effect on NO ...

Hirokawa, K.; O Shaughnessy, K.; Moore, K.; Ramrakha, P.; Wilkins, M. R.

1994-01-01

301

Aminoguanidine-provoked leukocyte adherence to rat mesenteric venules: role of constitutive nitric oxide synthase inhibition.  

OpenAIRE

1. The effects of aminoguanidine on neutrophil adherence to venules and on the diameter of arterioles in the mesenteric vascular bed of the pentobarbitone-anaesthetized rat have been compared with those of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). 2. Administration of L-NAME (1-10 mg kg-1, i.v.) caused a dose-dependent increase in leukocyte adherence and a reduction in leukocyte rolling velocity in postcapillary venules of the rat mesentery over 1 h. 3. L...

Lopez-belmonte, J.; Whittle, B. J.

1995-01-01

302

Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer  

OpenAIRE

Abstract Background We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the...

Srivastava Anurag; Shukla Nootan K; DattaGupta Siddartha; Sawhney Meenakshi; Kaur Jatinder; Ralhan Ranju

2010-01-01

303

Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.  

Science.gov (United States)

Basic enzyme: The tetraprenyl-?-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-?-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. PMID:23554321

Sato, Tsutomu; Yamaga, Hiroaki; Kashima, Shoji; Murata, Yusuke; Shinada, Tetsuro; Nakano, Chiaki; Hoshino, Tsutomu

2013-05-10

304

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

Scientific Electronic Library Online (English)

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

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

2013-06-01

305

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

Directory of Open Access Journals (Sweden)

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.

L. Vargas

2013-06-01

306

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

Scientific Electronic Library Online (English)

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

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

2013-12-01

307

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

Science.gov (United States)

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

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

2006-09-01

308

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

Science.gov (United States)

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

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

2005-12-01

309

Defining the Potassium Binding Region in an Apple Terpene Synthase*S?  

OpenAIRE

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating...

Green, Sol; Squire, Christopher J.; Nieuwenhuizen, Niels J.; Baker, Edward N.; Laing, William

2009-01-01

310

Hyaluronan synthase in trabecular meshwork cells  

OpenAIRE

Background/aims: Hyaluronan is present in the trabecular meshwork where it is involved in the pathophysiology of aqueous outflow environment. In this study, the expression and regulation of hyaluronan synthase (HAS), which is the enzyme synthesising hyaluronan, in trabecular meshwork cells were investigated.

Usui, T.; Nakajima, F.; Ideta, R.; Kaji, Y.; Suzuki, Y.; Araie, M.; Miyauchi, S.; Heldin, P.; Yamashita, H.

2003-01-01

311

Characteristics and function of cardiac mitochondrial nitric oxide synthase.  

Science.gov (United States)

We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H(2)DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca(2+) uptake by exposure to different cytoplasmic Ca(2+) concentrations ([Ca(2+)](i) = 1, 2 and 5 microm) resulted in a dose-dependent increase of NO production by mitochondria when L-arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca(2+) uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of L-arginine, mitochondrial NO production during stimulation of Ca(2+) uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit L-arginine availability resulted in 50% inhibition of Ca(2+)-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-N(5)-(1-iminoethyl)ornithine (L-NIO) or iNOS inhibitor N-(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca(2+)-induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of L-arginine, mitochondrial Ca(2+) uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by L-arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH(4); 100 microm) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca(2+) uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l-arginine and BH(4). In conclusion, our data show the importance of L-arginine and BH(4) for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS. PMID:19103678

Dedkova, Elena N; Blatter, Lothar A

2009-02-15

312

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 susceptíveis aos herbicidas inibidores da acetolactase sintase  

Directory of Open Access Journals (Sweden)

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 condições de casa-de-vegetação com o objetivo de comparar a capacidade competitiva de biotipos resistentes e suscetíveis 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 média 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 suscetível. A competição chamada de intraespecífíca foi mais importante que a competição interespecífica para o trigo, porém o inverso foi verdadeiro para os biotípos resistentes e susceptíveis de kochia. Os resultados do índice de diferenciação ecológica indicaram que trigo e qualquer um dos dois biotípos de kochia estudados foram limitados apenas parcialmente pelos mesmos recursos de crescimento. No entanto, o crescimento dos biotípos resistentes e susceptíveis de kochia foram limitados pelos mesmos fatores de crescimento.

P.J. Christoffoleti

1994-08-01

313

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 susceptíveis aos herbicidas inibidores da acetolactase sintase  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Experimentos foram instalados em condições de casa-de-vegetação com o objetivo de comparar a capacidade competitiva de biotipos resistentes e suscetíveis 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 média 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 suscetível. A competição chamada de intraespecífíca foi mais importante que a competição interespecífica para o trigo, porém o inverso foi verdadeiro para os biotípos resistentes e susceptíveis de kochia. Os resultados do índice de diferenciação ecológica indicaram que trigo e qualquer um dos dois biotípos de kochia estudados foram limitados apenas parcialmente pelos mesmos recursos de crescimento. No entanto, o crescimento dos biotípos resistentes e susceptíveis de kochia foram limitados pelos mesmos fatores de crescimento. Abstract in english 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.

P.J., Christoffoleti; P., Westra.

1994-08-01

314

Geranyl diphosphate synthase large subunit, and methods of use  

Energy Technology Data Exchange (ETDEWEB)

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, 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 large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Croteau, Rodney B. (Pullman, WA); Burke, Charles C. (Moscow, ID); Wildung, Mark R. (Colfax, WA)

2001-10-16

315

Montelukast exerts no acute direct effect on NO synthases.  

Science.gov (United States)

The cysteinyl leukotrienes (CysLTs) LTC(4), LTD(4) and LTE(4) are potent proinflammatory lipid mediators that play a central role in inflammation, contraction and remodelling of airways observed in asthmatics. Montelukast, a competitive inhibitor of the cysteinyl leukotriene-1 (CysLT(1)) receptor attenuates asthmatic airway inflammation, contraction and remodelling. As a number of studies have shown that montelukast reduced exhaled nitric oxide (NO) levels, a marker of inflammation that correlates with the severity of asthma, we investigated whether or not a direct inhibition of NO synthase (NOS) by montelukast takes place. In an ex vivo rat lung perfusion and ventilation model the NOS-dependent vasodilation effect after lipopolysaccharide (LPS) infusion was assessed with and without montelukast. Functional organ bath studies using isolated aortic rings from the same species aimed to assess effects of montelukast on the inducible and endothelial NOS isoenzymes (i- and eNOS) as well as on iNOS expression. Neuronal NOS (nNOS) was assessed by field stimulated rabbit corpus cavernosum, and isolated human iNOS enzyme activity was assessed for potential inhibition. Montelukast failed to cause vasoconstriction in LPS challenged rat lung, or to inhibit i- and eNOS activity as well as iNOS expression in aortic rings from the same species. Also the assays for nNOS in rabbit corpus cavernosum and on isolated human iNOS enzyme gave no evidence for a direct inhibition by montelukast in physiological and supraphysiological concentrations up to 10(-4)M. We therefore conclude that montelukast has no acute NOS inhibitor action. Its effect on exhaled NO is therefore probably indirectly mediated by a modulation of the asthmatic airway inflammation. PMID:16815057

Hamacher, Jürg; Eichert, Katja; Braun, Clemens; Grebe, Thomas; Strub, Andreas; Lucas, Rudolf; Eltze, Manfrid; Wendel, Albrecht

2007-01-01

316

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

2012-07-25

317

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase.  

Science.gov (United States)

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo. PMID:17623038

Jekabsone, Aiste; Neher, Jonas J; Borutaite, Vilmante; Brown, Guy C

2007-10-01

318

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre-eclampsia  

Science.gov (United States)

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ?30 mmHg in RUPP rats, and 1400 W attenuated this increase by ?50% (P iNOS expression and aortic nitrotyrosine levels (P iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation. PMID:23890248

Amaral, Lorena M; Pinheiro, Lucas C; Guimaraes, Danielle A; Palei, Ana CT; Sertório, Jonas T; Portella, Rafael L; Tanus-Santos, Jose E

2013-01-01

319

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

Science.gov (United States)

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

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

2010-10-01

320

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

Science.gov (United States)

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

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

2007-12-01

321

Genomic organization of plant terpene synthases and molecular evolutionary implications.  

Science.gov (United States)

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

Trapp, S C; Croteau, R B

2001-01-01

322

Immunolocalisation of spermidine synthase in Solanum tuberosum.  

Science.gov (United States)

Spermidine synthase (SPDS) catalyses the formation of spermidine, which is an essential polyamine and widespread in living organisms. Spermidine is formed from putrescine by transfer of an aminopropyl group from decarboxylated S-adenosylmethionine. Spermidine is also a precursor to further polyamines, such as spermine and thermospermine, most of which contribute to tolerance against drought and salinity in plants. Thermospermine is indispensible for vascular tissue growth. Plant spermidine synthases have been cloned from several angiosperms; organ-specific gene expression levels are known for Arabidopsis only. In this study, immunolocalisation of SPDS in potato (Solanum tuberosum) organs is presented. Polyclonal antibodies for SPDS from potato produced in rabbits were purified by affinity chromatography. Cross-reaction with potato putrescine N-methyltransferase was eliminated. Accumulation of SPDS protein in the phloem region of vascular tissues throughout the potato plant is demonstrated. PMID:22445073

Sichhart, Yvonne; Dräger, Birgit

2013-07-01

323

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

Science.gov (United States)

Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 ?m. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ?100 ?m, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 ?g h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

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

2014-12-26

324

Chitin synthase homologs in three ectomycorrhizal truffles  

OpenAIRE

Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii. A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the...

Delpero, Massimiliano; Bonfante, Paola; Lanfranco, Luisa

1995-01-01

325

Nitric oxide synthase in the pineal gland  

OpenAIRE

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 ...

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

1996-01-01

326

Nitric oxide synthases in heart failure.  

OpenAIRE

SIGNIFICANCE: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca(2+) homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. RECENT ADVANCES: Major strides in un...

Carnicer, R.; Crabtree, Mj; Sivakumaran, V.; Casadei, B.; Kass, Da

2013-01-01

327

Gating NO Release from Nitric Oxide Synthase  

OpenAIRE

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work has indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped flow UV-vis methods, reactions were triggered by mixing reduced enzyme/N-hydroxy-L-arginine complex with aerated buffer solution. NO release kinetics were obtained fo...

Whited, Charlotte A.; Warren, Jeffrey J.; Lavoie, Katherine D.; Weinert, Emily E.; Agapie, Theodor; Winkler, Jay R.; Gray, Harry B.

2012-01-01

328

Use of a Glycolipid Inhibitor to Ameliorate Renal Cancer in a Mouse Model  

OpenAIRE

In a xenograft model wherein, live renal cancer cells were implanted under the kidney capsule in mice, revealed a 30-fold increase in tumor volume over a period of 26 days and this was accompanied with a 32-fold increase in the level of lactosylceramide (LacCer). Mice fed D- threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase (LCS: ?-1,4-GalT-V), showed marked reduction in tumor volume. This was accompanied ...

Chatterjee, Subroto; Alsaeedi, Nezar; Hou, Jennifer; Bandaru, Veera Venkata Ratnam; Wu, Lan; Halushka, Marc K.; Pili, Roberto; Ndikuyeze, Georges; Haughey, Norman J.

2013-01-01

329

Role of oxidative stress and inducible nitric oxide synthase in morphine-induced tolerance and dependence in mice. Effect of alpha-lipoic acid.  

Science.gov (United States)

In this study, the possible role of oxidative stress and nitric oxide (NO) synthase isoforms in the development of morphine tolerance and dependence, and effect of alpha-lipoic acid on these parameters were investigated in mice. The development of morphine tolerance as measured by the hot plate test and dependence, as assessed by naloxone-precipitated withdrawal manifestations, produced an increase in brain glutamate and malondialdehyde (MDA) levels and NO production as well as a decrease in brain intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity. Also, the development of these syndromes increased inducible but not neuronal NO synthase mRNA and protein expressions in mice brain. Co-administration of alpha-lipoic acid (?-LA) inhibited the development of morphine tolerance and dependence, their associated biochemical alterations, except elevation of brain glutamate level, and their associated increase in brain inducible NO synthase mRNA and protein expressions. The inhibitory effect of ?-LA on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent administration of the NMDA receptor antagonist, dizocilpine, the antioxidant, N-acetylcysteine or the selective inducible NO synthase inhibitor, aminoguanidine. On the other hand, this inhibitory effect of ?-LA was not changed by concurrent administration of the selective neuronal NO synthase inhibitor, 7-nitroindazole but antagonized by concurrent administration of the NO precursor, L-arginine. These results suggest that ?-LA through inhibition of morphine-induced oxidative stress and increase in the expression and activity of inducible NO synthase in the brain can attenuate the development of morphine tolerance and dependence. PMID:23470902

Abdel-Zaher, Ahmed O; Mostafa, Mostafa G; Farghaly, Hanan S M; Hamdy, Mostafa M; Abdel-Hady, Randa H

2013-06-15

330

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

OpenAIRE

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...

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

1999-01-01

331

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.  

Science.gov (United States)

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites. PMID:10556516

Yamaguchi, T; Kurosaki, F; Suh, D Y; Sankawa, U; Nishioka, M; Akiyama, T; Shibuya, M; Ebizuka, Y

1999-11-01

332

Folate binding site of flavin-dependent thymidylate synthase  

OpenAIRE

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH2H4folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of ...

Koehn, Eric M.; Perissinotti, Laura L.; Moghram, Salah; Prabhakar, Arjun; Lesley, Scott A.; Mathews, Irimpan I.; Kohen, Amnon

2012-01-01

333

The cellulose synthase superfamily in fully sequenced plants and algae  

OpenAIRE

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

Xu Ying; Huang Jinling; Yin Yanbin

2009-01-01

334

Cellulose synthase interacting protein: A new factor in cellulose synthesis  

OpenAIRE

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...

Gu, Ying; Somerville, Chris

2010-01-01

335

Bacterial infection induces nitric oxide synthase in human neutrophils.  

OpenAIRE

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

Wheeler, M. A.; Smith, S. D.; Garci?a-carden?a, G.; Nathan, C. F.; Weiss, R. M.; Sessa, W. C.

1997-01-01

336

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

Science.gov (United States)

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-N?-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. PMID:23704776

Hong, Chang-Won; Kim, Young-Mee; Pyo, Hongryull; Lee, Joon-Ho; Kim, Suwan; Lee, Sunyoung; Noh, Jae Myoung

2013-01-01

337

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

International Nuclear Information System (INIS)

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-N?-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)

338

Proton pump inhibitors  

Science.gov (United States)

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

339

Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background We reported increased levels of Phosphatidyl Inositol synthase (PI synthase, (enzyme that catalyses phosphatidyl inositol (PI synthesis-implicated in intracellular signaling and regulation of cell growth in smokeless tobacco (ST exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC and premalignant lesions (leukoplakia, and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST exposure. Methods Tissue microarray (TMA Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Results Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000. Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005 and tobacco consumption (p = 0.03, OR = 9.0. Exposure of oral cell systems to smokeless tobacco (ST in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K and cyclin D1 levels. Conclusion Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco.

Srivastava Anurag

2010-04-01

340

Nitric oxide synthase inhibition reverts muscarinic receptor down-regulation induced by pilocarpine- and kainic acid-evoked seizures in rat fronto-parietal cortex.  

Science.gov (United States)

We investigated how nitric oxide (NO) synthase inhibitor modulates muscarinic receptor expression in epileptic rats. We found that subchronic treatment (4 days) with N?-nitro-l-arginine reduced the down-regulation of muscarinic receptors induced by pilocarpine and kainic acid in rat fronto-parietal cortex, notwithstanding the dramatic potentiation of seizures induced by both convulsants. Furthermore, functional experiments in fronto-parietal cortex slices, showed that N?-nitro-l-arginine reduces the down-regulating effect of pilocarpine on carbachol-induced phosphoinositol hydrolysis. Finally, N?-nitro-l-arginine greatly potentiated the induction of basic fibroblast growth factor (FGF2) by pilocarpine. These data suggest a potential role of NO in a regulatory feedback loop to control muscarinic receptor signal during seizures. The dramatic potentiation of convulsions by NO synthase inhibitors in some animal models of seizures could derive from preventing this feedback loop. PMID:24246145

Capannolo, Marta; Ciccarelli, Carmela; Molteni, Raffaella; Fumagalli, Fabio; Rocchi, Cristina; Romeo, Stefania; Fasciani, Irene; Aloisi, Gabriella; Zani, Bianca M; Riva, Marco A; Maggio, Roberto

2014-01-01

341

Involvement of neuronal nitric oxide synthase in desensitisation of µ-opioid receptors in the rat locus coeruleus.  

Science.gov (United States)

Nitric oxide (NO) has been recently shown to enhance µ-opioid receptor (MOR) desensitisation in locus coeruleus (LC) neurons. The aim of this study was to evaluate by single-unit extracellular recordings in rat brain slices whether the neuronal NO synthase is involved in MOR desensitisation in LC neurons. As expected, a high concentration of the opioid agonist Met(5)-enkephalin (ME; 10 µM, 10 min) strongly desensitised the inhibition induced by a test application of ME (0.8 µM, 1 min), whereas lower ME concentrations (1 and 3 µM) only weakly desensitised it. The neuronal NO synthase inhibitors 7-nitroindazole (10-100 µM), S-methyl-L-thiocitrulline (0.01-10 µM) and N(?)-propyl-L-arginine (1-10 µM) attenuated ME (10 µM)-induced opioid desensitisation, although the endothelial NO synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine (3-30 µM) failed to change it. The NO donor sodium nitroprusside (1 mM), but not its inactive analog potassium ferricyanide (1 mM), enhanced the ME (3 µM)-induced desensitisation and prevented the effect of S-methyl-L-thiocitrulline (10 µM). Sodium nitroprusside (1 mM) failed to change the desensitisation of ?2-adrenoceptors by noradrenaline (100 µM, 10 min). These results suggest the contribution of NO and a neuronal type of NO synthase in homologous MOR desensitisation in rat LC neurons. PMID:24961237

Santamarta, María T; Llorente, Javier; Mendiguren, Aitziber; Pineda, Joseba

2014-10-01

342

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells  

Energy Technology Data Exchange (ETDEWEB)

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Chen, Taosheng, E-mail: taosheng.chen@stjude.org [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States)

2010-01-01

343

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells  

International Nuclear Information System (INIS)

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

344

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells 1  

Science.gov (United States)

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. PMID:19995556

Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

2009-01-01

345

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells.  

Science.gov (United States)

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. PMID:19995556

Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

2010-01-01

346

Thiol oxidation of mitochondrial FO-c subunits: a way to switch off antimicrobial drug targets of the mitochondrial ATP synthase.  

Science.gov (United States)

A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents "the enzyme of life" of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the common drug binding site of the enzyme is somehow destabilized, thus weakening the enzyme-drug interactions and making the ATP synthase insensitive to drug inhibition. On these bases we hypothesize that the selective oxidation of these cysteine thiols can be exploited to desensitize the mitochondrial ATP synthase to drugs which target FO and maintain their inhibitory potency on bacterial ATP synthases. According to our hypothesis, this strategy could represent an intriguing tool to prevent adverse effects of antimicrobial drugs in mammals, thus enhancing the number of natural and synthetic compounds which can be used in therapy. To this aim studies should be addressed to the identification and formulation of compounds and/or treatments able to selectively oxidize the crucial cysteine thiols of c-subunits without affecting the overall functionality of the mitochondrial ATP synthase and other thiol containing proteins. PMID:24932580

Nesci, S; Ventrella, V; Trombetti, F; Pirini, M; Pagliarani, A

2014-08-01

347

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

Science.gov (United States)

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

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

2001-12-01

348

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

OpenAIRE

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

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

1996-01-01

349

Atorvastatin prevents hypoxia-induced inhibition of endothelial nitric oxide synthase expression but does not affect heme oxygenase-1 in human microvascular endothelial cells  

OpenAIRE

Beneficial cardiovascular effects of statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are particularly assigned to the modulation of inflammation. Endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) are listed among the crucial protective, anti-inflammatory genes in the vasculature. Here we show that atorvastatin at pharmacologically relevant concentration (0.1 ?M) enhanced the expression of eNOS in human microvascular endothelial cells (...

Loboda, Agnieszka; Jazwa, Agnieszka; Jozkowicz, Alicja; Dorosz, Jerzy; Balla, Jozsef; Molema, Grietje; Dulak, Jozef

2006-01-01

350

Glycogen Synthase Kinase-3? Plays a Pro-Apoptotic Role in ?-Adrenergic Receptor-Stimulated Apoptosis in Adult Rat Ventricular Myocytes: Role of ?1 Integrins  

OpenAIRE

?-adrenergic receptor (?-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVM). ?1 integrin signaling plays a protective role in ?-AR-stimulated apoptosis. Glycogen synthase kinase-3? (GSK-3?), a multifunctional serine/threonine kinase, negatively regulates cardiac hypertrophy. Here we show that ?-AR stimulation (isoproterenol; 15 min) increases tyr216 phosphorylation and GSK-3? activity. Inclusion of LiCl, inhibitor of GSK-3?, in the reaction mix or expression o...

Menon, Bindu; Johnson, Jennifer N.; Ross, Robert S.; Singh, Mahipal; Singh, Krishna

2006-01-01

351

Glycogen Synthase Kinase 3? Inhibition Prevents Monocyte Migration across Brain Endothelial Cells via Rac1-GTPase Suppression and Down-Regulation of Active Integrin Conformation  

OpenAIRE

Glycogen synthase kinase (GSK) 3? has been identified as a regulator of immune responses. We demonstrated previously that GSK3? inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3? inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3? inhibitors resulted in a de...

Rom, Slava; Fan, Shongshan; Reichenbach, Nancy; Dykstra, Holly; Ramirez, Servio H.; Persidsky, Yuri

2012-01-01

352

Inhibition of Glycogen Synthase Kinase 3? Promotes Tight Junction Stability in Brain Endothelial Cells by Half-Life Extension of Occludin and Claudin-5  

OpenAIRE

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3? (GSK3?) inhibition has on primary human brain endothelial cells. Here we show that GSK3? inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electric...

Ramirez, Servio H.; Fan, Shongshan; Dykstra, Holly; Rom, Slava; Mercer, Aaron; Reichenbach, Nancy L.; Gofman, Larisa; Persidsky, Yuri

2013-01-01

353

Nitric oxide synthase inhibition enhances the tumor vascular-damaging effects of combretastatin a-4 3-o-phosphate at clinically relevant doses.  

OpenAIRE

PURPOSE: The therapeutic potential of combining the prototype tumor vascular-disrupting agent combretastatin A-4 3-O-phosphate (CA-4-P) with systemic nitric oxide synthase (NOS) inhibition was investigated preclinically. EXPERIMENTAL DESIGN: Vascular response (uptake of (125)I-labeled iodoantipyrine; laser Doppler flowmetry) and tumor response (histologic necrosis; cytotoxicity and growth delay) were determined. RESULTS: Inducible NOS selective inhibitors had no effect on blood flow in the P2...

Tozer, Gm; Prise, Ve; Lewis, G.; Xie, S.; Wilson, I.; Hill, Sa

2009-01-01

354

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

International Nuclear Information System (INIS)

1,2-anhydroglucitol-6P, a known inhibitor of glucose-6P isomerase, behaved as a fructose-6P site-directed irreversible inhibitor of bacterial glucosamine-6P synthase. The lack of reproducibility of the aldolase-mediated condensation of dihydroxyacetone phosphate and glycidaldehyde followed by borohydride reduction previously described prompted us to develop a chemical route to this compounds and its radiolabelled counterpart. The compound was synthesized in 13 steps from D-arabinose with a 6% overall yield. Tritium introduction was performed at step 11 (3 ? 4) allowing isolation of the title compound of high specific radioactivity. (author)

355

AcEST: DK959414 [AcEST  

Lifescience Database Archive (English)

Full Text Available TST39A01NGRL0004_K01 672 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0004_K01. 5' end seq ... |ILVI_BUCBP Acetolactate synthase large subunit OS=Buch ... 87 8e-17 sp|P57321|ILVI_BUCAI Acetolactate synt ... hase large subunit OS=Buch ... 84 5e-16 sp|O85293|ILVI_BUCAP Acetolactate synt ... hase large subunit OS=Buch ... 84 9e-16 sp|Q04789|ILVX_BACSU Acetolactate synt ... |ILVI_BUCSC Acetolactate synthase large subunit OS=Buch ... 78 5e-14 sp|P00893|ILVI_ECOLI Acetolactate synt ...

356

No?·NO from NO?synthase  

OpenAIRE

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we ther...

Schmidt, Harald?H ?H ?W; Hofmann, Heinrich; Schindler, Ursula; Shutenko, Zhanna?S; Cunningham, David?D; Feelisch, Martin

1996-01-01

357

Primary structure of porcine heart citrate synthase.  

OpenAIRE

The sequence of 437 amino acid residues of porcine heart citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO leads to acetyl-CoA), EC 4. 1. 3. 7] has been determined by the alignment of fragments generated by cleavage with cyanogen bromide and with trypsin. Isolation of the peptides was facilitated by recent developments in the high-performance liquid chromatography of peptide mixtures. The alignment of these peptides was consistent with that previously deduced from fragments derived ...

Bloxham, D. P.; Parmelee, D. C.; Kumar, S.; Wade, R. D.; Ericsson, L. H.; Neurath, H.; Walsh, K. A.; Titani, K.

1981-01-01

358

Structural classification and properties of ketoacyl synthases  

OpenAIRE

Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and t...

Chen, Yingfei; Kelly, Erin E.; Masluk, Ryan P.; Nelson, Charles L.; Cantu, David C.; Reilly, Peter J.

2011-01-01

359

Sphingomyelin Synthases Regulate Protein Trafficking and Secretion  

OpenAIRE

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...

Subathra, Marimuthu; Qureshi, Asfia; Luberto, Chiara

2011-01-01

360

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

Science.gov (United States)

Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% ?-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases. PMID:25230152

Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

2014-10-15

361

Ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in Bacteria and Archaea, which contain inositol phospholipid.  

Science.gov (United States)

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes. PMID:24269814

Morii, Hiroyuki; Ogawa, Midori; Fukuda, Kazumasa; Taniguchi, Hatsumi

2014-01-01

362

Strictosidine synthase: mechanism of a Pictet-Spengler catalyzing enzyme.  

Science.gov (United States)

The Pictet-Spengler reaction, which yields either a beta-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a beta-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the beta-carboline product is proposed from these data. PMID:18081287

Maresh, Justin J; Giddings, Lesley-Ann; Friedrich, Anne; Loris, Elke A; Panjikar, Santosh; Trout, Bernhardt L; Stöckigt, Joachim; Peters, Baron; O'Connor, Sarah E

2008-01-16

363

Characterisation of the tryptophan synthase alpha subunit in maize  

OpenAIRE

Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase ???? heterotetramer. Plants have evolved multiple ? (TSA) and ? (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the...

Gierl Alfons; Frey Monika; Letzel Thomas; Fießelmann Andreas; Weigang Linda; Kriechbaumer Verena; Glawischnig Erich

2008-01-01

364

Intermediacy of eudesmane cation during catalysis by aristolochene synthase.  

Science.gov (United States)

Aristolochene synthase from Penicillium roqueforti (PR-AS) catalyzes the formation of the bicyclic sesquiterpene (+)-aristolochene (5) from farnesyl diphosphate (1, FDP) in two mechanistically distinct cyclization reactions. The first reaction transforms farnesyl diphosphate to the uncharged intermediate (S)-(-)-germacrene A (3) through a macrocyclization process that links C1 and C10 upon magnesium ion-assisted diphosphate ester activation. In the second reaction mediated by PR-AS, a protonation induced cyclization has been suggested to generate the highly reactive trans-fused eudesmane cation 4 as a consequence of the precise folding of the enzyme-bound germacrene A intermediate. This contribution describes the use of the transition state analogue inhibitor 4-aza-eudesm-11-ene to explore the intermediacy of cation 4 as an on-path intermediate in the biosynthesis of aristolochene. 4-Aza-eudesm-11-ene as the hydrochloride salt 6 was stereospecifically synthesized in seven steps and 37% overall yield starting from chiral enamine 9. The synthetic sequence featured a highly regio- and stereoselective deracemization reaction of 9 that gave rise to the corresponding Michael adduct in >95% diastereomeric excess as evidenced by optical rotation and NMR measurements. 6 acts as a potent competitive inhibitor of PR-AS (K(i) = 0.35 +/- 0.12 microM) independent of the presence of diphosphate (K(i) = 0.24 +/- 0.09 microM). The failure of exogenous PP(i) to enhance the binding affinity of 6 for PR-AS could be interpreted against an eudesmyl cation/diphosphate anion pair mechanism as the enzymatic strategy to stabilize the highly reactive eudesmane cation 4. In addition, these observations seem to rule out simple favorable electrostatic and/or hydrogen bonding interactions between the active site anchored diphosphate ion and the ammonium ion 6 as the binding mode. Ammonium ion 6 seems to act as a genuine mimic of eudesmane cation (4) that most likely binds the active site of PR-AS in a productive conformation resembling that adapted by 4 during the PR-AS-catalyzed synthesis of 5. PMID:20095558

Faraldos, Juan A; Kariuki, Benson; Allemann, Rudolf K

2010-02-19

365

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase  

OpenAIRE

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS?/?) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibrobl...

Szabo?, Csaba; Vira?g, La?szlo?; Cuzzocrea, Salvatore; Scott, Gwen S.; Hake, Paul; O’connor, Michael P.; Zingarelli, Basilia; Salzman, Andrew; Kun, Ernest

1998-01-01

366

Effect of 7-nitro indazole on neurotransmission in the rat vas deferens: mechanisms unrelated to inhibition of nitric oxide synthase.  

OpenAIRE

1. The effect of the nitric oxide synthase (NOS) inhibitor, 7-nitro indazole (7-NI), on sympathetic and purinergic neurotransmission in the rat isolated vas deferens preparation has been studied. 2. 7-NI (50-200 microM) caused a dose- and frequency-dependent inhibition of the phasic (predominantly purinergic) contractile response of the rat vas deferens to electrical (field) stimulation (100 V, 0.5 ms). Greatest inhibition occurred at lower frequencies of stimulation (0.1-10 Hz). The sustaine...

Allawi, H. S.; Wallace, P.; Pitcher, A.; Gaffen, Z.; Bland-ward, P. A.; Moore, P. K.

1994-01-01

367

Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells  

OpenAIRE

Fatty acid synthase (FAS) activity is a potential therapeutic target to treat cancer and obesity. Here, we have identified a molecular link between FAS and HER2 (erbB-2) oncogene, a marker for poor prognosis that is overexpressed in 30% of breast and ovarian cancers. Pharmacological FAS inhibitors cerulenin and C75 were found to suppress p185HER2 oncoprotein expression and tyrosine-kinase activity in breast and ovarian HER2 overexpressors. Similarly, p185HER2 expression was dramatically down-...

Menendez, Javier A.; Vellon, Luciano; Mehmi, Inderjit; Oza, Bharvi P.; Ropero, Santiago; Colomer, Ramon; Lupu, Ruth

2004-01-01

368

Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat.  

OpenAIRE

1. We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock. 2. Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite cau...

Wu, C. C.; Thiemermann, C.; Vane, J. R.

1995-01-01

369

The Wnt Pool of Glycogen Synthase Kinase 3? Is Critical for Trophic-Deprivation-Induced Neuronal Death?  

OpenAIRE

Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3?, but not GSK-3?, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and...

Hongisto, Vesa; Vainio, Jenni C.; Thompson, Ro?isi?n; Courtney, Michael J.; Coffey, Eleanor T.

2008-01-01

370

Involvement of Calpain-Calpastatin in Cigarette Smoke–Induced Inhibition of Lung Endothelial Nitric Oxide Synthase  

OpenAIRE

We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5–10% CSE for 2–24 h. eNOS gene trans...

Cui, Zhaoqiang; Han, Zhaosheng; Li, Zhaozhong; Hu, Hanbo; Patel, Jawaharlal M.; Antony, Veena; Block, Edward R.; Su, Yunchao

2005-01-01

371

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

OpenAIRE

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

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

2009-01-01

372

Loss of LRPPRC causes ATP synthase deficiency.  

Science.gov (United States)

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

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

2014-05-15

373

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

International Nuclear Information System (INIS)

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

374

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1991-01-01

375

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

Directory of Open Access Journals (Sweden)

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

Rossi Adriano G

2006-09-01

376

Glycogen synthase kinase-3 as a therapeutic target for cognitive dysfunction in neuropsychiatric disorders.  

Science.gov (United States)

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is involved in a broad range of cellular processes including cell proliferation, apoptosis and inflammation. It is now also increasingly acknowledged as having a role to play in cognitive-related processes such as neurogenesis, synaptic plasticity and neural cell survival. Cognitive impairment represents a major debilitating feature of many neurodegenerative and psychiatric disorders, including Alzheimer's disease, mood disorders, schizophrenia and fragile X syndrome, as well as being a result of traumatic brain injury or cranial irradiation. Accordingly, GSK-3 has been identified as an important therapeutic target for cognitive impairment, and recent preclinical studies have yielded important evidence demonstrating that GSK-3 inhibitors may be useful therapeutic interventions for restoring cognitive function in some of these brain disorders. The current review summarises the role of GSK-3 as a regulator of cognitive-dependent functions, examines current preclinical and clinical evidence of the potential of GSK-3 inhibitors as therapeutic agents for cognitive impairments in neuropsychiatric disorders, and offers some insight into the current obstacles that are impeding the clinical use of selective GSK-3 inhibitors in the treatment of cognitive impairment. PMID:25380674

O'Leary, Olivia; Nolan, Yvonne

2015-01-01

377

Functional Contribution of Chorismate Synthase, Anthranilate Synthase, and Chorismate Mutase to Penetration Resistance in Barley-Powdery Mildew Interactions  

Science.gov (United States)

Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2) and chorismate mutase 1 (HvCM1) occupy pivotal branch-points downstream of the s...

378

Modeling of eicosanoid fluxes reveals functional coupling between cyclooxygenases and terminal synthases.  

Science.gov (United States)

Eicosanoids, including prostaglandins (PG) and leukotrienes, are lipid mediators derived from arachidonic acid. A quantitative and biochemical level understanding of eicosanoid metabolism would aid in understanding the mechanisms that govern inflammatory processes. Here, we present a combined experimental and computational approach to understanding the biochemical basis of eicosanoid metabolism in macrophages. Lipidomic and transcriptomic measurements and analyses reveal temporal and dynamic changes of the eicosanoid metabolic network in mouse bone marrow-derived macrophages (BMDM) upon stimulation of the Toll-like receptor 4 with Kdo2-Lipid A (KLA) and stimulation of the P2X7 purinergic receptor with adenosine 5'-triphosphate. Kinetic models were developed for the cyclooxygenase (COX) and lipoxygenase branches of arachidonic acid metabolism, and then the rate constants were estimated with a data set from ATP-stimulated BMDM, using a two-step matrix-based approach employing a constrained least-squares method followed by nonlinear optimization. The robustness of the model was validated through parametric sensitivity, uncertainty analysis, and predicting an independent dataset from KLA-primed ATP-stimulated BMDM by allowing the parameters to vary within the uncertainty range of the calculated parameters. We analyzed the functional coupling between COX isozymes and terminal enzymes by developing a PGH2-divided model. This provided evidence for the functional coupling between COX-2 and PGE2 synthase, between COX-1/COX-2 and PGD2 synthase, and also between COX-1 and thromboxane A2 synthase. Further, these functional couplings were experimentally validated using COX-1 and COX-2 selective inhibitors. The resulting fluxomics analysis demonstrates that the "multi-omics" systems biology approach can define the complex machinery of eicosanoid networks. PMID:24559999

Kihara, Yasuyuki; Gupta, Shakti; Maurya, Mano R; Armando, Aaron; Shah, Ishita; Quehenberger, Oswald; Glass, Christopher K; Dennis, Edward A; Subramaniam, Shankar

2014-02-18

379

Crystal structure of the acyltransferase domain of the iterative polyketide synthase in enediyne biosynthesis.  

Science.gov (United States)

Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT(DYN10)) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 ? resolution unveils a ?/? hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a ?/? fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude ?-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser(651) residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates. PMID:22589546

Liew, Chong Wai; Nilsson, Martina; Chen, Ming Wei; Sun, Huihua; Cornvik, Tobias; Liang, Zhao-Xun; Lescar, Julien

2012-06-29

380

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

International Nuclear Information System (INIS)

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7,500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to subunit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degree C. The monomer is formed upon heating with SDS to 121 degree C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m-chlorophenylhydrazone. These resuyanide m-chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter

381

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

Energy Technology Data Exchange (ETDEWEB)

The ATP synthase (F{sub 1}F{sub 0}) 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 F{sub 1} ATPase, subunits of M{sub r} 26,000 (a), 23,000 (b), and 7,500 (c) have been purified. The ATPase activity of F{sub 1}F{sub 0} was specifically activated about 10-fold by Na{sup +} ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with ({sup 14}C)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{sup +} ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na{sup +} 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{sup +} transport and also that it is a primary event and not accomplished by a H{sup +}-translocating ATP synthase in combination with a Na{sup +}/H{sup +} antiporter.

Laubinger, W.; Dimroth, P. (Technischen Universitaet Muenchen (West Germany))

1988-09-20

382

Relaxation of rat thoracic aorta induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation.  

OpenAIRE

1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), ...

Moritoki, H.; Hisayama, T.; Takeuchi, S.; Kondoh, W.; Imagawa, M.

1994-01-01

383

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

Science.gov (United States)

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers. PMID:25454716

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

2014-11-01

384

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

DEFF Research Database (Denmark)

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers.

Hempel, Casper; Kohnke, Hannes

2014-01-01

385

Comparative evaluation of the effect of tricyclic antidepressants on inducible nitric oxide synthase expression in neuropathic pain model.  

Science.gov (United States)

The analgesic effect of acute i.p. administration of amitriptyline (norepinepherine and serotonin reuptake inhibitor), clomipramine (serotonin reuptake inhibitor) and desipramine (norepinepherine reuptake inhibitor) was studied in chronic constriction injury (CCI) model of sciatic nerve in rats and mRNA and protein expression of inducible nitric oxide synthase (iNOS) were also investigated. Acute treatment with amitriptyline and clomipramine produced antinociceptive effects after sciatic nerve injury and blockade of norepinephrine reuptake using desipramine did not demonstrate antinociceptive effects. The antinociceptive effect of amitriptyline, not clomipramine, was augmented by the selective iNOS inhibitor, aminoguanidine. Amitriptyline inhibited iNOS mRNA and protein expression in cerebellum and hippocampus. However, desipramine altered neither iNOS expression at mRNA level nor at post-transcriptional level. Based on our experimental findings, we conclude that the analgesic effect of the dual norepinepherine and serotonin reuptake inhibitor, amitriptyline, is partially due to inhibition of central iNOS. PMID:22584260

Farghaly, Hanan Sayed Mohamed; Abdel-Zaher, Ahmed Osman; Mostafa, Mostafa Galal; Kotb, Hassan Ibrahim

2012-08-15

386

Leukaemia Inhibitory Factor Stimulates Proliferation of Olfactory Neuronal Progenitors via Inducible Nitric Oxide Synthase  

Science.gov (United States)

Neurogenesis continues in the adult brain and in the adult olfactory epithelium. The cytokine, leukaemia inhibitory factor and nitric oxide are both known to stimulate neuronal progenitor cell proliferation in the olfactory epithelium after injury. Our aim here was to determine whether these observations are independent, specifically, whether leukaemia inhibitory factor triggers neural precursor proliferation via the inducible nitric oxide synthase pathway. We evaluated the effects of leukaemia inhibitory factor on inducible form of nitric oxide synthase (iNOS) expression, and cell proliferation in olfactory epithelial cell cultures and olfactory neurosphere-derived cells. Leukaemia inhibitory factor induced expression of iNOS and increased cell proliferation. An iNOS inhibitor and an anti-leukaemia inhibitory factor receptor blocking antibody inhibited leukaemia inhibitory factor-induced cell proliferation, an effect that was reversed by a NO donor. Altogether, the results strongly suggest that leukaemia inhibitory factor induces iNOS expression, increasing nitric oxide levels, to stimulate proliferation of olfactory neural precursor cells. This finding sheds light on neuronal regeneration occurring after injury of the olfactory epithelium. PMID:23024784

Lopez-Arenas, Estefania; Mackay-Sim, Alan; Bacigalupo, Juan; Sulz, Lorena

2012-01-01

387

Validation of spermidine synthase as a drug target in African trypanosomes.  

Science.gov (United States)

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements. PMID:17916066

Taylor, Martin C; Kaur, Harparkash; Blessington, Bernard; Kelly, John M; Wilkinson, Shane R

2008-01-15

388

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

Directory of Open Access Journals (Sweden)

Full Text Available Backgrounds: 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 N?-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 (PPP 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. Keywords: Catalase, L-NAME, Malondialdehyde, Oxidative stress, Propolis, Rat

Oguz Cakir

2013-06-01

389

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

Science.gov (United States)

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

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

2015-01-01

390

Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target  

DEFF Research Database (Denmark)

To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.

Yang, Zhiping; Huang, Yuh-Chin T

2014-01-01

391

Inhibition of glycogen synthase kinase-3? by falcarindiol isolated from Japanese Parsley (Oenanthe javanica).  

Science.gov (United States)

A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1? erg3? pdr1? pdr3?) was discovered as an inhibitor of glycogen synthase kinase-3? (GSK-3?). Falcarindiol inhibited GSK-3? in an ATP noncompetitive manner with a Ki value of 86.9 ?M using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 ?M. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer's disease. PMID:23895038

Yoshida, Jun; Seino, Hiroko; Ito, Yoshiaki; Nakano, Toshimitsu; Satoh, Takumi; Ogane, Yoshiko; Suwa, Saori; Koshino, Hiroyuki; Kimura, Ken-Ichi

2013-08-01

392

Exogenous iron and ?-irradiation induce NO-synthase synthesis in mouse liver  

International Nuclear Information System (INIS)

Protein synthesis inhibitor (cycloheximide, CHI) and exogenous antioxidant (phenazan) suppress the synthesis of NO in mouse liver in vivo which is induced by administration to the animals of ?-irradiation, bacterial lipopolysaccharide (LPS), or Fe2+-citrate together with LPS. Biosynthesis of NO was monitored by the ESR signal of paramagnetic mononitrosyl iron complexes with the exogenous ligand diethyldithiocarbamate (MNIC-DETC) 30 min after addition of the ligand. The complexes arise from NO binding to DETC complexes with exogenous and endogenous Fe2+, which act as selective NO traps. The enhancement of NO biosynthesis after ?-irradiation or LPS or LPS + Fe2+-citrate is apparently due to the induction of the synthesis of NO-synthase, which is inhibited by cycloheximide. This process is triggered by reactive oxygen species, presumably through the activation of the transcription factor protein NFkB. The accumulation of free radical oxygen species is inhibited by the antioxidant phenazan

393

Famotidine inhibits glycogen synthase kinase-3?: an investigation by docking simulation and experimental validation.  

Science.gov (United States)

Famotidine was investigated as an inhibitor of glycogen synthase kinase-3? (GSK-3?) in an attempt to explain the molecular mechanism of its hypoglycemic side effects. The investigation included simulated docking experiments, in vitro enzyme inhibition assay, glycogen sparing studies using animal models and single dose oral glucose tolerance test (OGTT). Docking studies showed how famotidine is optimally fit within the binding pocket of GSK-3? via numerous attractive interactions with some specific amino acids. Experimentally, famotidine could inhibit GSK-3? (IC?? = 1.44 ?M) and increased significantly liver glycogen spares in fasting animal models. Moreover, a single oral dose of famotidine was shown to decrease the glycemic response curve after 75 g OGTT. PMID:22512725

Mohammad, Mohammad; Al-Masri, Ihab M; Issa, Ala; Al-Ghussein, Mohamed A S; Fararjeh, Mohammad; Alkhatib, Hatim; Taha, Mutasem O; Bustanji, Yasser

2013-08-01

394

Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16  

OpenAIRE

Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of...

Dudnik, Alexey; Bigler, Laurent; Dudler, Robert

2014-01-01

395

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

OpenAIRE

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...

Li, Shundai; Lei, Lei; Somerville, Chris R.; Gu, Ying

2012-01-01

396

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

International Nuclear Information System (INIS)

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

397

Characterisation of the tryptophan synthase alpha subunit in maize  

Directory of Open Access Journals (Sweden)

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

Gierl Alfons

2008-04-01

398

Effects of inhibition of neuronal nitric oxide synthase on basal retinal blood flow regulation.  

Science.gov (United States)

Nitric oxide (NO) has been observed to regulate blood flow under basal and stimulated conditions in the retina. Recent evidence suggests that NO produced by neuronal nitric oxide synthase (nNOS) may regulate blood flow in addition to that produced by endothelial nitric oxide synthase (eNOS). The objective of the current study was to investigate the contribution of NO produced by nNOS in the regulation of basal retinal blood flow. A non-specific NOS inhibitor N (G)-nitro-l-arginine methyl ester (l-NAME) and the specific nNOS inhibitors 1-(2-trifluoromethylphenyl) imidazole (TRIM) and (4S)-N-(4-amino-5 [aminoethyl] aminopentyl)-N-nitroguanidine (AAAN) were injected into the vitreous (intravitreal) of Long-Evans rats. Vessel diameters, velocities and volumetric blood flow rates (VBF) in the retinal circulation were determined prior to and in 30-min intervals for 4-4.5h after injection. In addition, the basal amount of nNOS in the rat retina was quantified using a specific enzyme linked immunoassay (ELISA). Treatment with l-NAME and TRIM significantly decreased diameters and VBF. Compared with saline, treatment with l-NAME and TRIM produced a significant (p<0.001) decrease of approximately 12-17% in vessel diameters. Treatment with AAAN significantly decreased vessel diameters and venous VBF. Compared with saline AAAN produced a significant decrease of approximately 7% in arterial (p<0.001) and 5% in venous (p=0.011) diameters, respectively. The amount of nNOS in the rat retina was 0.17+/- 0.0147 pmol mg(-1) of dry retina. The results suggest that though inhibition of nNOS decreases basal diameters, constant VBF is maintained in the retinal circulation. PMID:19646435

Tummala, Shanti R; Benac, Sanja; Tran, Harry; Vankawala, Anand; Zayas-Santiago, Astrid; Appel, Alyssa; Kang Derwent, Jennifer J

2009-11-01

399

Triptan-induced enhancement of neuronal nitric oxide synthase in trigeminal ganglion dural afferents underlies increased responsiveness to potential migraine triggers.  

Science.gov (United States)

Migraine is a common neurological disorder often treated with triptans. Triptan overuse can lead to increased frequency of headache in some patients, a phenomenon termed medication overuse headache. Previous preclinical studies have demonstrated that repeated or sustained triptan administration for several days can elicit persistent neural adaptations in trigeminal ganglion cells innervating the dura, prominently characterized by increased labelling of neuronal profiles for calcitonin gene related peptide. Additionally, triptan administration elicited a behavioural syndrome of enhanced sensitivity to surrogate triggers of migraine that was maintained for weeks following discontinuation of drug, a phenomenon termed 'triptan-induced latent sensitization'. Here, we demonstrate that triptan administration elicits a long-lasting increase in identified rat trigeminal dural afferents labelled for neuronal nitric oxide synthase in the trigeminal ganglion. Cutaneous allodynia observed during the period of triptan administration was reversed by NXN-323, a selective inhibitor of neuronal nitric oxide synthase. Additionally, neuronal nitric oxide synthase inhibition prevented environmental stress-induced hypersensitivity in the post-triptan administration period. Co-administration of NXN-323 with sumatriptan over several days prevented the expression of allodynia and enhanced sensitivity to stress observed following latent sensitization, but not the triptan-induced increased labelling of neuronal nitric oxide synthase in dural afferents. Triptan administration thus promotes increased expression of neuronal nitric oxide synthase in dural afferents, which is critical for enhanced sensitivity to environmental stress. These data provide a biological basis for increased frequency of headache following triptans and highlight the potential clinical utility of neuronal nitric oxide synthase inhibition in preventing or treating medication overuse headache. PMID:20627971

De Felice, Milena; Ossipov, Michael H; Wang, Ruizhong; Dussor, Gregory; Lai, Josephine; Meng, Ian D; Chichorro, Juliana; Andrews, John S; Rakhit, Suman; Maddaford, Shawn; Dodick, David; Porreca, Frank

2010-08-01

400

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

Science.gov (United States)

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

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

2013-06-01