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

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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); pyraz...

Fabiano André Petter; Alan Mario Zuffo; Leandro Pereira Pacheco

2013-01-01

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

Science.gov (United States)

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

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

2005-01-01

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

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Bidens pilosa and Amaranthus quitensis are major weeds infesting soybean [Glycine max L (Merrill)] fields in Brazil and Argentina. The repetitive use of acetolactate synthase (ALS EC 4.1.3.18) inhibiting herbicides in São Gabriel do Oeste, MS, Brazil and in the provinces of Córdoba and Tucumã, Argentina, has selected for resistant (R) biotypes of these weeds. Research work was developed to study the management, growth, biochemistry, and genetics of these R weed biotypes. In a field experim...

2003-01-01

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

Science.gov (United States)

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

Muhitch, M J

1988-01-01

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

Science.gov (United States)

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

Aviv, Dvora; Amsellem, Ziva; Gressel, Jonathan

2002-12-01

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

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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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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.

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

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

International Nuclear Information System (INIS)

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

1991-01-01

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New Aspects on Inhibition of Plant Acetolactate Synthase by Chlorsulfuron and Imazaquin 1  

Science.gov (United States)

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

Durner, Jorg; Gailus, Valerie; Boger, Peter

1991-01-01

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

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

Durner, J.; Gailus, V.; Boeger, P. (Univ. Konstanz (West Germany))

1991-04-01

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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  

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

Plaza Guido A.

2006-12-01

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

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Bidens pilosa and Amaranthus quitensis are major weeds infesting soybean [Glycine max L (Merrill)] fields in Brazil and Argentina. The repetitive use of acetolactate synthase (ALS EC 4.1.3.18) inhibiting herbicides in São Gabriel do Oeste, MS, Brazil and in the provinces of Córdoba and Tucumã, Argentina, has selected for resistant (R) biotypes of these weeds. Research work was developed to study the management, growth, biochemistry, and genetics of these R weed biotypes. In a field experim...

Patrícia Andrea Monquero; Pedro Jacob Christoffoleti; Helaine Carrer

2003-01-01

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Lack of Cross-Resistance of Imazaquin-Resistant Xanthium strumarium Acetolactate Synthase to Flumetsulam and Chlorimuron.  

Science.gov (United States)

Acetolactate synthase (ALS) was isolated from a field population of cocklebur (Xanthium strumarium) that developed resistance to the herbicide Scepter following three consecutive years of application. The active ingredient of Scepter, imazaquin, gave an inhibitor concentration required to produce 50% inhibition of the enzyme activity that was more than 300 times greater for the resistant enzyme than for the wild-type cocklebur ALS. Tests with flumetsulam and chlorimuron show that the resistant ALS was not cross-resistant to these two other classes of ALS inhibitors.

Schmitzer, P. R.; Eilers, R. J.; Cseke, C.

1993-01-01

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Lack of Cross-Resistance of Imazaquin-Resistant Xanthium strumarium Acetolactate Synthase to Flumetsulam and Chlorimuron.  

Science.gov (United States)

Acetolactate synthase (ALS) was isolated from a field population of cocklebur (Xanthium strumarium) that developed resistance to the herbicide Scepter following three consecutive years of application. The active ingredient of Scepter, imazaquin, gave an inhibitor concentration required to produce 50% inhibition of the enzyme activity that was more than 300 times greater for the resistant enzyme than for the wild-type cocklebur ALS. Tests with flumetsulam and chlorimuron show that the resistant ALS was not cross-resistant to these two other classes of ALS inhibitors. PMID:12231935

Schmitzer, P. R.; Eilers, R. J.; Cseke, C.

1993-09-01

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

Scientific Electronic Library Online (English)

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

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

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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.

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

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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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Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.  

Science.gov (United States)

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that Km and Vmax did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F1, F2 and F3 generations. F1 hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F2 generation following chlorsulfuron application. A segregation ratio of 1?2?1 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F3 families, derived from intermediate F2 individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS. PMID:24169770

Boutsalis, P; Powles, S B

1995-07-01

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

Science.gov (United States)

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

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

2006-03-01

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

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

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

G.A. Gelmini

2002-08-01

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

Scientific Electronic Library Online (English)

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

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

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A double mutant allele, csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics.  

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A comparison is made of the kinetic characteristics of acetolactate synthase (EC 4.1.3.18) in extracts from Columbia wild type and four near-isogenic, herbicide-resistant mutants of Arabidopsis thaliana (L.) Heynh. The mutants used were the chlorsulfuron-resistant GH50 (csr1-1), the imazapyr-resistant GH90 (csr1-2), the triazolopyrimidine-resistant Tzp5 (csr1-3) and the multiherbicide-resistant, double mutant GM4.8 (csr1-4), derived from csr1-1 and csr1-2 by intragenic recombination (G. Mourad et al. 1994, Mol. Gen. Genet. 243, 178-184). Kmapp and Vmax values for the substrate pyruvate were unaffected by any of the mutations giving rise to herbicide resistance. Feedback inhibition by L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile) of acetolactate synthase extracted from wild type and mutants fitted a mixed competitive pattern most closely. Ki values for L-Val, L-Leu and L-Ile inhibition were not significantly different from wild type in extracts from csr1-1, csr1-2, and csr1-3. Ki values were significantly higher than wild type by two- and five-fold, respectively, for csr1-4 with L-Val and L-Leu but not L-Ile. GM4.8 (csr1-4) plants were also highly resistant in their growth to added L-Val and L-Leu.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7767237

Mourad, G; Williams, D; King, J

1995-01-01

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

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

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

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

2013-01-01

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

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

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

2013-12-01

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

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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.

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

30

Novel method to detect a construct-specific sequence of the acetolactate synthase gene in genetically-modified flax CDC Triffid (FP967).  

Science.gov (United States)

During the fall of 2009, a trace of unauthorized genetically modified (GM) flax (Linum usitatissimum L.) line, CDC Triffid, which is resistant to sulfonylurea herbicides, was detected in many countries including Japan. A method to reliably identify the CDC Triffid line was urgently required. We developed a novel construct-specific real-time polymerase chain reaction (PCR) method to identify the mutant acetolactate synthase gene in the CDC Triffid line. We confirmed that the method can detect 0.001% GM flax in DNA mixing solution. The study shows that the developed method is specific, sensitive and reliable way to monitor a trace of CDC Triffid. PMID:20190423

Nakamura, Kosuke; Akiyama, Hiroshi; Yamada, Chihiro; Satoh, Rie; Makiyama, Daiki; Sakata, Kozue; Kawakami, Hiroshi; Mano, Junichi; Kitta, Kazumi; Teshima, Reiko

2010-01-01

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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

32

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

Science.gov (United States)

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

Preston, C; Powles, S B

2002-01-01

33

Inhibitors of specific ceramide synthases.  

Science.gov (United States)

Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed. PMID:21945810

Schiffmann, Susanne; Hartmann, Daniela; Fuchs, Sina; Birod, Kerstin; Ferreiròs, Nerea; Schreiber, Yannick; Zivkovic, Aleksandra; Geisslinger, Gerd; Grösch, Sabine; Stark, Holger

2012-02-01

34

Pharmacological inhibitors of glycogen synthase kinase 3.  

Science.gov (United States)

Three closely related forms of glycogen synthase kinase 3 (GSK-3alpha, GSK-3beta and GSK-3beta2) have a major role in Wnt and Hedgehog signaling pathways and regulate the cell-division cycle, stem-cell renewal and differentiation, apoptosis, circadian rhythm, transcription and insulin action. A large body of evidence supports speculation that pharmacological inhibitors of GSK-3 could be used to treat several diseases, including Alzheimer's disease and other neurodegenerative diseases, bipolar affective disorder, diabetes, and diseases caused by unicellular parasites that express GSK-3 homologues. The toxicity, associated side-effects and concerns regarding the absorption, distribution, metabolism and excretion of these inhibitors affect their clinical potential. More than 30 inhibitors of GSK-3 have been identified. Seven of these have been co-crystallized with GSK-3beta and all localize within the ATP-binding pocket of the enzyme. GSK-3, as part of a multi-protein complex that contains proteins such as axin, presenilin and beta-catenin, contains many additional target sites for specific modulation of its activity. PMID:15559249

Meijer, Laurent; Flajolet, Marc; Greengard, Paul

2004-09-01

35

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

Scientific Electronic Library Online (English)

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

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

36

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

37

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

International Nuclear Information System (INIS)

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

1987-01-01

38

Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 poc...

Hevener, Kirk E.; Yun, Mi-kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephen W.; Lee, Richard E.

2010-01-01

39

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

Scientific Electronic Library Online (English)

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

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

40

Inhibitors of glycogen synthase 3 kinase  

Energy Technology Data Exchange (ETDEWEB)

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

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

2000-01-01

 
 
 
 
41

Antifolate inhibitors of thymidylate synthase as anticancer drugs.  

Science.gov (United States)

Inhibitors of thymidylate synthase (TS) play an essential role in the pharmacological management of several tumors. Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs, with Pemetrexed, unlike Raltitrexed, undergoing further intense clinical development. Other antifolate TS inhibitors, recently/currently tested in clinical studies, that show encouraging anticancer activities are Plevitrexed, GW7904L and Nolatrexed. A new prospect among antifolates, demonstrating a very desirable pattern of pharmacological properties, is BGC 945 that showed promising antitumor activities and has been nominated for clinical development. In this paper, apart from reviewing their biochemical and pharmacological properties, up-to-date characteristics of clinical development of all the mentioned agents are presented. In addition, trends and perspectives for developing improved antifolate inhibitors of TS and future drugs are discussed. Drug resistance is the main barrier to more effective treatment of cancers with antifolates; therefore, mechanisms of antifolate resistance and currently applied approaches to overcome it are also pointed out in the review. PMID:20854257

Jarmu?a, A

2010-11-01

42

Aldosterone synthase inhibitors in cardiovascular and renal diseases.  

Science.gov (United States)

Aldosterone is involved in various cardiovascular pathologies, including hypertension, heart failure, atherosclerosis and fibrosis. Mineralocorticoid receptor (MR)-dependent and -independent, genomic and non-genomic processes mediate its complex effects. Spironolactone and eplerenone, both MR antagonists, are the only commercially available compounds targeting directly the actions of aldosterone. However, due to the poor selectivity (spironolactone), low potency (eplerenone) and the fact that only MR-dependent effects of aldosterone can be inhibited, these drugs have limited clinical use. An attractive approach to abolish potentially all of aldosterone-mediated pathologies is the inhibition of aldosterone synthase. This review summarizes current knowledge on the complex effects mediated by aldosterone, potential advantages and disadvantages of aldosterone inhibition and novel directions in the development of aldosterone synthase inhibitors. PMID:24493871

Namsolleck, Pawel; Unger, Thomas

2014-02-01

43

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

Science.gov (United States)

The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors. PMID:24570839

Hargovan, Milan; Ferro, Albert

2014-01-01

44

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

45

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)

2012-05-01

46

Human hematopoietic prostaglandin D synthase inhibitor complex structures.  

Science.gov (United States)

In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH(2) in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD(2). We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 Å resolution. When complexed with H-PGDS, Cibacron Blue had an IC(50) value of 40 nM and APAS 2.1 ?M. The Cibacron Blue molecule was stabilized by four hydrogen bonds and ?-? stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH(2) substrate ?-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC(50) values obtained for the two inhibitors. PMID:22418579

Kado, Yuji; Aritake, Kosuke; Uodome, Nobuko; Okano, Yousuke; Okazaki, Nobuo; Matsumura, Hiroyoshi; Urade, Yoshihiro; Inoue, Tsuyoshi

2012-04-01

47

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.

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

48

Biochemistry: Acetohydroxyacid Synthase  

Directory of Open Access Journals (Sweden)

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

Pham Ngoc Chien

2010-02-01

49

Biochemistry: Acetohydroxyacid Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Pham Ngoc Chien

2010-01-01

50

Design, synthesis, and enzyme kinetics of novel benzimidazole and quinoxaline derivatives as methionine synthase inhibitors.  

Science.gov (United States)

Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyltetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radiometric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase (IC??: 20 ?M, 18 ?M, 9 ?M, respectively). Kinetic analysis of these compounds using Lineweaver-Burk plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j. Docking study into a homology model of the rat methionine synthase gave insights into the molecular determinants of the activity of this class of compounds. The identification of these drug-like inhibitors could lead the design of the next generation modulators of methionine synthase. PMID:24268539

Elshihawy, Hosam; Helal, Mohamed A; Said, Mohamed; Hammad, Mohamed A

2014-01-01

51

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

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In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this cat...

Vi?tec?ek, Jan; Lojek, Antoni?n; Valacchi, Giuseppe; Kubala, Luka?s?

2012-01-01

52

Potent, Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) vs. (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors we have introduced a CF2 moiety geminal to an amino group in the long tail of one of t...

Xue, Fengtian; Li, Huiying; Delker, Silvia L.; Fang, Jianguo; Marta?sek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2010-01-01

53

Acetohydroxyacid synthase: a target for antimicrobial drug discovery.  

Science.gov (United States)

Acetohydroxyacid synthase (AHAS) (EC 2.2.1.6) (also known as acetolactate synthase) is the first common enzyme in the branched chain amino acid (BCAA) biosynthesis pathway. This pathway is present in microorganisms and in plants but not in animals, making it an attractive target for both drug and herbicide discovery. The function of AHAS is to catalyze the conversion of two molecules of pyruvate to 2-acetolactate or to convert one molecule of pyruvate and a molecule of 2-ketobutyrate into 2-aceto-2- hydroxybutyrate. Three cofactors are required for the activity of AHAS: thiamine diphosphate (ThDP), Mg(2+) and flavin-adenine dinucleotide (FAD). AHAS is the target for several classes of commercial herbicides that include the sulfonylurea and imidazolinone families. These herbicides are potent and selective inhibitors of AHAS with Ki values that can be in the low nM range. Such compounds also exhibit low application rates as herbicides (typically ~3 g ha(-1)) and have low mammalian toxicity (LD50 values typically >4g/kg), thereby highlighting their utility and effectiveness as biocidal agents. However, somewhat surprisingly given the central importance of AHAS in the metabolism of microorganisms, no inhibitors of this enzyme have been commercialized into antimicrobial agents. Here we provide an overview of the biochemical characterization of AHASs from bacterial and fungal sources, analyse the structural features of these enzymes that are criticial to catalysis andprovide the current data on AHAS inhibitors that have potential to be developed into antimicrobial therapeutics. PMID:23688082

Pue, Nason; Guddat, Luke W

2014-01-01

54

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

55

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

56

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)

1986-05-01

57

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

Science.gov (United States)

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

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

2013-09-01

58

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

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

2000-01-01

59

The Role of Zinc in Isoform Selective Inhibitor Binding to Neuronal Nitric Oxide Synthase  

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In previous studies (Delker et al. (2010), J. Am. Chem. Soc. 132, 5437-5442) we determined the crystal structures of neuronal nitric oxide synthase (nNOS) complexed with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S, S)-cis inhibitors an unexpected “flipped” orientation was observed for...

2010-01-01

60

Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors.  

Science.gov (United States)

The present work reports the effects of caspofungin, a ?-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ?-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ?-glucan synthase inhibitor against this fungus. PMID:24614372

Fernandes, Chantal; Anjos, Jorge; Walker, Louise A; Silva, Branca M A; Cortes, Luísa; Mota, Marta; Munro, Carol A; Gow, Neil A R; Gonçalves, Teresa

2014-05-01

 
 
 
 
61

Human thromboxane synthase: comparative modeling and docking evaluation with the competitive inhibitors Dazoxiben and Ozagrel.  

Science.gov (United States)

Abstract Thromboxane synthase (TXAS) is a P450 epoxygenase that synthesizes thromboxane A2 (TXA2), a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. This enzyme plays an important role in several human diseases, including myocardial infarction, stroke, septic shock, asthma and cancer. Despite of the increasing interest on developing TXAS inhibitors, the structure and activity of TXAS are still not totally elucidated. In this study, we used a comparative molecular modeling approach to construct a reliable model of TXAS and analyze its interactions with Dazoxiben and Ozagrel, two competitive inhibitors. Our results were compatible with experimental published data, showing feasible cation-? interaction between the iron atom of the heme group of TXAS and the basic nitrogen atom of the imidazolyl group of those inhibitors. In the absence of the experimental structure of thromboxane synthase, this freely available model may be useful for designing new antiplatelet drugs for diseases related with TXA2. PMID:23914925

Sathler, Plínio Cunha; Santana, Marcos; Lourenço, André Luiz; Rodrigues, Carlos Rangel; Abreu, Paula; Cabral, Lúcio Mendes; Castro, Helena Carla

2014-08-01

62

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

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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...

1990-01-01

63

Fatty acid synthase inhibitor C75 ameliorates experimental colitis.  

Science.gov (United States)

Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic ?-methylene-?-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or dimethyl sulfoxide (DMSO) (vehicle) was administered intraperitoneally from d 2 to 6. Clinical parameters were monitored daily. Mice were euthanized on d 8 for histological evaluation and measurements of colon length, chemokine, cytokine and inflammatory mediator expression. C75 significantly reduced body weight loss from 23% to 15% on d 8, compared with the vehicle group. The fecal bleeding, diarrhea and colon histological damage scores in the C75-treated group were significantly lower than scores in the vehicle animals. Colon shortening was significantly improved after C75 treatment. C75 protected colon tissues from DSS-induced apoptosis by inhibiting caspase-3 activity. Macrophage inflammatory protein 2, keratinocyte-derived chemokine, myeloperoxidase activity and proinflammatory cytokines (tumor necrosis factor-?, interleukin [IL]-1? and IL-6) in the colon were significantly downregulated in the C75-treated group, compared with the vehicle group. Treatment with C75 in colitis mice inhibited the elevation of FASN, cyclooxygenase-2 and inducible nitric oxide synthase expression as well as I?B degradation in colon tissues. C75 administration alleviates the severity of colon damage and inhibits the activation of inflammatory pathways in DSS-induced colitis. Thus, inhibition of FASN may represent an attractive therapeutic potential for treating IBD. PMID:24306512

Matsuo, Shingo; Yang, Weng-Lang; Aziz, Monowar; Kameoka, Shingo; Wang, Ping

2014-01-01

64

Serotonergic mediation of the antidepressant-like effects of nitric oxide synthase inhibitors.  

Science.gov (United States)

Recent studies indicate that nitric oxide (NO) synthase inhibitors have antidepressant-like potential in various animal models. In the present study the behavioural activity of the NO synthase inhibitors, N(G)-nitro-L-arginine (L-NA) and 7-nitroindazole (7-NI), were assessed in a modified rat forced swimming test (FST). Both L-NA and 7-NI, dose dependently reduced immobility and increased swimming behaviour in the rat FST. This behavioural profile parallels the one previously shown with selective serotonin re-uptake inhibitors and serotonergic agonists. Thus, we examined the role of serotonin mediating the behavioural effects of L-NA and 7-NI in the rat FST. Depletion of endogenous serotonin using para-chlorophenylalanine (pCPA; 3 x 150 mg/kg, i.p.) completely blocked L-NA (20 mg/kg, i.p.) and 7-NI (20 mg/kg, i.p.)-induced reductions in immobility and increases in swimming behaviour during the FST. In conclusion these observations suggest that NO synthase inhibitors elicit their antidepressant-like activity in the modified swimming test through a serotonin dependent mechanism. PMID:12668047

Harkin, A; Connor, T J; Walsh, M; St John, N; Kelly, J P

2003-04-01

65

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

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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

66

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

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Introduction: Increase in the nitric oxide in the spermatic veins of men by varicocele has been reported. Although Several studies have considered the relationship between varicocele and semen NO concentrations, no study on the effects of nitric oxide synthase inhibitor (L-NAME) on epididymal sperm count, motility and morphology which are important in fertility of the individual has been reported. The aim of study was to evaluate the effects of nitric oxide synthase inhibitor (L-NAME) on epid...

Bahmanzadeh M; Abolhassani F.; Amidi F.; Ejtemaiemehr Sh.; Salehi M.; Abbasi M.

2008-01-01

67

Analogues of 2-aminopyridine-based selective inhibitors of neuronal nitric oxide synthase with increased bioavailability  

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Overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) has been linked to several neurodegenerative diseases. We have recently designed potent and isoform selective inhibitors of nNOS, but the lead compound contains several basic functional groups. A large number of charges and hydrogen bond donors can impede the ability of molecules to cross the blood brain barrier and thereby limit the effectiveness of potential neurological therapeutics. Replacement of secondary amines in ...

2009-01-01

68

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

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

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

2008-01-01

69

Virtual screening reveals allosteric inhibitors of the Toxoplasma gondii thymidylate synthase-dihydrofolate reductase.  

Science.gov (United States)

The parasite Toxoplasma gondii can lead to toxoplasmosis in those who are immunocompromised. To combat the infection, the enzyme responsible for nucleotide synthesis thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a suitable drug target. We have used virtual screening to determine novel allosteric inhibitors at the interface between the two TS domains. Selected compounds from virtual screening inhibited TS activity. Thus, these results show that allosteric inhibition by small drug-like molecules can occur in T. gondii TS-DHFR and pave the way for new and potent species-specific inhibitors. PMID:24440298

Sharma, Hitesh; Landau, Mark J; Sullivan, Todd J; Kumar, Vidya P; Dahlgren, Markus K; Jorgensen, William L; Anderson, Karen S

2014-02-15

70

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

Scientific Electronic Library Online (English)

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

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

71

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase.  

Science.gov (United States)

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase. PMID:24726306

Zhou, Xiang; Ferree, Sarah D; Wills, Veronica S; Born, Ella J; Tong, Huaxiang; Wiemer, David F; Holstein, Sarah A

2014-05-01

72

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

Science.gov (United States)

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

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

2013-10-01

73

Heme-Coordinating Inhibitors of Neuronal Nitric Oxide Synthase. Iron-Thioether Coordination is Stabilized by Hydrophobic Contacts Without Increased Inhibitor Potency  

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The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge...

Martell, Jeffrey D.; Li, Huiying; Doukov, Tzanko; Marta?sek, Pavel; Roman, Linda J.; Soltis, Michael; Poulos, Thomas L.; Silverman, Richard B.

2010-01-01

74

Anmindenols A and B, Inducible Nitric Oxide Synthase Inhibitors from a Marine-Derived Streptomyces sp.  

Science.gov (United States)

Anmindenols A (1) and B (2), inhibitors of inducible nitric oxide synthase (iNOS), were isolated from a marine-derived bacterium Streptomyces sp. Their chemical structures were elucidated by interpreting various spectroscopic data, including IR, MS, and NMR. Anmindenols A and B are sesquiterpenoids possessing an indene moiety with five- and six-membered rings derived from isoprenyl units. The absolute configuration of C-4 in anmindenol B was determined by electronic circular dichroism (ECD) of a dimolybdenum complex. Anmindenols A (1) and B (2) inhibited nitric oxide production in stimulated RAW 264.7 macrophage cells with IC50 values of 23 and 19 ?M, respectively. PMID:24878306

Lee, Jihye; Kim, Hiyoung; Lee, Tae Gu; Yang, Inho; Won, Dong Hwan; Choi, Hyukjae; Nam, Sang-Jip; Kang, Heonjoong

2014-06-27

75

Synthesis and biological evaluation of nonsymmetrical aromatic disulfides as novel inhibitors of acetohydroxyacid synthase.  

Science.gov (United States)

46 Novel nonsymmetrical aromatic disulfides containing [1,3,4]thiadiazole or [1,3,4]oxadiazole groups were synthesized and their biological activities were evaluated as inhibitors of acetohydroxyacid synthase (AHAS, EC 2.2.1.6). Besides their strong in vitro inhibition against plant AHAS, compounds 3e and 3f also display 80-100% post-emergence herbicidal activities in greenhouse bioassay at 1500g /ha dosage. The assay of exogenous branched-chain amino acids supplementation on rape root growth of 3e suggests that the herbicidal activity has relationship with AHAS inhibition. PMID:23726033

Li, Zai-Shun; Wang, Wei-Min; Lu, Wei; Niu, Cong-Wei; Li, Yong-Hong; Li, Zheng-Ming; Wang, Jian-Guo

2013-07-01

76

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in R...

Panda, Koustubh; Chawla-sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C.; Parkinson, John F.; Stuehr, Dennis J.

2005-01-01

77

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

Science.gov (United States)

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation. PMID:18849972

Garcin, Elsa D; Arvai, Andrew S; Rosenfeld, Robin J; Kroeger, Matt D; Crane, Brian R; Andersson, Gunilla; Andrews, Glen; Hamley, Peter J; Mallinder, Philip R; Nicholls, David J; St-Gallay, Stephen A; Tinker, Alan C; Gensmantel, Nigel P; Mete, Antonio; Cheshire, David R; Connolly, Stephen; Stuehr, Dennis J; Aberg, Anders; Wallace, Alan V; Tainer, John A; Getzoff, Elizabeth D

2008-11-01

78

Identification of potent and selective glucosylceramide synthase inhibitors from a library of N-alkylated iminosugars.  

Science.gov (United States)

Glucosylceramide synthase (GCS) is an important target for clinical drug development for the treatment of lysosomal storage disorders and a promising target for combating type 2 diabetes. Iminosugars are useful leads for the development of GCS inhibitors; however, the effective iminosugar type GCS inhibitors reported have some unwanted cross-reactivity toward other glyco-processing enzymes. In particular, iminosugar type GCS inhibitors often also inhibit to some extent human acid glucosylceramidase (GBA1) and the nonlysosomal glucosylceramidase (GBA2), the two enzymes known to process glucosylceramide. Of these, GBA1 itself is a potential drug target for the treatment of the lysosomal storage disorder, Gaucher disease, and selective GBA1 inhibitors are sought after as potential chemical chaperones. The physiological importance of GBA2 in glucosylceramide processing in relation to disease states is less clear, and here, selective inhibitors can be of use as chemical knockout entities. In this communication, we report our identification of a highly potent and selective N-alkylated l-ido-configured iminosugar. In particular, the selectivity of 27 for GCS over GBA1 is striking. PMID:24900289

Ghisaidoobe, Amar; Bikker, Pieter; de Bruijn, Arjan C J; Godschalk, Frithjof D; Rogaar, Eva; Guijt, Marieke C; Hagens, Peter; Halma, Jerre M; Van't Hart, Steven M; Luitjens, Stijn B; van Rixel, Vincent H S; Wijzenbroek, Mark; Zweegers, Thor; Donker-Koopman, Wilma E; Strijland, Anneke; Boot, Rolf; van der Marel, Gijs; Overkleeft, Herman S; Aerts, Johannes M F G; van den Berg, Richard J B H N

2011-02-10

79

Identification of Potent and Selective Glucosylceramide Synthase Inhibitors from a Library of N-Alkylated Iminosugars  

Science.gov (United States)

Glucosylceramide synthase (GCS) is an important target for clinical drug development for the treatment of lysosomal storage disorders and a promising target for combating type 2 diabetes. Iminosugars are useful leads for the development of GCS inhibitors; however, the effective iminosugar type GCS inhibitors reported have some unwanted cross-reactivity toward other glyco-processing enzymes. In particular, iminosugar type GCS inhibitors often also inhibit to some extent human acid glucosylceramidase (GBA1) and the nonlysosomal glucosylceramidase (GBA2), the two enzymes known to process glucosylceramide. Of these, GBA1 itself is a potential drug target for the treatment of the lysosomal storage disorder, Gaucher disease, and selective GBA1 inhibitors are sought after as potential chemical chaperones. The physiological importance of GBA2 in glucosylceramide processing in relation to disease states is less clear, and here, selective inhibitors can be of use as chemical knockout entities. In this communication, we report our identification of a highly potent and selective N-alkylated l-ido-configured iminosugar. In particular, the selectivity of 27 for GCS over GBA1 is striking.

2010-01-01

80

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.  

Science.gov (United States)

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

Begley, Darren W; Edwards, Thomas E; Raymond, Amy C; Smith, Eric R; Hartley, Robert C; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D; Myler, Peter J; Staker, Bart L; Stewart, Lance J

2011-09-01

 
 
 
 
81

Recent advances toward improving the bioavailability of neuronal nitric oxide synthase inhibitors.  

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Overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) has been highly correlated with numerous neurodegenerative diseases and stroke. Given its role in human diseases, nNOS is an important target for therapy that deserves further attention. During the last decade, a large number of organic scaffolds have been investigated to develop selective nNOS inhibitors, resulting in two principal classes of compounds, 2-aminopyridines and thiophene-2- carboximidamides. The former compounds were investigated in detail by our group, exhibiting great potency and excellent selectivity; however, they suffer from poor bioavailability, which hampers their therapeutic potential. Here we present a review of various strategies adopted by our group to improve the bioavailability of 2-aminopyridine derivatives and describe recent advances in thiophene-2-carboximidamide based nNOS-selective inhibitors, which exhibit promising pharmacological profiles. PMID:23578024

Huang, He; Silverman, Richard B

2013-01-01

82

[Hematopoietic prostaglandin D synthase inhibitors for the treatment of duchenne muscular dystrophy].  

Science.gov (United States)

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle disease, characterized by progressive skeletal muscle atrophy and weakness. DMD is caused by mutations in the dystrophin gene, which encodes for the cytoskeletal protein dystrophin. DMD is one of the most common types of muscular dystrophies, affecting approximately 1 in 3,500 boys. There is no complete cure for this disease. Clinical trials for gene transfer therapy as a treatment for DMD have been performed but mainly in animal models. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) was found to be induced in grouped necrotic muscle fibers of DMD patients and animal models, mdx mice, and DMD dogs. We found an orally active H-PGDS inhibitor (HQL-79) and determined the 3D structure of the inhibitor-human H-PGDS complex by X-ray crystallography. Oral administration of HQL-79 markedly suppressed prostaglandin D2 (PGD2) production, reduced necrotic muscle volume, and improved muscle strength in mdx dystrophic mice. Based on the high-resolution 3D structures of the inhibitor-H-PGDS complex, we designed alternative H-PGDS inhibitors, which were 100- to 3000-times more potent than HQL-79, as assessed by in vitro and in vivo analyses. We used these novel inhibitors for the treatment of DMD dogs and confirmed that oral administration of these inhibitors prevented skeletal muscle atrophy and weakness by decreasing PGD2 production. These results indicate that PGD2, synthesized by H-PGDS, is involved in the expansion of muscle necrosis in DMD. Thus, inhibition of H-PGDS by using inhibitors is a novel therapy for DMD. PMID:22068479

Kamauchi, Shinya; Urade, Yoshihiro

2011-11-01

83

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

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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

84

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

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

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

2011-03-01

85

Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase  

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The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurologica...

Endres, Matthias; Laufs, Ulrich; Huang, Zhihong; Nakamura, Tadashi; Huang, Paul; Moskowitz, Michael A.; Liao, James K.

1998-01-01

86

In silico design, synthesis, and screening of novel deoxyhypusine synthase inhibitors targeting HIV-1 replication.  

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The human enzyme deoxyhypusine synthase (DHS) is an important host cell factor that participates in the post-translational hypusine modification of eukaryotic initiation factor 5A (eIF-5A). Hypusine-modified eIF-5A plays a role in a number of diseases, including HIV infection/AIDS. Thus, DHS represents a novel and attractive drug target. So far, four crystal structures are available, and various substances have been tested for inhibition of human DHS. Among these inhibitors, N-1-guanyl-1,7-diaminoheptane (GC7) has been co-crystallized in the active site of DHS. However, despite its potency, GC7 is not selective enough to be used in drug applications. Therefore, new compounds that target DHS are needed. Herein we report the in silico design, chemical synthesis, and biological evaluation of new DHS inhibitors. One of these inhibitors showed dose-dependent inhibition of DHS in vitro, as well as suppression of HIV replication in cell cultures. Furthermore, the compound exhibited no cytotoxic effects at active concentrations. Thus, this designed compound demonstrated proof of principle and represents a promising starting point for the development of new drug candidates to specifically interfere with DHS activity. PMID:24616161

Schroeder, Marcus; Kolodzik, Adrian; Pfaff, Katharina; Priyadarshini, Poornima; Krepstakies, Marcel; Hauber, Joachim; Rarey, Matthias; Meier, Chris

2014-05-01

87

Thiolactomycin-based ?-Ketoacyl-AcpM Synthase A (KasA) Inhibitors  

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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.

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

2013-01-01

88

Phenanthrenoids from Juncus acutus L., new natural lipopolysaccharide-inducible nitric oxide synthase inhibitors.  

Science.gov (United States)

The novel natural product juncutol (1), 1,4,7-trimethyl-8,9-dihydro-4H-cyclopenta[def]phenanthrene-2,6-diol, along with the three related metabolites juncusol (2), dehydrojuncusol (3), and 6-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene-2-ol (4), were isolated from the rhizomes of Juncus acutus L. (Juncaceae) growing in Egypt. The structural identity of 1 was determined on the basis of spectroscopic analyses, including 2D NMR spectroscopy. The inhibitory effect of these natural products on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide-stimulated RAW264.7 macrophage cells was determined for the first time. The unprecedented symmetrical compound juncutol (1) was found to be the most potent inhibitor against the induction of the proinflammatory iNOS protein. PMID:17666857

Behery, Fathi Abdelmohsen Abdelhalim; Naeem, Zain Elabdin Metwally; Maatooq, Galal Taha; Amer, Mohamed Mahmoud Abdelfattah; Wen, Zhi-Hong; Sheu, Jyh-Horng; Ahmed, Atallah Fouad

2007-08-01

89

Differential Activity of NO Synthase Inhibitors as Chemopreventive Agents in a Primary Rat Tracheal Epithelial Cell Transformation System1  

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A model to study the effectiveness of potential chemopreventive agents that inhibit neoplastic process by different mechanisms has been used to test the efficacy of seven nitric oxide synthase (NOS) inhibitors. Five selective inducible NOS (iNOS) inhibitors: S-methyl isothiourea (S-MITU), S-2-aminoethyl isothiourea (S-2-AEITU), S-ethyl isothiourea (S-EITU), aminoguanidine (AG), 2-amino-4-methyl pyridine (2-AMP), and two non selective general NOS inhibitors: l-N6-(1-iminoethyl) lysine (IEL) an...

Sharma, Sheela; Wilkinson, Betty P.; Gao, Pu; Steele, Vernon E.

2002-01-01

90

2-Alkylaminoethyl-1,1-Bisphosphonic Acids Are Potent Inhibitors of the Enzymatic Activity of Trypanosoma cruzi Squalene Synthase  

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As part of our efforts aimed at searching for new antiparasitic agents, the effect of representative 2-alkylaminoethyl-1,1-bisphosphonic acids on Trypanosoma cruzi squalene synthase (TcSQS) was investigated. These compounds had proven to be potent inhibitors of T. cruzi. This cellular activity had been associated with an inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase. 2-Alkylaminoethyl-1,1-bisphosphonic acids appear to have a dual action, since they also inhibit TcSQS at the nanomolar range.

Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Szajnman, Sergio H.

2012-01-01

91

Endogenous nitric-oxide synthase inhibitor ADMA after acute brain injury.  

Science.gov (United States)

Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment. PMID:24663083

Jung, Carla S; Wispel, Christian; Zweckberger, Klaus; Beynon, Christopher; Hertle, Daniel; Sakowitz, Oliver W; Unterberg, Andreas W

2014-01-01

92

Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury  

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

Carla S. Jung

2014-03-01

93

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

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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

94

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

95

Cyclooxygenase competitive inhibitors alter tyrosyl radical dynamics in prostaglandin H synthase-2.  

Science.gov (United States)

Reaction of prostaglandin H synthase (PGHS) isoforms 1 or 2 with peroxide forms a radical at Tyr385 that is required for cyclooxygenase catalysis and another radical at Tyr504, whose function is unknown. Both tyrosyl radicals are transient and rapidly dissipated by reductants, suggesting that cyclooxygenase catalysis might be vulnerable to suppression by intracellular antioxidants. Our initial hypothesis was that the two radicals are in equilibrium and that their proportions and stability are altered upon binding of fatty acid substrate. As a test, we examined the effects of three competitive inhibitors (nimesulide, flurbiprofen, and diclofenac) on the proportions and stability of the two radicals in PGHS-2 pretreated with peroxide. Adding nimesulide after ethyl peroxide led to some narrowing of the tyrosyl radical signal detected by EPR spectroscopy, consistent with a small increase in the proportion of the Tyr504 radical. Neither flurbiprofen nor diclofenac changed the EPR line width when added after peroxide. In contrast, the effects of cyclooxygenase inhibitors on the stability of the preformed tyrosyl radicals were dramatic. The half-life of total tyrosyl radical was 4.1 min in the control, >10 h with added nimesulide, 48 min with flurbiprofen, and 0.8 min with diclofenac. Stabilization of the tyrosyl radicals was evident even at substoichiometric levels of nimesulide. Thus, the inhibitors had potent, structure-dependent, effects on the stability of both tyrosyl radicals. This dramatic modulation of tyrosyl radical stability by cyclooxygenase site ligands suggests a mechanism for regulating the reactivity of PGHS tyrosyl radicals with cellular antioxidants. PMID:19894761

Wu, Gang; Tsai, Ah-Lim; Kulmacz, Richard J

2009-12-22

96

Syntheses and herbicidal activity of new triazolopyrimidine-2-sulfonamides as acetohydroxyacid synthase inhibitor.  

Science.gov (United States)

The triazolopyrimidine-2-sulfonanilide, discovered from preparing bioisosteres of the sulfonylurea herbicides, is an important class of acetohydroxyacid synthase (AHAS, EC 4.1.3.18) inhibitors. At least over ten triazolopyrimidine sulfonanilides have been commercialized as herbicides for the control of broadleaf weeds and grass with cereal crop selectivity. Herein, a series of triazolopyrimidine-2-sulfonanilides were designed and synthesized with the aim of discovery of new herbicides with higher activity. The assay results of the inhibition activity of the synthesized compounds against Arabidopsis thatiana AHAS indicated that some compounds showed a little higher activity against flumetsulam (FS), the first commercial triazolopyrimidine-2-sulfonanilide-type herbicide. The ki values of two promising compounds 3d and 8h are respectively, 1.61 and 1.29 microM, while that of FS is 1.85 microM. Computational simulation results indicated the ester group of compound 3d formed hydrogen bonds with the surrounding residues Arg'198 and Ser653, which accounts for its 11.5-folds higher AHAS inhibition activity than Y6610. Further green house assay showed that compound 3d has comparable herbicidal activity as FS. Even at the concentration of 37.5g.ai/ha, 3d showed excellent herbicidal activity against Galium aparine, Cerastium arvense, Chenopodium album, Amaranthus retroflexus, and Rmumex acetasa, moderate herbicidal activity against Polygonum humifusum, Cyperus iria, and Eclipta prostrate. The combination of in vitro and in vivo assay indicated that 3d could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study. PMID:20598554

Chen, Chao-Nan; Chen, Qiong; Liu, Yu-Chao; Zhu, Xiao-Lei; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

2010-07-15

97

Synthesis and evaluation of tetrahedral intermediate mimic inhibitors of 3-deoxy-d-manno-octulosonate 8-phosphate synthase.  

Science.gov (United States)

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the first committed step in the biosynthesis of 3-deoxy-d-manno-octulosonate (KDO), an important component of the lipopolysaccharide of Gram-negative bacteria. The pathway for KDO biosynthesis has been identified as a potential target of antibacterial drug design. The reaction catalysed by KDO8P synthase is an aldol-like condensation between phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A5P) and proceeds through a bisphosphorylated tetrahedral intermediate. In this study a bisphosphate analogue of the tetrahedral intermediate was synthesised and was found to inhibit the metal-dependent KDO8P synthase from Neisseriameningitidis and the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans with inhibition constants in the low micromolar range. Additionally, monophosphorylated inhibitors were synthesised to determine the relative importance of the two phosphate groups of this bisphosphate analogue for enzyme inhibition. The removal of either of these two phosphate groups gave less potent inhibitors for both enzymes. PMID:22204912

Harrison, Aidan N; Reichau, Sebastian; Parker, Emily J

2012-01-15

98

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

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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

99

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

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Highly potent and selective inhibitors of neuronal nitric oxide synthase (nNOS) possessing a 2-aminopyridine group were recently designed and synthesized in our laboratory and were shown to have significant in vivo efficacy. In this work, analogs of our lead compound possessing 2- and 4-aminothiazole rings in place of the aminopyridine were synthesized. The less basic aminothiazole rings will be less protonated at physiological pH than the aminopyridine ring, and so the molecule will carry a ...

2009-01-01

100

Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine.  

Science.gov (United States)

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP. PMID:10772721

Gao, W Y; Johns, D G; Mitsuya, H

2000-01-01

 
 
 
 
101

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

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

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

1998-01-01

102

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels  

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Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhib...

Giovannetti, E.; Backus, H. H. J.; Wouters, D.; Ferreira, C. G.; Houten, V. M. M.; Brakenhoff, R. H.; Poupon, M-f; Azzarello, A.; Pinedo, H. M.; Peters, G. J.

2007-01-01

103

Fatty Acid Synthase Inhibitors Modulate Energy Balance via Mammalian Target of Rapamycin Complex 1 Signaling in the Central Nervous System  

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OBJECTIVE—Evidence links the hypothalamic fatty acid synthase (FAS) pathway to the regulation of food intake and body weight. This includes pharmacological inhibitors that potently reduce feeding and body weight. The mammalian target of rapamycin (mTOR) is an intracellular fuel sensor whose activity in the hypothalamus is also linked to the regulation of energy balance. The purpose of these experiments was to determine whether hypothalamic mTOR complex 1 (mTORC1) signaling is involved in me...

Proulx, Karine; Cota, Daniela; Woods, Stephen C.; Seeley, Randy J.

2008-01-01

104

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor.  

Science.gov (United States)

AG-331 (N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino- benz[cd]indole glucuronate) is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects of purine incorporation. In addition, H35 cells treated with 10 microM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breast-cancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated. PMID:8004755

O'Connor, B M; Webber, S; Jackson, R C; Galivan, J; Rhee, M S

1994-01-01

105

Zymosan suppresses leukotriene C? synthase activity in differentiating monocytes: antagonism by aspirin and protein kinase inhibitors.  

Science.gov (United States)

Cysteinyl leukotrienes (cysLTs) are potent proinflammatory mediators with particular relevance for asthma. However, control of cysLT biosynthesis in the time period after onset of acute inflammation has not been extensively studied. As a model for later phases of inflammation, we investigated regulation of leukotriene (LT) C(4) synthase (LTC(4)S) in differentiating monocytes, exposed for several days to fungal zymosan. Incubations with LTA(4) revealed 20-fold increased LTC(4)S activity during differentiation of monocytic Mono Mac 6 (MM6) cells, which was reduced by 80% in the presence of zymosan (25 ?g/ml, 96 h). Zymosan (48 h) similarly attenuated LTC(4)S activity of primary human monocyte-derived macrophages and dendritic cells. Several findings indicate phosphoregulation of LTC(4)S: increased activity during MM6 cell differentiation correlated with reduced phosphorylation of 70-kDa ribosomal protein S6 kinase (p70S6K), which could phosphorylate purified LTC(4)S; the p70S6K inhibitor rapamycin (20 nM) doubled LTC(4)S activity of undifferentiated MM6 cells, and protein kinase A and C inhibitors (H-89, CGP-53353, and staurosporine) reversed the zymosan-induced suppression of LTC(4)S activity. Finally, zymosan (48 h) up-regulated PGE(2) biosynthesis, and aspirin (10 ?M) or prostaglandin E(2) (PGE(2)) receptor antagonists counteracted the zymosan effect. Our results suggest a late PGE(2)-mediated phosphoregulation of LTC(4)S during microbial exposure, which may contribute to resolution of inflammation, with implications for aspirin hypersensitivity. PMID:21228223

Esser, Julia; Gehrmann, Ulf; Salvado, M Dolores; Wetterholm, Anders; Haeggström, Jesper Z; Samuelsson, Bengt; Gabrielsson, Susanne; Scheynius, Annika; Rådmark, Olof

2011-04-01

106

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

107

The Interaction of Hydroxymandelate Synthase with the 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor: NTBC.  

Science.gov (United States)

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of polycyclic non-ribosomal peptide antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O(2), and also conducts a dioxygenation reaction. The difference between the two lies in the insertion of the second oxygen atom, HMS directing this atom onto the benzylic carbon of the substrate while HPPD hydroxylates the aromatic C1 carbon. We have shown that HMS will bind NTBC, a herbicide/therapeutic whose mode of action is based on the inhibition of HPPD. This occurs despite the difference in residues at the active site of HMS from those known to contact the inhibitor in HPPD. Moreover, the minimal kinetic mechanism for association of NTBC to HMS differs only slightly from that observed with HPPD. The primary difference is that three charge-transfer species are observed to accumulate during association. The first reversible complex forms with a weak dissociation constant of 520 microM, the subsequent two charge-transfer complexes form with rate constants of 2.7 s(-1) and 0.67 s(-1). As was the case for HPPD, the final complex has the most intense charge-transfer, is not observed to dissociate, and is unreactive towards dioxygen. PMID:18496607

Conrad, John A; Moran, Graham R

2008-03-01

108

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg [...] /kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

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

109

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

Directory of Open Access Journals (Sweden)

Full Text Available It has been demonstrated that nitric oxide (NO has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight, a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g and rats with fever induced by lipopolysaccharide (LPS (100 µg/kg body weight administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02. The coinjection of LPS and 7-NI was followed by a significant (P<0.02 hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

Perotti C.A.A.

1999-01-01

110

Discovery of a compound that acts as a bacterial PyrG (CTP synthase) inhibitor.  

Science.gov (United States)

PyrG (CTP synthase) catalyses the conversion of UTP to CTP, an essential step in the pyrimidine metabolic pathway in a variety of bacteria, including those causing community-acquired respiratory tract infections (RTIs). In this study, a luminescence-based ATPase assay of PyrG was developed and used to evaluate the inhibitory activity of 2-(3-[3-oxo-1,2-benzisothiazol-2(3H)-yl]phenylsulfonylamino) benzoic acid (compound G1). Compound G1 inhibited PyrG derived from Streptococcus pneumoniae with a 50?% inhibitory concentration value of 0.091 µM, and the inhibitory activity of compound G1 was 13 times higher than that of acivicin (1.2 µM), an established PyrG inhibitor. The results of saturation transfer difference analysis using nuclear magnetic resonance spectroscopy suggested that these compounds compete with ATP and/or UTP for binding to Strep. pneumoniae PyrG. Finally, compound G1 was shown to have antimicrobial activity against several different bacteria causing RTIs, such as Staphylococcus aureus and Haemophilus influenzae, suggesting that it is a prototype chemical compound that could be harnessed as an antimicrobial drug with a novel structure to target bacterial PyrG. PMID:22700553

Yoshida, Tatsuhiko; Nasu, Hatsumi; Namba, Eiko; Ubukata, Osamu; Yamashita, Makoto

2012-09-01

111

[Biological and pathophysiological roles of endogenous methylarginines as inhibitors of nitric oxide synthase].  

Science.gov (United States)

Protein arginine N-methyltransferases (PRMTs) catalyse the methylation of guanidinonitrogen(s) of arginine to produce NG-monomethyl-L-arginine (L-NMMA), asymmetric NG,NG-dimethyl-L-arginine (ADMA) and symmetric NG,NG-dimethyl-L-arginine (SDMA), which are subsequently released into the cytoplasm following proteolysis. Free intracellular L-NMMA and ADMA, but not SDMA, are inhibitors of all three isoforms of nitric oxide synthases (nNOS, eNOS and iNOS). L-NMMA and ADMA, but not SDMA, are actively metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and methylamine (and dimethylamine). Free methylarginines are detectable in cell cytosol, plasma and tissues. Elevated ADMA has been detected in the plasma of patients or experimental animals with hypercholesterolemia, renal failure, atherosclerosis, hypertension, thrombotic microangiopathy, peripheral arterial occlusive disease and in the regenerated endothelial cells after angioplasty. Moreover, in the non-cardiovascular field, ADMA was increased in the urethral tissue following ischemia and in the plasma of patients with schizophrenia and multiple sclerosis. Altered biosynthesis of NO has been implicated in the pathogenesis of these diseases, and it is possible to consider that the accumulation of endogenous L-NMMA and ADMA underlies the impaired NO generation and increased O2- production. We described herein the biosynthesis, transmembrane transport, metabolic pathway and possible pathophysiological roles of endogenous methylarginines. PMID:11862754

Masuda, Hitoshi; Azuma, Hiroshi

2002-01-01

112

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

Science.gov (United States)

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

Cross, Penelope J; Parker, Emily J

2013-09-17

113

Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors.  

Science.gov (United States)

Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of l-[(3) H]-citrulline produced from l -[(3) H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of l-citrulline with o-phthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay. PMID:24687974

Maccallini, Cristina; Di Matteo, Mauro; Ammazzalorso, Alessandra; D'Angelo, Alessandra; De Filippis, Barbara; Di Silvestre, Sara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Amoroso, Rosa

2014-06-01

114

Indirubin core structure of glycogen synthase kinase-3 inhibitors as novel chemotype for intervention with 5-lipoxygenase.  

Science.gov (United States)

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 ?M), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention. PMID:24697244

Pergola, Carlo; Gaboriaud-Kolar, Nicolas; Jestädt, Nadine; König, Stefanie; Kritsanida, Marina; Schaible, Anja M; Li, Haokun; Garscha, Ulrike; Weinigel, Christina; Barz, Dagmar; Albring, Kai F; Huber, Otmar; Skaltsounis, Alexios L; Werz, Oliver

2014-05-01

115

Design of selective neuronal nitric oxide synthase inhibitors for the prevention and treatment of neurodegenerative diseases.  

Science.gov (United States)

Nitric oxide (NO), which is produced from L-arginine by the nitric oxide synthase (NOS) family of enzymes, is an important second-messenger molecule that regulates several physiological functions. In endothelial cells, it relaxes smooth muscle, which decreases blood pressure. Macrophage cells produce NO as an immune defense system to destroy pathogens and microorganisms. In neuronal cells, NO controls the release of neurotransmitters and is involved in synaptogenesis, synaptic plasticity, memory function, and neuroendocrine secretion. NO is a free radical that is commonly thought to contribute to oxidative damage and molecule and tissue destruction, and thus it is somewhat surprising that it has so many significant beneficial physiological effects. However, the cell is generally protected from NO's toxic effects, except under certain pathological conditions in which excessive NO is produced. In that case, tissue damage and oxidative stress can result, leading to a wide variety of diseases, including rheumatoid arthritis, Alzheimer's disease, and Parkinson's disease, among others. In this Account, we describe research aimed at identifying small molecules that can selectively inhibit only the neuronal isozyme of NOS, nNOS. By targeting only nNOS, we attained the beneficial effects of lowering excess NO in the brain without the detrimental effects of inhibition of the two isozymes found elsewhere in the body (eNOS and iNOS). Initially, in pursuit of this goal, we sought to identify differences in the second sphere of amino acids in the active site of the isozymes. From this study, the first class of dual nNOS-selective inhibitors was identified. The moieties important for selectivity in the best lead compound were determined by structure modification. Enhancement provided highly potent, nNOS-selective dipeptide amides and peptidomimetics, which were active in a rabbit model for fetal neurodegeneration. Crystal structures of these compounds bound to NOS isozymes showed a one-amino-acid difference between nNOS and eNOS in the second sphere of amino acids; this was the difference that we were searching for from the beginning of this project. With the aid of these crystal structures, we developed a new fragment-based de novo design method called "fragment hopping", which allowed the design of a new class of nonpeptide nNOS-selective inhibitors. These compounds were modified to give low nanomolar, highly dual-selective nNOS inhibitors, which we recently showed are active in a rabbit model for the prevention of neurobehavioral symptoms of cerebral palsy. These compounds could also have general application in other neurodegenerative diseases for which excess NO is responsible. PMID:19154146

Silverman, Richard B

2009-03-17

116

Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus.  

Science.gov (United States)

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively. PMID:12441645

Senda, Mineo; Kasai, Atsushi; Yumoto, Setsuzo; Akada, Shinji; Ishikawa, Ryuji; Harada, Takeo; Niizeki, Minoru

2002-10-01

117

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.  

Science.gov (United States)

Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death. PMID:18603020

Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

2008-10-01

118

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

119

Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase.  

Science.gov (United States)

5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS. PMID:9521774

Lobo, A P; Nair, M G; Changchien, L; Weichsel, A; Montfort, W R; Maley, F

1998-03-31

120

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  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

 
 
 
 
121

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

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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

122

Effects of the glucolipid synthase inhibitor, P4, on functional and phenotypic parameters of murine myeloma cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This study describes the effects of the glucolipid synthase inhibitor P4, (DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol), on various functional and phenotypic parameters of 5T33 murine myeloma cells. Cell recovery was reduced by >85% following incubation of the cells for 3 days in the presence of 4 ?M P4 (the IC50 concentration). Both cytostatic and cytotoxic inhibition was observed with tumour cell metabolic activity and clonogenic potential reduced to 42% and 14% of controls...

Manning, L. S.; Radin, N. S.

1999-01-01

123

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

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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

124

A phase II study in advanced breast cancer: ZD1694 ('Tomudex') a novel direct and specific thymidylate synthase inhibitor.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

ZD1694 ('Tomudex'), a novel, direct and specific thymidylate synthase (TS) inhibitor, was developed in a collaborative research programme between Zeneca Pharmaceuticals and the Institute of Cancer Research (UK) and entered clinical trials in 1991; phase II studies began in 1992, using 3.0 mg m-2 every 3 weeks as a short 15 min infusion. Forty-six patients entered a phase II study of ZD1694 in advanced breast cancer. A total of 74% of patients had received prior systemic therapy (either as adj...

1996-01-01

125

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

Science.gov (United States)

Abstract 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

126

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 Ca"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 "3"2P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated "3"2P-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

1986-05-01

127

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

128

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

International Nuclear Information System (INIS)

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

2007-08-15

129

Synthesis and evaluation of 5-substituted 2'-deoxyuridine monophosphate analogues as inhibitors of flavin-dependent thymidylate synthase in Mycobacterium tuberculosis.  

Science.gov (United States)

A series of 5-substituted 2'-deoxyuridine monophosphate analogues has been synthesized and evaluated as potential inhibitors of mycobacterial ThyX, a novel flavin-dependent thymidylate synthase in Mycobacterium tuberculosis. A systematic SAR study led to the identification of compound 5a, displaying an IC(50) value against mycobacterial ThyX of 0.91 ?M. This derivative lacks activity against the classical mycobacterial thymidylate synthase ThyA (IC(50) > 50 ?M) and represents the first example of a selective mycobacterial FDTS inhibitor. PMID:21657202

Kögler, Martin; Vanderhoydonck, Bart; De Jonghe, Steven; Rozenski, Jef; Van Belle, Kristien; Herman, Jean; Louat, Thierry; Parchina, Anastasia; Sibley, Carol; Lescrinier, Eveline; Herdewijn, Piet

2011-07-14

130

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

International Nuclear Information System (INIS)

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

2001-01-01

131

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

Directory of Open Access Journals (Sweden)

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

ElaineDel Bel

2011-06-01

132

Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors : SAR and structural insights  

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Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-in...

2011-01-01

133

The renal effects of the water-soluble, non-folylpolyglutamate synthetase-dependent thymidylate synthase inhibitor ZD9331 in mice.  

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ZD9331 is a novel, potent thymidylate synthase (TS) inhibitor which does not require polyglutamation by folylpolyglutamate synthetase (FPGS) for its activity. In contrast to Tomudex (ZD1694), ZD9331 may therefore be active against tumours with low FPGS activity. ZD9331 shows anti-tumour activity by both 24-h infusion and bolus administration in the murine thymidine kinase-deficient (TK -/-) lymphoma L5178Y. In view of the history of renal toxicity with some earlier TS inhibitors and the possi...

Walton, M. I.; Mitchell, F.; Aherne, G. W.; Medlow, C. J.; Boyle, F. T.; Jackman, A. L.

1998-01-01

134

Protein inhibitor of neuronal nitric oxide synthase (PIN) is a new regulator of glucose-induced insulin secretion.  

Science.gov (United States)

We previously showed that pancreatic beta-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting beta-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nomega-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery. PMID:17130471

Lajoix, Anne-Dominique; Badiou, Stéphanie; Péraldi-Roux, Sylvie; Chardès, Thierry; Dietz, Samuel; Aknin, Cindy; Tribillac, Florence; Petit, Pierre; Gross, René

2006-12-01

135

Structure-based design of novel inhibitors of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.  

Science.gov (United States)

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) is the phosphorylated precursor of KDO, an essential sugar of the lipopolysaccharide of Gram negative bacteria. KDO8P is produced by a specific synthase (KDO8PS) by condensing arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP), with release of inorganic phosphate. As KDO8PS is present in bacteria and plants, but not in mammalian cells, and mutations that inactivate KDO8PS also block cell replication, KDO8PS is a promising target for the design of new antimicrobials that act by blocking lipopolysaccharide biosynthesis. Previous studies have shown that a compound mimicking an intermediate of the condensation reaction is a good ligand and a powerful inhibitor. Here we report on the crystallographic investigation of the binding to KDO8PS of new derivatives of this original inhibitor. The structures of the enzyme in complex with these compounds, and also with the PEP analogs, 2-phosphoglyceric acid (2-PGA) and Z-methyl-PEP, point to future strategies for the design of novel inhibitors of KDO8PS. PMID:14675946

Xu, Xingjue; Wang, Jian; Grison, Claude; Petek, Sylvian; Coutrot, Philippe; Birck, Matthew R; Woodard, Ronald W; Gatti, Domenico L

2003-01-01

136

The Interaction of Hydroxymandelate Synthase with the 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor: NTBC  

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Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of polycyclic non-ribosomal peptide antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O2, and also conducts a dioxygenation reaction. The difference between the two lies in the insertion of the second oxygen atom, HMS directing this atom onto the benzylic carbon of the su...

Conrad, John A.; Moran, Graham R.

2008-01-01

137

Synthesis of Bisubstrate Inhibitors of Porphobilinogen Synthase from Pseudomonas aeruginosa  

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Porphobilinogen synthase (PBGS) synthesizes porphobilinogen 2 (PBG), the common precursor of all natural tetrapyrroles, through an asymmetric condensation of two molecules of 5-aminolevulinic acid 1 (ALA). Symmetrically linked dimers 7-11 derived from levulinic acid 3 (?-oxovaleric acid) have been synthesized to mimic the assumed bisubstrate bound to the active site of the enzyme. Their inhibition potential was characterized by determination of the IC50<...

Gacond, Sabine; Fre?re, Frederic; Nentwich, Merle; Faurite, Jean-philippe; Frankenberg-dinkel, Nicole; Neier, Reinhard

2010-01-01

138

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase  

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Thymidylate deprivation brings about “thymineless death” in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. ...

Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D.; Waldman, Alan S.

2008-01-01

139

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

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

2002-01-01

140

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

 
 
 
 
141

Amelioration of virulent Babesia bovis infection in calves by administration of the nitric oxide synthase inhibitor aminoguanidine.  

Science.gov (United States)

Calves undergoing initial infection with a virulent strain of the haemoprotozoan parasite Babesia bovis were treated with aminoguanidine (AG), an inhibitor of the inducible form of nitric oxide synthase (iNOS). The mean maximum parasitaemia of the AG treated calves was significantly lower than that of the control cattle. In addition, the febrile response and decrease in packed cell volume (PCV) observed during acute infection were significantly ameliorated in the AG treated cattle relative to the controls. However, AG had no effect on the multiplication of B. bovis in the microaerophilous stationary-phase (MASP) in-vitro culture system. These results provide evidence of a role for nitric oxide (NO) produced in response to acute infection in the pathology of bovine babesiosis. PMID:9767611

Gale, K R; Waltisbuhl, D J; Bowden, J M; Jorgensen, W K; Matheson, J; East, I J; Zakrzewski, H; Leatch, G

1998-09-01

142

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

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

Graham R. Lawton

2009-06-01

143

Studies on the decarboxylation of acetolactate in milk products  

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The effect of different parameters on the decarboxylation of acetolactate (ALA) to diacetyl and acetoin were studied. The distillation volume and the milk solids concentration had no significant effect on decarboxylation of ALA, whereas breakdown of ALA increased with decreasing pH and increasing temperature. Oxygenation increased diacetyl production from ALA, but diacetyl was lost from the model system. Oxygenation did not have an effect on acetoin production from ALA. Metal ions (Cu2+, Fe2+...

Mohr, Britta

1997-01-01

144

Nitric oxide synthase activity and endogenous inhibitors in rats recovered from allergic encephalomyelitis  

Directory of Open Access Journals (Sweden)

Full Text Available We have previously reported that in comparison with normal rats, the presence of experimental allergic encephalomyelitis (EAE leads to decreased endogenous inhibitory activity (EIA of Ca2+-dependent nitric oxide synthase (NOS in both brain and serum, and increased expression of protein 3-nitrotyrosine (NT in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NOS, or NT expression. These results suggest that parallel to the reversal of the disease symptoms, a normalization of the production of nitric oxide and related species occurs.

SA Teixeira

2005-03-01

145

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

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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

146

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase.  

Science.gov (United States)

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex. PMID:2525127

Galivan, J; Rhee, M S; Johnson, T B; Dilwith, R; Nair, M G; Bunni, M; Priest, D G

1989-06-25

147

Nitric oxide synthase activity and endogenous inhibitors in rats recovered from allergic encephalomyelitis  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english We have previously reported that in comparison with normal rats, the presence of experimental allergic encephalomyelitis (EAE) leads to decreased endogenous inhibitory activity (EIA) of Ca2+-dependent nitric oxide synthase (NOS) in both brain and serum, and increased expression of protein 3-nitrotyr [...] osine (NT) in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NOS, or NT expression. These results suggest that parallel to the reversal of the disease symptoms, a normalization of the production of nitric oxide and related species occurs.

SA, Teixeira; AA, Varriano; AA, Dias; R, Martins Porto; MN, Muscará.

148

Inhibition of endothelial cell proliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor.  

Science.gov (United States)

Orlistat, an antiobesity drug, is cytostatic and cytotoxic to tumor cells. The antitumor activity of orlistat can be attributed to its ability to inhibit the thioesterase domain of fatty acid synthase (FAS). The objective of the present study was to test the effect of orlistat on endothelial cell proliferation and angiogenesis. Orlistat inhibits endothelial cell FAS, blocks the synthesis of fatty acids, and prevents endothelial cell proliferation. More significantly, orlistat inhibits human neovascularization in an ex vivo assay, which suggests that it may be useful as an antiangiogenic drug. The mechanism of these effects can be traced to the fact that orlistat prevents the display of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR/Flk1) on the endothelial cell surface. Thus, orlistat is an antiangiogenic agent with a novel mechanism of action. PMID:17012255

Browne, Cecille D; Hindmarsh, Elizabeth J; Smith, Jeffrey W

2006-10-01

149

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels.  

Science.gov (United States)

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors. PMID:17339891

Giovannetti, E; Backus, H H J; Wouters, D; Ferreira, C G; van Houten, V M M; Brakenhoff, R H; Poupon, M-F; Azzarello, A; Pinedo, H M; Peters, G J

2007-03-12

150

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 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

2004-10-01

151

Design and synthesis of new potent inhibitors of farnesyl pyrophosphate synthase.  

Science.gov (United States)

Predictive QSAR models for the inhibition activities of nitrogen-containing bisphosphonates (N-BPs) against farnesyl pyrophosphate synthase (FPPS) from Leishmania major (LeFPPS) were developed using a data set of 97 compounds. The QSAR models were developed through the use of Artificial Neural Networks and Random Forest learning procedures. The predictive ability of the models was tested by means of leave-one-out cross-validation; Q(2)values ranging from 0.45-0.79 were obtained for the regression models. The consensus prediction for the external evaluation set afforded high predictive power (Q(2)=0.76 for 35 compounds). The robustness of the QSAR models was also evaluated using a Y-randomization procedure. A small set of 6 new N-BPs were designed and synthesized applying the Michael reaction of tetrakis (trimethylsilyl) ethenylidene bisphosphonate with amines. The inhibition activities of these compounds against LeFPPS were predicted by the developed QSAR models and were found to correlate with their fungistatic activities against Candida albicans. The antifungal activities of N-BPs bearing n-butyl and cyclopropyl side chains exceeded the activities of Fluconazole, a triazole-containing antifungal drug. In conclusion, the N-BPs developed here present promising candidate drugs for the treatment of fungal diseases. PMID:24818603

Prokopenko, Volodymyr; Kovalishyn, Vasyl; Shevchuk, Michael; Kopernyk, Iryna; Metelytsia, Larysa; Romanenko, Vadim; Mogilevich, Sergey; Kukhar, Valery

2014-06-01

152

Sensitive Assay for Antifungal Activity of Glucan Synthase Inhibitors That Uses Germ Tube Formation in Candida albicans as an End Point  

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We implemented a simple, sensitive, objective, and rapid cellular assay to reveal the antifungal activity of a novel class of glucan synthase inhibitors. The assay, especially useful for early drug discovery, measures the transformation of Candida albicans from the yeast form to the hyphal form. Test compounds were ranked by potency (50% inhibitory concentration) and efficacy (percent inhibition of germ tube formation); the intra-assay coefficients of variation for these parameters were 17 an...

Brayman, Timothy G.; Wilks, John W.

2003-01-01

153

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

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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

154

L-arginine binding to human inducible nitric oxide synthase: an antisymmetric funnel route toward isoform-specific inhibitors?  

Science.gov (United States)

Nitric oxide (NO) is an important signaling molecule produced by a family of enzymes called nitric oxide synthases (NOS). Because NO is involved in various pathological conditions, the development of potent and isoform-selective NOS inhibitors is an important challenge. In the present study, the dimer of oxygenase domain of human iNOS (iNOSoxy) complexed to its natural substrate L-arginine (L-Arg) and both heme and tetrahydro-L-biopterin (BH4) cofactors was studied through multiple molecular dynamics simulations. Starting from the X-ray structure available for that complex (PDB: 1NSI ), a 16 ns equilibration trajectory was first obtained. Twelve dynamics of slow extraction of L-Arg out from the iNOSoxy active site were then performed. The steered molecular dynamics (SMD) approach was used starting from three different points of the reference trajectory for a total simulation time of 35 ns. A probable unbinding/binding pathway of L-Arg was characterized. It was suggested that a driving force directed the substrate toward the heme pocket. Key intermediate steps/residues along the access route to the active site were identified along this "funnel shape" pathway and compared to existing data. A quasi-normal mode analysis performed on the SMD data suggested that large collective motions of the protein may be involved in L-Arg binding and that opening the route to the active site in one monomer promoted an inverse, closing motion in the second monomer. Finally, our findings might help to rationalize the design of human iNOS isoform competitive inhibitors. PMID:21574590

Floquet, Nicolas; Hernandez, Jean-François; Boucher, Jean-Luc; Martinez, Jean

2011-06-27

155

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

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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

156

Phytotoxicity of Acetohydroxyacid Synthase Inhibitors Is Not Due to Accumulation of 2-Ketobutyrate and/or 2-Aminobutyrate.  

Science.gov (United States)

Acetohydroxyacid synthase (AHAS) is the site of action of herbicides of different chemical classes, such as imidazolinones, sulfonylureas, and triazolopyrimidines. Inhibition of AHAS causes the accumulation of 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) (the transamination product of 2-KB), and it has been proposed that the phytotoxicity of these inhibitors is due to this accumulation. Experiments were done to determine the relationship between accumulation of 2-KB and 2-AB and the phytotoxicity of imazaquin to maize (Zea mays). Imazaquin concentrations that inhibit growth of maize plants also cause the accumulation of 2-KB and 2-AB in the shoots. Supplementation of imazaquin-treated plants with isoleucine reduced the pools of 2-KB and 2-AB in the plant but did not protect plants from the growth inhibitory effects of imazaquin. Conversely, feeding 2-AB to maize plants increased 2-KB and 2-AB pools to much higher levels than those observed in imazaquin-treated plants, yet such high pools of 2-KB and 2-AB in the plant had no significant effect on growth. These results conclusively demonstrate that growth inhibition following imazaquin treatment is not due to accumulation of 2-KB and/or 2-AB in plants. Changes in the amino acid profiles after treatment with imazaquin suggest that starvation for the branched-chain amino acids may be the primary cause of growth retardation of maize.

Shaner, D. L.; Singh, B. K.

1993-01-01

157

Eliglustat tartrate, an orally active glucocerebroside synthase inhibitor for the potential treatment of Gaucher disease and other lysosomal storage diseases.  

Science.gov (United States)

Eliglustat tartrate (Genz-112638), currently under development by Genzyme Corp, is a glucocerebroside (glucosylceramide) synthase inhibitor for the treatment of Gaucher disease and other lysosomal storage disorders. Gaucher disease is an inherited defect of lysosomal functions caused by mutations in the GBA1 gene leading to accumulation of glucocerebroside, primarily in macrophages. Gaucher disease is characterized by visceromegaly and skeletal complications, including osteoporosis and painful episodes of osteonecrosis. In vitro studies demonstrated that, following exposure to eliglustat tartrate, the abundance of GM1 and GM3 gangliosides in cultured human erythroleukemia cells and murine melanoma cells was decreased. In vivo, eliglustat tartrate administered to Asp409Val/null mice lowered the concentrations of glucocerebroside in the liver, lung and spleen and reduced the number of Gaucher cells in the liver. In a phase Ib clinical trial in healthy volunteers, plasma glucocerebroside concentrations were decreased after dosing with eliglustat tartrate, and in phase II clinical trials in patients with type 1 (non-neuronopathic) Gaucher disease, spleen and liver volumes were diminished. Patients also demonstrated improved bone mineral density, correction of abnormal bone marrow signal with MRI and normalization of glucocerebroside and ganglioside GM3 levels. Eliglustat tartrate is orally active and, with potent effects on the primary identified molecular target for type 1 Gaucher disease and other glycosphingolipidoses, appears likely to fulfill high expectations for clinical efficacy. PMID:20872320

Cox, Timothy M

2010-10-01

158

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

Science.gov (United States)

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

Khan, Haseeb Ahmad

2012-11-01

159

Nitric oxide synthase inhibitors that interact with both heme propionate and tetrahydrobiopterin show high isoform selectivity.  

Science.gov (United States)

Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of ?-amino functionalized aminopyridine derivatives (3-8) were designed to probe the structure-activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors. PMID:24758147

Kang, Soosung; Tang, Wei; Li, Huiying; Chreifi, Georges; Martásek, Pavel; Roman, Linda J; Poulos, Thomas L; Silverman, Richard B

2014-05-22

160

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities  

Science.gov (United States)

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N?-monomethyl-L-arginine (L-NMMA), N?-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1? (rhIL-1?) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48?h in the presence of rhIL-1? with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na235SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1? challenge (0 to 250?U?ml?1 and 0 to 15?U?ml?1 respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1? (2500?U?ml?1 and 30?U?ml?1 respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000??M) on NO production by cartilage cells stimulated with IL-1? (25?U?ml?1 or 5?U?ml?1), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA?SMT>AG?L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1?, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME?L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000??M) were unable to correct nitrite levels or proteoglycan synthesis in IL-1? treated cells. L-arginine (5000??M) tended to reverse the correcting effect of L-NMMA (1000??M) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100??M) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1? on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1? on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

Cipolletta, Christine; Jouzeau, Jean-Yves; Gegout-Pottie, Pascale; Presle, Nathalie; Bordji, Karim; Netter, Patrick; Terlain, Bernard

1998-01-01

 
 
 
 
161

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

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Full Text Available 7-Nitroindazole (7-NI inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval, day 26: 16.75 (15.88-17.00; day 28: 0.00 (0.00-9.63; day 29: 13.75 (2.25-15.50; day 30: 0.5 (0.00-6.25; day 31: 4.00 (0.00-7.13, and day 34: 0.5 (0.00-14.63, Friedman followed by Wilcoxon test,vs day 26, P < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS. The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson’s disease.

N. Novaretti

2010-11-01

162

Amino-acid substitutions at the domain interface affect substrate and allosteric inhibitor binding in ?-isopropylmalate synthase from Mycobacterium tuberculosis.  

Science.gov (United States)

?-Isopropylmalate synthase (?-IPMS) is a multi-domain protein catalysing the condensation of ?-ketoisovalerate (?-KIV) and acetyl coenzyme A (AcCoA) to form ?-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and ?-IPMS is allosterically regulated by this amino acid. Existing crystal structures of ?-IPMS from Mycobacterium tuberculosis (MtuIPMS) indicate that this enzyme has a strikingly different domain arrangement in each monomer of the homodimeric protein. This asymmetry results in two distinct interfaces between the N-terminal catalytic domains and the C-terminal regulatory domains in the dimer. In this study, residues Arg97 and Asp444 across one of these unequal domain interfaces were substituted to evaluate the importance of protein asymmetry and salt bridge formation between this pair of residues. Analysis of solution-phase structures of wild-type and variant MtuIPMS indicates that substitutions of these residues have little effect on overall protein conformation, a result also observed for addition of the feedback inhibitor leucine to the wild-type enzyme. All variants had increased catalytic efficiency relative to wild-type MtuIPMS, and those with an Asp444 substitution displayed increased affinity for the substrate AcCoA. All variants also showed reduced sensitivity to leucine and altered biphasic reaction kinetics when compared with those of the wild-type enzyme. It is proposed that substituting residues at the asymmetric domain interface increases flexibility in the protein, particularly affecting the AcCoA binding site and the response to leucine, without penalty on catalysis. PMID:23500460

Huisman, Frances H A; Squire, Christopher J; Parker, Emily J

2013-04-01

163

The fatty acid synthase inhibitor orlistat reduces the growth and metastasis of orthotopic tongue oral squamous cell carcinomas.  

Science.gov (United States)

Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC. PMID:24362464

Agostini, Michelle; Almeida, Luciana Y; Bastos, Débora C; Ortega, Rose M; Moreira, Fernanda S; Seguin, Fabiana; Zecchin, Karina G; Raposo, Helena F; Oliveira, Helena C F; Amoêdo, Nivea D; Salo, Tuula; Coletta, Ricardo D; Graner, Edgard

2014-03-01

164

Structure-activity relationships of potent, selective inhibitors of neuronal nitric oxide synthase based on the 6-phenyl-2-aminopyridine structure.  

Science.gov (United States)

The synthesis and structure-activity relationships of a series of 6-phenyl-2-aminopyridines that potently and selectively inhibit the neuronal isoform of nitric oxide synthase (nNOS) are described. Compound 14bi from this series exhibits potent in vivo activity in harmaline-induced cGMP formation in rat cerebellum, a functional model of nNOS inhibition, and in the PCP-induced hypermotility model in the rat. These results suggest that 14bi may be a useful reagent for evaluating potential therapeutic applications of nNOS inhibitors in the central nervous system. PMID:14998342

Lowe, John A; Qian, Weimin; Drozda, Susan E; Volkmann, Robert A; Nason, Deane; Nelson, Robert B; Nolan, Charles; Liston, Dane; Ward, Karen; Faraci, Steve; Verdries, Kim; Seymour, Pat; Majchrzak, Michael; Villalobos, Anabella; White, W Frost

2004-03-11

165

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

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

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

1995-01-01

166

A Novel Lumazine Synthase Inhibitor Derived from Oxidation of 1,3,6,8-Tetrahydroxy-2,7-naphthyridine to a Tetraazaperylenehexaone Derivative  

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Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-Ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66 ± 13 ?M in Tris buffer and 22 ± 4 ?M in pho...

Zhang, Yanlei; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Georg, Gunda I.; Ye, Qi-zhuang; Velde, David Vander; Fanwick, Phillip E.; Song, Yunlong; Cushman, Mark

2007-01-01

167

Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury  

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The exact functional role of nitric oxide (NO) in liver injury is currently a source of controversy. NO is enzymatically synthesized by nitric oxide synthase (NOS). In this study, we assessed the role of inducible NOS (iNOS) in carbon tetrachloride (CCl4)- induced acute liver injury using inhibitors of iNOS, and wani thN Oor dwointhoor.utA thdeu litN IOCSR imnhiicbei tworesr e(5 i-nmjeecthteydl iswoitthhi oCuCrela4 hemisulfate [SMT] and l-N6-(1-iminoethyl)-lysine [...

Tipoe, G. L.; Leung, T. M.; Liong, E.; So, H.; Leung, K. M.; Lau, T. Y. H.; Tom, W. M.; Fung, M. L.; Fan, S. T.; Nanji, A. A.

2006-01-01

168

Effects of chronic treatment with nitric oxide synthase inhibitors on regional haemodynamic responses to vasodilators in conscious Brattleboro rats.  

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1. The effects of acute inhibition of nitric oxide (NO) synthase on cardiovascular responses to vasodilator challenges have already been described. We now report the responses to vasodilators during and after chronic NO synthase inhibition. 2. In conscious Brattleboro rats, the regional haemodynamic effects of 3 min infusions of acetylcholine (4 micrograms min-1), sodium nitroprusside (15 micrograms min-1) or adrenaline (0.2 micrograms min-1) were assessed (from areas under or over curves (AU...

Gardiner, S. M.; Kemp, P. A.; Bennett, T.

1993-01-01

169

The renal and systemic hemodynamic effects of a nitric oxide-synthase inhibitor are reversed by a selective endothelin(a) receptor antagonist in men.  

Science.gov (United States)

There is evidence for an interaction between nitric oxide (NO) and endothelin (ET) at the level of the renal vasculature. We hypothesized that acute renal effects of systemic NO synthase inhibition (NG-monomethyl-l-arginine, L-NMMA) may be blunted by coadministration of a specific ET(A) receptor antagonist (BQ-123) in healthy humans. Fifteen healthy young male subjects participated in this randomized, double-blind, placebo-controlled 3-way crossover study. These sodium-repleted volunteers received L-NMMA alone, or BQ-123 alone, or L-NMMA with a subsequent coinfusion of BQ-123. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined with the PAH and inulin clearance method, respectively. Mean arterial pressure (MAP) and pulse rate were measured noninvasively at baseline and every 15 min after the start of the study period. L-NMMA alone reduced RPF (-22%, P < 0.001) and GFR (-8%, P < 0.009) and increased MAP (+10%, P < 0.001). BQ-123 alone did not affect these parameters. However, coinfusion of BQ-123 blunted the effects of L-NMMA on RPF (P < 0.001), GFR (P < 0.001), and MAP (P = 0.006). Peripheral and renal hemodynamic effects of acute systemic NO synthase inhibition are at least partially reversed by ET(A) receptor blockade with BQ-123. This indicates a functional antagonism between specific ET(A) receptor antagonist and NO synthase inhibitors at the level of the renal vasculature. PMID:11485375

Schmidt, A; Bayerle-Eder, M; Pleiner, H; Zeisner, C; Wolzt, M; Mayer, G; Schmetterer, L

2001-08-01

170

Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity.  

Science.gov (United States)

Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis. PMID:19589393

Fugelstad, Johanna; Bouzenzana, Jamel; Djerbi, Soraya; Guerriero, Gea; Ezcurra, Inés; Teeri, Tuula T; Arvestad, Lars; Bulone, Vincent

2009-10-01

171

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 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.)

2006-09-01

172

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

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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

173

Sensitivity of Aspergillus nidulans to the cellulose synthase inhibitor dichlobenil: insights from wall-related genes' expression and ultrastructural hyphal morphologies.  

Science.gov (United States)

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely ?-, ?-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes' expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage ?-1,3;1,4 glucan synthase celA together with the ?-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

Guerriero, Gea; Silvestrini, Lucia; Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

2013-01-01

174

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes' Expression and Ultrastructural Hyphal Morphologies  

Science.gov (United States)

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely ?-, ?-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage ?-1,3;1,4 glucan synthase celA together with the ?-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.

Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

2013-01-01

175

The discovery of novel, potent and highly selective inhibitors of inducible nitric oxide synthase (iNOS).  

Science.gov (United States)

By careful analysis of experimental X-ray ligand crystallographic protein data across several inhibitor series we have discovered a novel, potent and selective series of iNOS inhibitors exemplified by compound 8. PMID:21398123

Cheshire, David R; Åberg, Anders; Andersson, Gunilla M K; Andrews, Glen; Beaton, Haydn G; Birkinshaw, Timothy N; Boughton-Smith, Nigel; Connolly, Stephen; Cook, Tony R; Cooper, Anne; Cooper, Sally L; Cox, David; Dixon, John; Gensmantel, Nigel; Hamley, Peter J; Harrison, Richard; Hartopp, Paul; Käck, Helena; Leeson, Paul D; Luker, Timothy; Mete, Antonio; Millichip, Ian; Nicholls, David J; Pimm, Austen D; St-Gallay, Steve A; Wallace, Alan V

2011-04-15

176

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

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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...

Schönitzer Veronika; Weiss Ingrid M

2007-01-01

177

Structures of Aquifex aeolicus KDO8P synthase in complex with R5P and PEP, and with a bisubstrate inhibitor: role of active site water in catalysis.  

Science.gov (United States)

We have determined the crystal structures of the metalloenzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase from Aquifex aeolicus in complex with phosphoenolpyruvate (PEP) and ribose 5-phosphate (R5P), and with a bisubstrate inhibitor that mimics the postulated linear reaction intermediate. R5P, which is not a substrate for KDO8P synthase, binds in a manner similar to that of arabinose 5-phosphate (A5P), which is the natural substrate. The lack of reactivity of R5P appears to be primarily a consequence of the loss of a water molecule coordinated to Cd(2+) and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OH(R5P), which is oriented in a different direction from C2-OH(A5P). The bisubstrate inhibitor binds with its phosphate and phosphonate moieties occupying the positions of the phosphate groups of A5P and PEP, respectively. One of the inhibitor hydroxyls replaces water as a ligand of Cd(2+). The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2(PEP), forming a tetrahedral-like intermediate with a buildup of negative charge at C3(PEP). The ensuing condensation of C3(PEP) with C1(A5P) would be favored by a proton transfer from the phosphate moiety of PEP to the aldehyde carbonyl of A5P to generate the hydroxyl. Overall, the process can be described as a syn addition of water and A5P to the si side of PEP. PMID:11747443

Wang, J; Duewel, H S; Woodard, R W; Gatti, D L

2001-12-25

178

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

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

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

1997-01-01

179

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

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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

180

Investigating the efficacy of pamidronate, a chemical inhibitor of farnesyl pyrophosphate synthase, in the inhibition of influenza virus infection in vitro and in vivo.  

Science.gov (United States)

Influenza A virus has caused significant pandemics in the past decades, including the H1N1?2009 pandemic. Viperin is an interferon?inducible protein that acts as a broad?spectrum antiviral protein via the inhibition of farnesyl pyrophosphate synthase (FPPS). To mimic this activity of viperin, the present study investigated the effectiveness of a commercially available FPPS inhibitor (pamidronate) as an inhibitor of influenza virus infection in vitro and in vivo. HeLaM cells were treated with pamidronate to determine its effect on the replication of influenza virus A/H1N1/WSN/1933. C57BL/6 mice were also subjected to intratracheal pamidronate treatment regimes prior to and following lethal influenza challenge. Treatment with the FPPS inhibitor in vitro resulted in a considerable reduction in the viral titer of ~1 log and diminished lipid raft formation without cellular toxicity, thus mimicking the antiviral effect of viperin. However, pamidronate lacked efficacy in vivo and was associated with increased pulmonary damage, most likely due to the complexity of drug?host interactions in the infected mice. Further studies are warranted on pamidronate treatment in other infectious diseases that are more susceptible to FPPS inhibition. PMID:24154548

Tan, Kai Sen; Ng, Wai Chii; Seet, Ju Ee; Olfat, Farzad; Engelward, Bevin P; Chow, Vincent T K

2014-01-01

 
 
 
 
181

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

182

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/?-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma.  

Science.gov (United States)

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/?-catenin pathway, recombinant WNT3A and stabilized ?-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/?-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/?-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/?-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

Chen, Eleanor Y; DeRan, Michael T; Ignatius, Myron S; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M; Lobbardi, Riadh M; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M

2014-04-01

183

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

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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.

2012-01-01

184

Zymosan suppresses leukotriene C-4 synthase activity in differentiating monocytes: antagonism by aspirin and protein kinase inhibitors  

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Cysteinyl leukotrienes (cysLTs) are potent proinflammatory mediators with particular relevance for asthma. However, control of cysLT biosynthesis in the time period after onset of acute inflammation has not been extensively studied. As a model for later phases of inflammation, we investigated regulation of leukotriene (LT) C-4 synthase (LTC4S) in differentiating monocytes, exposed for several days to fungal zymosan. Incubations with LTA(4) revealed 20-fold increased LTC4S activity during diff...

2011-01-01

185

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 u...

Scho?nitzer, Veronika; Weiss, Ingrid M.

2010-01-01

186

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2013-11-15

187

Design and synthesis of N-2,6-difluorophenyl-5-methoxyl-1,2,4-triazolo[1,5-a]-pyrimidine-2-sulfonamide as acetohydroxyacid synthase inhibitor.  

Science.gov (United States)

Triazolopyrimidine-2-sulfonamide belongs to a herbicide group called acetohydroxyacid synthase inhibitors. With the aim to discover new triazolopyrimidine sulfonanilide compounds with high herbicidal activity and faster degradation rate in soil, the methyl group of Flumetsulam (FS) was modified into a methoxy group to produce a new herbicidal compound, N-2,6-difluorophenyl-5-methoxy-1,2,4-triazolo[1,5-a]pyrimidine-2-sulfonamide (experimental code: Y6610). The enzymatic kinetic results indicated that compound Y6610 and FS have k(i) values of 3.31x10(-6) M and 3.60x10(-7) M against Arabidopsis thaliana AHAS, respectively. The 10-fold lower enzyme-inhibiting activity of Y6610 was explained rationally by further computational simulations and binding free energy calculations. In addition, compound Y6610 was found to display the same level in vivo post-emergent herbicidal activity as FS against some broad-leaf weeds and good safety to rice, maize, and wheat at the dosages of 75-300 gai/ha. Further determination of the half-lives in soil revealed that the half-life in soil of Y6610 is 3.9 days shorter than that of FS. The experimental results herein showed that compound Y6610 could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study. PMID:19342247

Chen, Chao-Nan; Lv, Li-Li; Ji, Feng-Qin; Chen, Qiong; Xu, Hui; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

2009-04-15

188

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

International Nuclear Information System (INIS)

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

2013-11-15

189

Plasma concentrations of asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, are elevated in sickle cell patients but do not increase further during painful crisis.  

Science.gov (United States)

Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, are elevated in the clinically asymptomatic state of sickle cell disease (SCD). However, the role of ADMA during vaso-occlusive complications has not been defined. ADMA concentrations were determined in HbSS (n = 43) and HbSC (n = 25) patients with healthy blood donors (HbAA) as controls. In the clinically asymptomatic state ADMA concentrations were elevated in sickle cell patients as compared to healthy controls (HbSS 0.70 micromol/L, HbSC 0.54 micromol/L, HbAA 0.39 micromol/L) (P < 0.001). Yet plasma ADMA concentrations did not increase further at presentation with a painful crisis implicating no role of primary importance during vaso-occlusive crises. PMID:18383318

Landburg, Precious P; Teerlink, Tom; Muskiet, Frits A J; Duits, Ashley J; Schnog, John-John B

2008-07-01

190

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)

2008-01-01

191

Structural Basis for the Design of Potent and Species-specific Inhibitors of 3-hydroxy-3-methylglutaryl CoA Synthases  

Energy Technology Data Exchange (ETDEWEB)

3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

Pojer,F.; Ferrer, J.; Richard, S.; Nagegowda, D.; Chye, M.; Bach, T.; Noel, J.

2006-01-01

192

Antitumor effect of orlistat, a fatty acid synthase inhibitor, is via activation of caspase-3 on human colorectal carcinoma-bearing animal.  

Science.gov (United States)

We established a HT-29/tk-luc human colorectal carcinoma-bearing animal model for the study of the inhibition effect and mechanism of orlistat, a fatty acid synthase (FASN) inhibitor. The results showed that orlistat caused cell cycle arrest at G1 phase, and triggered apoptosis through caspase-3 activation. The tumor inhibition effect of orlistat may also due to the inhibition of fatty acid synthesis without altering FASN activity. The tumor size of orlistat-treated mice in vivo was significantly smaller than that of the controls with 55% inhibition. The therapeutic efficacy was further confirmed with the bioluminescent imaging and nuclear molecular imaging with ¹³¹I-FIAU/gamma scintigraphy and ¹¹C-acetate/microPET. We suggest that FASN is a potential target for the treatment of human colorectal carcinoma. PMID:21723078

Chuang, Hui-Yen; Chang, Ya-Fang; Hwang, Jeng-Jong

2011-07-01

193

Potential physiological functions of acceptor products of dextransucrase with cellobiose as an inhibitor of mutansucrase and fungal cell synthase.  

Science.gov (United States)

A series of oligosaccharides (cellobio-oligosaccharides) ranging from degrees of polymer 3 to 6 were synthesized by Leuconostoc mesenteroides B-512 FMCM in the presence of cellobiose. The major oligosaccharides were the trisaccharides, ?-d-glucopyranosyl-(1?2)-?-d-glucopyranosyl-(1?4)-d-glucopyranose and ?-d-glucopyranosyl-(1?6)-?-d-glucopyranosyl-(1?4)-d-glucopyranose. These cellobio-oligosaccharides were inhibitory on mutansucrase, an enzyme that causes dental caries. They were also found to be effective antifungal agents against Aspergillus terreus acting by inhibiting ?-(1?3)-glucan synthase, which is required for fungal cell wall formation. PMID:20929235

Kim, Misook; Day, Donal F; Kim, Doman

2010-11-10

194

Regulation of inducible nitric oxide synthase by rapid cellular turnover and cotranslational down-regulation by dimerization inhibitors  

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Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of many disorders. iNOS is notably distinguished from constitutive NOSs by its production of large amounts of NO for a prolonged period; hence, it was termed the high-output NOS. Understanding how cells regulate iNOS is a prerequisite for strategies aimed at modulating NO synthesis. iNOS is thought to be regulated primarily at the transcriptional level in response to cytokines...

Kolodziejski, Pawel J.; Koo, Ja-seok; Eissa, N. Tony

2004-01-01

195

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

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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

196

Synthesis and evaluation of 3-modified 1D-myo-inositols as inhibitors and substrates of phosphatidylinositol synthase and inhibitors of myo-inositol uptake by cells.  

Science.gov (United States)

A number of 3-substituted 1D-myo-inositols were synthesized and evaluated as substrates for phosphatidylinositol synthase and uptake by intact cells. 1D-3-Amino-, -3-chloro-, and -3-(acetylthio)-3-deoxy-myo-inositols were all synthesized by nucleophilic displacement of the 6-O-(trifluoromethyl)sulfonyl group of 1L-1,2:3,4-di-O-cyclohexylidene-5-O-methyl-6-O-[(trifluoromethyl)-sulfon yl] - chiro-inositol (which was prepared from L-quebrachitol), respectively, by reaction with LiN3, followed by reduction of the azido function, and with LiCl and KSAc to give the O-protected compounds. O-Demethylation using BBr3 and concomitant acetal hydrolysis furnished the free-hydroxy 3-amino- and 3-chloro-3-deoxy-1D-myo-inositols. The 3-mercapto analogue was obtained by removal of the acetal groups of the acetylthio analogue, followed by acetylation and purification of the peracetate, and subsequent O-demethylation and deacetylation. The 3-deoxy derivative was synthesized from the 6-O-(imidazol-1-ylthiocarbonyl) compound via Barton-McCombie deoxygenation. The 3-azido derivative was directly synthesized from 1L-1-O-tosyl-chiro-inositol via displacement with azide. The 3-keto analogue was prepared by Pt-catalyzed air oxidation of 1L-chiro-inositol. The compounds were all evaluated as substrates for phosphatidylinositol (PtdIns) synthase from mouse brain. The 3-NH2, 3-F, 3-deoxy, and 3-keto analogues all showed activity as substrates, as measured by liberation of cytidine monophosphate. These compounds also showed inhibition of the reaction of myo-[3H]inositol with PtdIns synthase. These results taken together indicate that these compounds are likely to be incorporated into phospholipids. As a further indication that these compounds might be useful as probes for the PtdIns pathway, it was demonstrated that the 3-NH2, 3-F, and 3-deoxy compounds are taken up by intact fibroblast cells as evidenced by their competing with myo-[3H]inositol uptake. PMID:8246231

Johnson, S C; Dahl, J; Shih, T L; Schedler, D J; Anderson, L; Benjamin, T L; Baker, D C

1993-11-12

197

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

Science.gov (United States)

Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 ? resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

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

2011-04-01

198

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

199

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

Science.gov (United States)

...preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173...preparation derived from a recombinant Bacillus subtilis. The food additive alpha-acetolactate...enzyme preparation derived from a modified Bacillus subtilis strain that contains the...

2010-01-01

200

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

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

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

1999-01-01

 
 
 
 
201

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

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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 th...

Suborov, Evgeny; Smetkin, Alexey Anatolievich; Kondratyev, Timofey; Valkov, Andrey Yurjevich; Kuzkov, Vsevolod; Kirov, Mikhail; Bjertnaes, Lars J.

2012-01-01

202

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.

2009-06-19

203

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

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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. PMID:23872097

Gilmour, Peter S; O'Shea, Patrick J; Fagura, Malbinder; Pilling, James E; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F; Kavanagh, Stefan; Hall, Peter A; Escott, K Jane

2013-10-15

204

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.

205

G619, a dual thromboxane synthase inhibitor and thromboxane A2 receptor antagonist, reduces myocardial damage and polymorphonuclear leukocyte accumulation following coronary artery occlusion and reperfusion in rats.  

Science.gov (United States)

We investigated the effect of G 619, a dual thromboxane synthase inhibitor and thromboxane A2 (TxA2) receptor antagonist, in pentobarbital-anaesthetized rats subjected to left main coronary artery ligation (1 h) followed by reperfusion (1 h; MI/R). Sham-operated rats were used as controls (sham MI/R). Survival rate, myocardial necrosis, myocardial myeloperoxidase (MPO) activity (investigated as an index of leukocyte adhesion and accumulation) and serum creatine phosphokinase (CPK) activity were studied. MI/R injury significantly reduced survival rate (45%), caused a marked myocardial necrosis, increased serum CPK activity (sham MI/R = 35 +/- 12 U/ml; MI/R = 205 +/- 13 U/ml) and produced an increase in myocardial MPO activity in the area at risk and in the necrotic area (6.3 +/- 0.5 and 6.6 +/- 0.9 U x 10(-3)/g tissue, respectively). The administration of G 619 significantly increased survival rate, lowered the area of necrosis, blunted the increase in serum CPK activity and reduced the increase in MPO activity in both the area at risk and the necrotic area. These data are consistent with an involvement of TxA2 in MI/R injury and suggest that G 619 may represent a novel therapeutic approach to the treatment of acute myocardial infarction. PMID:8415867

Squadrito, F; Ioculano, M; Altavilla, D; Zingarelli, B; Canale, P; Campo, G M; Saitta, A; Oriti, S; Spignoli, G; Caputi, A P

1993-09-01

206

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

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

Zhao Xuan

2013-01-01

207

The NO donor sodium nitroprusside reverses the negative effects on hepatic arterial flow induced by endotoxin and the NO synthase inhibitor L-NAME.  

Science.gov (United States)

In previous studies we have observed that the nitric oxide synthase inhibitor L-NAME induces a profound deterioration of liver circulation in experimental endotoxemia. Using the same porcine model we now have evaluated the possibility of modulating these effects with the nitric oxide donor sodium nitroprusside. Infusion of endotoxin led to a gradual deterioration of hemodynamic parameters, including liver blood flow. The decreases in portal blood flow paralleled and matched the decreases in cardiac output, and no compensatory increase in hepatic arterial flow occurred. L-NAME had detrimental effects on hemodynamics, including the liver circulation. The latter effects could, however, partially be reversed by sodium nitroprusside. Hepatic arterial flow increased from 1.9 to 7.2 ml/kg/min, with a concomitant decrease in hepatic arterial resistance from 5,364 to 1,746 dyn s/cm5 kg. A control group exhibited no significant change in either flow or resistance. The response to sodium nitroprusside was rapid and vigorous, and probably largely due to relaxation of the hepatic arterioles, and not to abatement of intrahepatic edema or plugging of the sinusoids. Furthermore, we conclude that the endotoxin-induced dysfunction of the hepatic arterial buffer response may be due to a selective inhibition of vascular endothelial function. PMID:8880121

Gundersen, Y; Saetre, T; Scholz, T; Carlsen, H; Kjekshus, H; Smiseth, O A; Lilleaasen, P; Aasen, A O

1996-01-01

208

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

209

Modulation of thymidilate synthase and p53 expression by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with 5FU or raltitrexed.  

Science.gov (United States)

Despite the introduction of several novel anticancer agents almost 50% of colorectal cancer (CRC) patients die for cancer suggesting the necessity of new therapeutical approaches. In this study we demonstrated that the HDAC inhibitor vorinostat exerted potent antiproliferative effect in a panel of mut- and wt-p53 human CRC cell lines. Moreover, in combination with 5-fluorouracil modulated by folinic acid (5FU-FA) or with Raltitrexed (RTX), both commonly used in the treatment of this disease, it showed a clear schedule-dependent synergistic antiproliferative interaction as demonstrated by calculating combination indexes. Only simultaneous, or 24 h pretreatment with vorinostat followed by either agent, produced synergistic effect paralleled by evident cell cycle perturbations with major S-phase arrest. Moreover, we provided for the first time evidences that vorinostat can overcome resistance to both 5FU and RTX. Downmodulation of Thymidilate synthase (TS) protein induced by vorinostat within 24 h, represented a key factor in enhancing the effects of both drugs in sensitive as well as resistant tumor cells. Furthermore, p53, whose wild-type expression is critical for sensitivity to 5FU and RTX, was upregulated by vorinostat in wt- and downregulated in mut-p53 cells, suggesting an additional mechanism of the antiproliferative synergistic interactions observed. Overall these data add new insights in the mechanism of vorinostat antitumor effect and suggested that the association of vorinostat plus 5FU-FA and/or RTX should be clinically explored. PMID:19270508

Di Gennaro, Elena; Bruzzese, Francesca; Pepe, Stefano; Leone, Alessandra; Delrio, Paolo; Subbarayan, Pochi R; Avallone, Antonio; Budillon, Alfredo

2009-05-01

210

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

Science.gov (United States)

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

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

2014-07-01

211

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

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

212

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

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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

213

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.

2012-12-15

214

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

215

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

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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

216

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

217

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

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Full Text Available INTRODUCTION: The role of ovarian hormones and nitric oxide in learning and memory has been widely investigated. OBJECTIVE: The present study was carried out to evaluate the effect of the nitric oxide synthase (NOS inhibitor, N (G-nitro-L-arginine methyl ester (L-NAME, on the ability of estradiol to improve learning in OVX rats using the Morris water maze. METHODS: Forty rats were divided into five groups: (1 ovariectomized (OVX, (2 ovariectomized-estradiol (OVX-Est, (3 ovariectomized-L-NAME 10 (OVX-LN 10, (4 ovariectomized-L-NAME 50 (OVX-LN 50 and (5 ovariectomized-estradiol-L-NAME 50 (OVX-Est-LN 50. The animals in the OVX-Est group were treated with a weekly injection of estradiol valerate (2 mg/kg; i.m.. The OVX-LN 10 and OVX-LN 50 groups were treated with daily injections of 10 and 50 mg/kg L-NAME (i.p., respectively. The animals in the OVX-Est-LN 50 group received a weekly injection of estradiol valerate and a daily injection of 50 mg/kg L-NAME. After 8 weeks, all animals were tested in the Morris water maze. RESULTS: The animals in the OVX-Est group had a significantly lower latency in the maze than the OVX group (p<0.001. There was no significant difference in latency between the OVX-LN 10 and OVX-LN 50 groups in comparison with the OVX group. The latency in the OVX-Est-LN 50 group was significantly higher than that in the OVX-Est group (p<0.001. CONCLUSION: These results show that L-NAME treatment attenuated estradiol-mediated enhancement of spatial learning and memory in OVX rats, but it had no significant effect in OVX rats without estrogen, suggesting an interaction of nitric oxide and estradiol in these specific brain functions.

Hamid Azizi-Malekabadi

2011-01-01

218

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  

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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

219

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-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

2006-10-15

220

Thromboxane synthase immunohistochemistry in inflammatory bowel disease  

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Background: Thromboxanes are produced in excess in inflammatory bowel disease. Preliminary reports suggest that ridogrel, a thromboxane synthase inhibitor, is anti-inflammatory and may have therapeutic benefits in patients with ulcerative colitis.

Carty, E.; Nickols, C.; Feakins, R. M.; Rampton, D. S.

2002-01-01

 
 
 
 
221

Enhancement of the anti-inflammatory and anti-arthritic effects of theophylline by a low dose of a nitric oxide donor or non-specific nitric oxide synthase inhibitor  

Science.gov (United States)

Background and purpose: Although there are many new specific phosphodiesterase inhibitors with anti-inflammatory activity, none have yet reached the market because of their low therapeutic efficacy. Our study was aimed to evaluate the anti-inflammatory and anti-arthritic effect of an established phosphodiesterase inhibitor, theophylline, and to investigate the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP) or NO synthase inhibitor, L-NG-monomethyl arginine (L-NMMA) on its actions. Experimental approach: The effects of theophylline alone and combined with SNP or L-NMMA on the pathogenesis of adjuvant-induced arthritis in rats were evaluated. Key results: Prophylactic or therapeutic doses of theophylline significantly ameliorated the pathogenesis of adjuvant arthritis in rats as evidenced by a significant decrease in the arthritis index, hind paws volume, ankle joint diameter, fever, body weight loss and hyperalgesia in a dose-dependent manner. Inflammatory cellular infiltrate in synovium of ankle joint and pannus formation were also markedly inhibited. Interleukin-10 (IL-10) levels were significantly increased in arthritic rats given theophylline alone or in combination with either SNP or L-NMMA. Co-administration of a low dose of SNP or L-NMMA enhanced significantly the anti-inflammatory and anti-arthritic effect of theophylline. In contrast, a high dose of SNP counteracted the anti-inflammatory and anti-arthritic effects of theophylline. Conclusions and Implication: These findings confirm the anti-inflammatory and anti-arthritic activities of theophylline and suggest a new approach to enhance the anti-inflammatory and anti-arthritic effects of theophylline would be to administer it in combination with a low dose of a NO donor or a non-specific NO synthase inhibitor.

Gomaa, Adel; Elshenawy, Mohsen; Afifi, Noha; Mohammed, Eman; Thabit, Romany

2009-01-01

222

Shedding of hyaluronate synthase from streptococci.  

Science.gov (United States)

Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state. PMID:2109602

Mausolf, A; Jungmann, J; Robenek, H; Prehm, P

1990-04-01

223

1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)  

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The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhib...

He, Xin; Reeve, Anne Mcelwee; Desai, Umesh R.; Kellogg, Glen E.; Reynolds, Kevin A.

2004-01-01

224

The Dominant Inhibitory Chalcone Synthase Allele C2-Idf (Inhibitor diffuse) From Zea mays (L.) Acts via an Endogenous RNA Silencing Mechanism  

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The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nat...

Della Vedova, Chris B.; Lorbiecke, Rene?; Kirsch, Helene; Schulte, Michael B.; Scheets, Kay; Borchert, Lutz M.; Scheffler, Brian E.; Wienand, Udo; Cone, Karen C.; Birchler, James A.

2005-01-01

225

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

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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.

2011-01-01

226

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

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

2012-01-01

227

Benzo[d]isothiazole 1,1-dioxide derivatives as dual functional inhibitors of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1.  

Science.gov (United States)

A series of 6-nitro-3-(m-tolylamino) benzo[d]isothiazole 1,1-dioxide analogues were synthesized and evaluated for their inhibition activity against 5-lipoxygenase (5-LOX) and microsomal prostaglandin E2 synthase (mPGES-1). These compounds can inhibit both enzymes with IC50 values ranging from 0.15 to 23.6?M. One of the most potential compounds, 3g, inhibits 5-LOX and mPGES-1 with IC50 values of 0.6?M, 2.1?M, respectively. PMID:24794107

Shang, Erchang; Wu, Yiran; Liu, Pei; Liu, Ying; Zhu, Wei; Deng, Xiaobing; He, Chong; He, Shan; Li, Cong; Lai, Luhua

2014-06-15

228

An Innovative Strategy for Dual Inhibitor Design and Its Application in Dual Inhibition of Human Thymidylate Synthase and Dihydrofolate Reductase Enzymes  

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Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover po...

Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Lee, Keun Woo

2013-01-01

229

Non-Bisphosphonate Inhibitors of Isoprenoid Biosynthesis Identified via Computer-Aided Drug Design  

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The relaxed complex scheme, a virtual-screening methodology that accounts for protein receptor flexibility, was used to identify a low-micromolar, non-bisphosphonate inhibitor of farnesyl diphosphate synthase. Serendipitously, we also found that several predicted farnesyl diphosphate synthase inhibitors were low-micromolar inhibitors of undecaprenyl diphosphate synthase. These results are of interest because farnesyl diphosphate synthase inhibitors are being pursued as both anti-infective and...

Durrant, Jacob D.; Cao, Rong; Gorfe, Alemayehu A.; Zhu, Wei; Li, Jikun; Sankovsky, Anna; Oldfield, Eric; Mccammon, J. Andrew

2011-01-01

230

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

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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

231

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.

2014-01-01

232

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  

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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

233

Imidazolinones and Acetohydroxyacid Synthase from Higher Plants  

Science.gov (United States)

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

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

1987-01-01

234

The dominant inhibitory chalcone synthase allele C2-Idf (inhibitor diffuse) from Zea mays (L.) acts via an endogenous RNA silencing mechanism.  

Science.gov (United States)

The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing. PMID:15956664

Della Vedova, Chris B; Lorbiecke, René; Kirsch, Helene; Schulte, Michael B; Scheets, Kay; Borchert, Lutz M; Scheffler, Brian E; Wienand, Udo; Cone, Karen C; Birchler, James A

2005-08-01

235

4-Hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-one as inhibitors of tyrosyl-tRNA synthase: Synthesis, molecular docking and antibacterial evaluation  

Science.gov (United States)

A series of novel 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors were synthesized. Of these compounds, 4-(naphthalen-1-ylmethyl)-5-oxo-2,5-dihydrothiophen-3-yl-2-(4-hydroxyphenyl)acetate (29) was the most potent. The binding model and structure-activity relationship indicate that replacement of phenyl acetate in the side chain of 29 with a substituent containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 29 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC50 value of 0.21 ?g/mL.

Sun, Juan; Liu, Jia-Jia; Zhou, Wei; Guo, Feng-Jiao; Wang, Xin-Yi; Zhu, Hai-Liang

2014-01-01

236

Antidepressant-like effect of nitric oxide synthase inhibitors and sildenafil against lipopolysaccharide-induced depressive-like behavior in mice.  

Science.gov (United States)

Inflammation, oxidative and nitrosative stress underlie depression being assessed in rodents by the systemic administration of lipopolysacharide (LPS). There is an increasing body of evidence of an involvement of nitric oxide (NO) pathway in depression, but this issue was not investigated in LPS-induced model. Thus, herein we evaluated the effects of NO-pathway-modulating drugs, named aminoguanidine, l-NAME, sildenafil and l-arginine, on the behavioral (forced swimming test [FST], sucrose preference [SPT] and prepulse inhibition [PPI] of the startle) and neurochemical (glutathione [GSH], lipid peroxidation, IL-1?) alterations in the prefrontal cortex, hippocampus and striatum as well as in BDNF levels in the hippocampus 24h after LPS (0.5mg/kg, i.p.) administration, a time-point related to depressive-like behavior. Twenty-four hours post LPS there was an increase in immobility time in the FST, decrease in sucrose preference and PPI levels accompanied by a decrease in GSH levels and an increase in lipid peroxidation, IL-1? and hippocampal BDNF levels suggestive of a depressive-like state. The pretreatment with the NOS inhibitors, l-NAME and aminoguanidine as well as sildenafil prevented the behavioral and neurochemical alterations induced by LPS, although sildenafil and l-NAME were not able to prevent the increase in hippocampal BDNF levels induced by LPS. The iNOS inhibitor, aminoguanidine, and imipramine prevented all behavioral and neurochemical alterations induced by LPS. l-arginine did not prevent the alterations in immobility time, sucrose preference and GSH induced by LPS. Taken together our results show that the NO-cGMP pathway is important in the modulation of the depressive-like alterations induced by LPS. PMID:24662848

Tomaz, V S; Cordeiro, R C; Costa, A M N; de Lucena, D F; Nobre Júnior, H V; de Sousa, F C F; Vasconcelos, S M M; Vale, M L; Quevedo, J; Macêdo, D

2014-05-30

237

Role of glycogen synthase kinase 3 beta (GSK3? in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA in MCF-7 breast cancer cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Histone deacetylase inhibitors (HDACIs have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1 complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3?, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3? in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA, a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3? in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA resulted in the dephosphorylation of Akt and GSK3?. Selective inhibition of GSK3? attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3? as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

Lam Eric

2006-10-01

238

The combination of epidermal growth factor and glycogen synthase kinase 3 inhibitor support long-term self-renewal of Sca-1 positive hepatic progenitor cells from normal adult mice  

Directory of Open Access Journals (Sweden)

Full Text Available Isolation and long-term maintenance of hepatic progenitor cells (HPCs from healthy, non-injured adult livers remains challenging due to the lack of specific surface markers for selection and a limited understanding of the mechanisms for maintaining self-renewal. Previously, we identified a Sca-1 positive, bipotent HPC population in the peri-portal region of adult liver, and found MAPK/ERK and Wnt/?-Catenin pathways to be synergistically involved in their proliferation. In this study, we report the long-term culture of Sca-1 positive HPCs with epidermal growth factor (EGF and CHIR99021, a small molecule inhibitor of glycogen synthase kinase 3 (GSK-3. Sca-1+ HPCs remain non-tumorigenic when passaged 35 times in vitro over 1 year. Flow cytometric analysis indicates that HPCs are positive for Sca-1 and putative liver progenitor cell markers, including CD13, CD24 and Prominin-1, but negative for hematopoietic/endothelial cell markers CD31, CD34, CD45, CD90 and CD117. Immunocyto-chemistry and RT-PCR indicate Sca-1+ HPCs express albumin (ALB, ?-fetoprotein (AFP, cytokeratin19 (CK19, Sox9 and a panel of special hepatic progenitor transcriptional factors. Moreover, Sca-1+ HPCs are able to differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions in vitro and can take part in liver repopulation in an acetaminophen (APAP induced liver injury mouse model. This study provides a paradigm to capture and maintain HPCs from naive liver tissue and offers a valuable cell model for investigating the molecular mechanisms underlying the cell lineage relationship in normal liver.

Cai-Xia Jin

2013-07-01

239

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

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

240

Computational study on the carboligation reaction of acetohidroxyacid synthase: new approach on the role of the HEThDP- intermediate.  

Science.gov (United States)

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl-thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP(-)). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP(-) has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP(-)) and a second pyruvate molecule. Our results suggest that additional acid-base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid-base properties of the HEThDP(-) intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP(-) intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. PMID:20225259

Jaña, Gonzalo; Jiménez, Verónica; Villà-Freixa, Jordi; Prat-Resina, Xavier; Delgado, Eduardo; Alderete, Joel

2010-05-15

 
 
 
 
241

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  

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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

242

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  

Directory of Open Access Journals (Sweden)

Full Text Available The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyltetrazole 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

2008-01-01

243

Discovery of a potent, orally bioavailable and highly selective human neuronal nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5-yl)thiophene-2-carboximidamide as a pre-clinical development candidate for the treatment of migraine.  

Science.gov (United States)

We recently reported a series of 1,6-disubstituted indoline-based thiophene amidine compounds (5) as selective neuronal nitric oxide synthase (nNOS) inhibitors to mitigate the cardiovascular liabilities associated with hERG K(+) channel inhibition (IC(50) = 4.7 ?M) with previously reported tetrahydroquinoline-based selective nNOS inhibitors (4). The extended structure-activity relationship studies within the indoline core led to the identification of 43 as a selection candidate for further evaluations. The in vivo activity in two different pain (spinal nerve ligation and migraine pain) models, the excellent physicochemical and pharmacokinetic properties, oral bioavailability (F(po) = 91%), and the in vitro safety profile disclosed in this report make 43 an ideal candidate for further evaluation in clinical applications related to migraine pain. PMID:22840695

Annedi, Subhash C; Maddaford, Shawn P; Ramnauth, Jailall; Renton, Paul; Rybak, Taras; Silverman, Sarah; Rakhit, Suman; Mladenova, Gabriela; Dove, Peter; Andrews, John S; Zhang, Dongqin; Porreca, Frank

2012-09-01

244

Development and binding mode assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-?-glutamyl-D-glutamic acid (BGC 945), a novel thymidylate synthase inhibitor that targets tumor cells.  

Science.gov (United States)

N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-?-glutamyl-d-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with ?-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through ?-folate receptor (?-FR) transport. The ?-FR, a cell-surface receptor glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2'-deoxyuridine-5'-monophosphate, and a model for a similar complex with human TS. PMID:23710599

Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O'Connell, Joseph D; Griner, Sarah; Finer-Moore, Janet S; Stroud, Robert M

2013-07-11

245

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

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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

246

Higher plant cellulose synthases  

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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

247

Discovery of cis-N-(1-(4-(methylamino)cyclohexyl)indolin-6-yl)thiophene-2-carboximidamide: a 1,6-disubstituted indoline derivative as a highly selective inhibitor of human neuronal nitric oxide synthase (nNOS) without any cardiovascular liabilities.  

Science.gov (United States)

A series of 1,6-disubstituted indoline derivatives were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS) designed to mitigate the cardiovascular liabilities associated with previously reported tetrahydroquinoline-based selective neuronal NOS inhibitors due to higher lipophilicity ( J. Med. Chem. 2011 , 54 , 5562 - 5575 ). This new series produced similar potency and selectivity among the NOS isoforms and was devoid of any cardiovascular liabilities associated with QT prolongation due to hERG activity or endothelial NOS mediated vasoconstriction effect. The SAR studies led to the identification of cis-45, which was shown to reverse thermal hyperalgesia in vivo in the spinal nerve ligation model of neuropathic pain with excellent safety profile (off-target activities at 80 CNS related receptors/ion channels/transporters). The results presented in this report make cis-45 as an ideal tool for evaluating the potential role of selective nNOS inhibitors in CNS related disorders where excess NO produced by nNOS is thought to play a crucial role. PMID:22175766

Annedi, Subhash C; Ramnauth, Jailall; Maddaford, Shawn P; Renton, Paul; Rakhit, Suman; Mladenova, Gabriela; Dove, Peter; Silverman, Sarah; Andrews, John S; Felice, Milena D; Porreca, Frank

2012-01-26

248

Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases.  

Science.gov (United States)

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and beta-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates. PMID:17083961

Baykal, Ahmet; Chakraborty, Sumit; Dodoo, Afua; Jordan, Frank

2006-12-01

249

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.

250

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

251

An Arabidopsis callose synthase.  

Science.gov (United States)

Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant. PMID:12081364

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

2002-08-01

252

Inhibition of chitin synthase 2 and antifungal activity of lignans from the stem bark of Lindera erythrocarpa.  

Science.gov (United States)

Potent chitin synthase 2 inhibitors, methyllinderone (1), linderone (2) and kanakugiol (3) were isolated from the stem bark of L. erythrocarpa Makino (Lauraceae). These compounds inhibited chitin synthase 2 with IC(50) values of 23.3, 21.4 and 23.8 microg/mL, respectively. Methyllinderone (1) and linderone (2) exhibited no inhibitory activities for chitin synthases 1 and 3 from S. cerevisiae, and chitin synthase 1 from Candida albicans up to the concentration of 280 microg/mL, while kanakugiol (3) exhibited very weak activity against chitin synthase 1 of C. albicans with an IC(50) of 160 microg/mL. All of the compounds showed moderate to weak antifungal activities against various pathogenic fungi (MIC: 8 - >128 microg/mL) including Cryptococcus neoformans, Aspergillus fumigatus, and Colletotrichum lagenarium. The results indicate that these compounds are specific inhibitors of chitin synthase 2 and can potentially serve as antifungal agents. PMID:17538872

Hwang, Eui Il; Lee, Yun Mi; Lee, Sang Myung; Yeo, Woon Hyung; Moon, Jae Sun; Kang, Tae Hoon; Park, Ki Duk; Kim, Sung Uk

2007-06-01

253

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

254

Dimers of mitochondrial ATP synthase form the permeability transition pore.  

Science.gov (United States)

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D; Petronilli, Valeria; Zoratti, Mario; Szabó, Ildikó; Lippe, Giovanna; Bernardi, Paolo

2013-04-01

255

Heme coordination of NO in NO synthase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A current question in nitric oxide (NO) biology is whether NO can act as a feedback inhibitor of NO synthase (NOS). We have approached this problem by examining the interaction of NO with neuronal NOS by optical absorption and resonance Raman scattering spectroscopies. Under an inert atmosphere NO coordinated to the heme iron in both the oxidized and reduced forms of NOS. The Soret and visible optical absorption transitions are detected at 436 and at 567 nm, respectively, in the Fe(2+)-NO hem...

Wang, J.; Rousseau, D. L.; Abu-soud, H. M.; Stuehr, D. J.

1994-01-01

256

Inhibition of glucosylceramide synthase stimulates autophagy flux in neurons.  

Science.gov (United States)

Aggregate-prone mutant proteins, such as ?-synuclein and huntingtin, play a prominent role in the pathogenesis of various neurodegenerative disorders; thus, it has been hypothesized that reducing the aggregate-prone proteins may be a beneficial therapeutic strategy for these neurodegenerative disorders. Here, we identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol and Genz-123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT-mammalian target of rapamycin (mTOR) signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on-target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant ?-synuclein in neurons, it does so, according to our data, through autophagy-independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease. Inhibition of GlcCer synthase enhances autophagy by inhibiting AKT-mTOR signaling, and increases the number and size of lysosomal/late endosomal structures. Furthermore, inhibition of GlcCer synthase decreased levels of mutant ?-synuclein in neurons, which may represent a potential therapeutic target for Parkinson's disease. PMID:24494600

Shen, Wei; Henry, Anastasia G; Paumier, Katrina L; Li, Li; Mou, Kewa; Dunlop, John; Berger, Zdenek; Hirst, Warren D

2014-06-01

257

A rapid, radiometric assay for sucrose synthase  

International Nuclear Information System (INIS)

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

1990-05-01

258

Folate binding site of flavin-dependent thymidylate synthase.  

Science.gov (United States)

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). 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 FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs. PMID:23019356

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

2012-09-25

259

Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production.  

Science.gov (United States)

The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis ?-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene. PMID:22153510

McAndrew, Ryan P; Peralta-Yahya, Pamela P; DeGiovanni, Andy; Pereira, Jose H; Hadi, Masood Z; Keasling, Jay D; Adams, Paul D

2011-12-01

260

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

Scientific Electronic Library Online (English)

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

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

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

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

A.M. Brighenti

2001-04-01

262

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

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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

263

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis  

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Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway fo...

Smanski, Michael J.; Yu, Zhiguo; Casper, Jeffrey; Lin, Shuangjun; Peterson, Ryan M.; Chen, Yihua; Wendt-pienkowski, Evelyn; Rajski, Scott R.; Shen, Ben

2011-01-01

264

Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides.  

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In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P...

Sakurai, H.; Kohsaka, H.; Liu, M. F.; Higashiyama, H.; Hirata, Y.; Kanno, K.; Saito, I.; Miyasaka, N.

1995-01-01

265

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

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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

266

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic  

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Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibiti...

Jaffe, Eileen K.; Kervinen, Jukka; Martins, Jacob; Stauffer, Fre?de?ric; Neier, Reinhard; Wlodawer, Alexander; Zdanov, Alexander

2010-01-01

267

Farnesyl diphosphate synthase assay.  

Science.gov (United States)

Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts. PMID:24777789

Arró, Montserrat; Manzano, David; Ferrer, Albert

2014-01-01

268

Imidazolinones and acetohydroxyacid synthase from higher plants: properties of the enzyme from maize suspension culture cells and evidence for the binding of imazapyr to acetohydroxyacid synthase in vivo.  

Science.gov (United States)

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

Muhitch, M J; Shaner, D L; Stidham, M A

1987-02-01

269

Thymidylate synthase pharmacogenetics.  

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Thymidylate synthase (TYMS) is an important target for chemotherapy drugs, such as 5-fluorouracil (5FU) and methotrexate. Over-expression of TYMS is linked to resistance to TYMS-targeted chemotherapy drugs. Currently there is no protocol for selecting cancer patients at risk for drug resistance prior to chemotherapy treatment. Three polymorphisms in the 5' and 3' untranslated regions (5'UTR and 3'UTR) of the thymidylate synthase gene have been shown to influence TYMS expression. Preliminary data has suggested a poorer response rate to 5FU or methotrexate is seen in patients with 3 copies of a 28 bp tandem repeat in the 5'UTR enhancer region (TSER polymorphism) and this relationship may be further clarified by the presence of a single nucleotide polymorphism (SNP) with the second repeat of the 3 repeat (TSER(*)3) allele. A 6 bp deletion in the 3'UTR of the TYMS gene has also been shown to affect TYMS RNA expression and has a significant association with poor outcome in 5FU treated patients. Evidence linking all 3 TYMS polymorphisms with TYMS expression and patient response to TYMS-targeted chemotherapy treatment will be highlighted. PMID:16267625

Marsh, Sharon

2005-12-01

270

Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.  

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Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors. PMID:22474324

Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

2012-05-25

271

Isolation of streptococcal hyaluronate synthase.  

Science.gov (United States)

Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography. PMID:3092808

Prehm, P; Mausolf, A

1986-05-01

272

78 FR 9317 - Glycine max  

Science.gov (United States)

...EPA-HQ-OPP-2012-0795; FRL-9376-4] Glycine max Herbicide-Resistant Acetolactate Synthase...a tolerance for residues of the Glycine max herbicide-resistant acetolactate synthase...permissible level for residues of Glycine max herbicide-resistant acetolactate...

2013-02-08

273

Classification of fungal chitin synthases.  

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Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced am...

Bowen, A. R.; Chen-wu, J. L.; Momany, M.; Young, R.; Szaniszlo, P. J.; Robbins, P. W.

1992-01-01

274

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

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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

275

Mechanism of Action and Inhibition of dehydrosqualene Synthase  

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'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

276

Impaired ovulation in mice with targeted deletion of the neuronal isoform of nitric oxide synthase.  

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BACKGROUND: Nitric oxide (NO) plays an important role in numerous reproductive processes. To date, most studies have assessed the role of NO by using nonspecific pharmacological inhibitors of the precursor to NO, nitric oxide synthase (NOS). These pharmacological NOS inhibitors suppress all isoforms of NOS; thus, the precise contribution of each isoform to female reproductive physiology is unknown. The purpose of this study was to determine the specific role of neuronal NOS (nNOS) in the regu...

Klein, S. L.; Carnovale, D.; Burnett, A. L.; Wallach, E. E.; Zacur, H. A.; Crone, J. K.; Dawson, V. L.; Nelson, R. J.; Dawson, T. M.

1998-01-01

277

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase  

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Thymidylate synthase (TS) is an important target of several chemotherapeutic agents, including 5-FU and raltitrexed (Tomudex). During TS inhibition, TTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). Thus, BER initiated by uracil DNA glycosylase (UDG) activity has been hypothesized to influence the toxicity induced by TS inhibitors. In this study we created a human cell line expressing the Ugi protein inhibitor...

Luo, Yuhong; Walla, Mike; Wyatt, Michael D.

2008-01-01

278

Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii.  

Science.gov (United States)

A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P?cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii. PMID:23271570

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

2013-03-01

279

An acyl-SAM analog as an affinity ligand for identifying quorum sensing signal synthases.  

Science.gov (United States)

N-Acylhomoserine lactones (AHLs) are quorum sensing signals produced by Gram-negative bacteria. We here report the affinity purification of AHL synthases using beads conjugated with an enzyme inhibitor, which was designed based on the catalytic intermediate acyl-SAM. PMID:24955553

Kai, Kenji; Fujii, Hiroki; Ikenaka, Rui; Akagawa, Mitsugu; Hayashi, Hideo

2014-07-01

280

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

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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

 
 
 
 
281

Hypoxia–Reoxygenation Triggers Coronary Vasospasm in Isolated Bovine Coronary Arteries via Tyrosine Nitration of Prostacyclin Synthase  

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The role of peroxynitrite in hypoxia–reoxygenation-induced coronary vasospasm was investigated in isolated bovine coronary arteries. Hypoxia–reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker. The inactivation of PGI2 synthase, as evidenced by ...

Zou, Ming-hui; Bachschmid, Markus

1999-01-01

282

Liver isozyme of glycogen synthase  

International Nuclear Information System (INIS)

The work described was aimed at comparing the liver isozymes of glycogen synthase in terms of primary structure and phosphorylation patterns, with the better studied muscle counterpart. Rat liver glycogen synthase was purified to apparent homogeneity. It was subjected to multiple phosphorylation by eight protein kinases. Phosphorylation sites were distributed between two CNBr-fragments, CB-1 (14,000) and CB-2 (28,000). Amino acid sequences of phosphopeptides of rabbit liver glycogen synthases modified by cyclic AMP-dependent protein kinase were determined and three phosphorylation sites were identified. A simple and effective procedure for determining the location of phosphorylation sites in phosphopeptides was also developed. The method employed measurement of [32P]inorganic phosphate release during Edman degradation cycles using the gas phase sequencer. Comparison of the liver and muscle isozymes has shown that similarities are more prominent than differences and isozymes share several important properties in multiple phosphorylation and hormonal regulation

1988-01-01

283

Drugs targeting nitric oxide synthase for migraine treatment.  

Science.gov (United States)

Introduction: Ample evidence that nitric oxide (NO) is a causative molecule in migraine has encouraged research to develop drugs that target the NO-cGMP cascade for migraine treatment. NO synthase (NOS) inhibition is an innovative therapeutic principle. Areas covered: This paper reviews the rationale underlying NOS inhibition in migraine treatment. It also provides a review on the efficacy and safety data for NOS inhibitors (nonselective NOS inhibitor L-N(G)-methyl-arginine hydrochloride [L-NMMA], selective inducible NOS [iNOS] inhibitors GW273629 and GW274150, combined neuronal NOS [nNOS] inhibitor and 5-HT1B/1D receptor agonist NXN-188) in acute or preventive migraine treatment. Expert opinion: The data highlighted herein, from four placebo-controlled trials and 1 open-labeled clinical trial using 4 different NOS inhibitors on a total of 705 patients, provide convincing efficacy data only for the nonselective NOS inhibitor L-NMMA. Unfortunately, this NOS inhibitor raises cardiovascular safety concerns and has an unfavorable pharmacokinetic profile. As experimental studies predicted, iNOS inhibitors are ineffective in migraine. Still, upcoming selective nNOS inhibitors are a hope for migraine treatment, with the nNOS isoform being most clearly involved in trigeminovascular transmission and central sensitization. Future studies should help to clarify whether NOS inhibition is equally fruitful in acute and preventive treatment. It should also clarify if nNOS inhibition holds promise as a therapeutic tool for the treatment of chronic migraine and other forms of headache. PMID:24818644

Barbanti, Piero; Egeo, Gabriella; Aurilia, Cinzia; Fofi, Luisa; Della-Morte, David

2014-08-01

284

INTERACTION BETWEEN GLYCOGENIN AND GLYCOGEN SYNTHASE  

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Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1 and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylati...

Skurat, Alexander V.; Dietrich, Amy D.; Roach, Peter J.

2006-01-01

285

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

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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, hymeglusin’s effects on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of inhibitor from culture media, a growth curve inflection point at 3.1 hr is observed (versu...

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

2012-01-01

286

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

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

P.J. Christoffoleti

2001-04-01

287

p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii.  

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Chalcone synthase and stilbene synthase are plant-specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization. PMID:10469148

Akiyama, T; Shibuya, M; Liu, H M; Ebizuka, Y

1999-08-01

288

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate.  

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Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis. PMID:1989499

Balinska, M; Rhee, M; Whiteley, J M; Priest, D G; Galivan, J

1991-01-01

289

Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo  

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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

290

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

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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

291

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase  

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Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit ?-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.

Jaffe, Eileen K.; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J.; Myler, Peter J.; Roos, David S.

2011-01-01

292

Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase and as potential antitumor agents.  

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Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent. PMID:16279780

Gangjee, Aleem; Lin, Xin; Kisliuk, Roy L; McGuire, John J

2005-11-17

293

Identification of avian wax synthases  

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Abstract Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. Resul...

Biester Eva-Maria; Hellenbrand Janine; Gruber Jens; Hamberg Mats; Frentzen Margrit

2012-01-01

294

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

Science.gov (United States)

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

Saxena, Praveen K.; King, John

1990-01-01

295

Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron : Evidence for Separable Sites of Action on the Target Enzyme.  

Science.gov (United States)

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

Saxena, P K; King, J

1990-11-01

296

Lipophilic iminosugars : synthesis and evaluation as inhibitors of glucosylceramide metabolism  

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The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and ?-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of these three enzymes among which several that improve upon the inhibition profile of the lead compound in this study. The successful use of lipophilic iminosugars in type 2 diabetes models and ...

Wennekes, Tom

2008-01-01

297

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
 

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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...

2013-01-01

298

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

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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

299

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

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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

300

Kinetic Characterization of Squalene Synthase from Trypanosoma cruzi: Selective Inhibition by Quinuclidine Derivatives?  

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The biosynthesis of sterols is a major route for the development of antitrypanosomals. Squalene synthase (SQS) catalyzes the first step committed to the biosynthesis of sterols within the isoprenoid pathway, and several inhibitors of the enzyme have selective antitrypanosomal activity both in vivo and in vitro. The enzyme from Trypanosoma cruzi is a 404-amino-acid protein with a clearly identifiable membrane-spanning region. In an effort to generate soluble recombinant enzyme, we have express...

Sealey-cardona, Marco; Cammerer, Simon; Jones, Simon; Ruiz-pe?rez, Luis M.; Brun, Reto; Gilbert, Ian H.; Urbina, Julio A.; Gonza?lez-pacanowska, Dolores

2007-01-01

 
 
 
 
301

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

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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

302

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

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

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

2006-01-01

303

Chitin synthase in Candida albicans: comparison of digitonin-permeabilized cells and spheroplast membranes.  

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The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid); partially prevented by the protease inhibitors antipain, leupeptin, and N alpha-tosyl-L-lysyl chloromethyl ketone; and also partially prevented by EDTA. Thus, a clostripain-like pro...

Georgopapadakou, N. H.; Smith, S. A.

1985-01-01

304

Retraction: Fatty Acid Synthase is a Novel Therapeutic Target in Multiple Myeloma  

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This study investigated the biological significance of the inhibition of fatty acid synthase (FAS) in multiple myeloma (MM) using the small molecule inhibitor Cerulenin. Cerulenin triggered growth inhibition in both MM cell lines and MM patient cells, and overcame the survival and growth advantages conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cells. It induced apoptosis in MM cell lines with only modest activation of caspase -8, -9, -3 and PARP; moreover, ...

Okawa, Yutaka; Hideshima, Teru; Ikeda, Hiroshi; Raje, Noopur; Vallet, Sonia; Kiziltepe, Tanyel; Yasui, Hiroshi; Enatsu, Sotaro; Pozzi, Samantha; Breitkreutz, Iris; Cirstea, Diana; Santo, Loredana; Richardson, Paul Gerard Guy; Anderson, Kenneth Carl

2008-01-01

305

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development  

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Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymi...

Rocca, Bianca; Spain, Lisa M.; Pure?, Ellen; Langenbach, Robert; Patrono, Carlo; Fitzgerald, Garret A.

1999-01-01

306

CREB DNA binding activity is inhibited by glycogen synthase kinase-3? and facilitated by lithium  

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The regulatory influences of glycogen synthase kinase-3? (GSK3?) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH-SY5Y cells. Activation of Akt (protein kinase B) with serum-increased phospho-serine-9-GSK3? (the inactive form of the enzyme), inhibited GSK3? activity, and increased CREB DNA binding activity. Inhibition of GSK3? by another paradigm, treatment with the selective inhibitor lithium, also increased CREB D...

Grimes, Carol A.; Jope, Richard S.

2001-01-01

307

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

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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

308

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

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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

309

Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy  

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Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in...

Sennlaub, Florian; Courtois, Yves; Goureau, Olivier

2001-01-01

310

Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy.  

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Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in...

Sennlaub, Florian; Courtois, Yves; Goureau, Olivier

2001-01-01

311

Acetaminophen-Induced Hepatotoxicity and Protein Nitration in Neuronal Nitric-Oxide Synthase Knockout Mice  

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In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and pro...

Agarwal, Rakhee; Hennings, Leah; Rafferty, Tonya M.; Letzig, Lynda G.; Mccullough, Sandra; James, Laura P.; Macmillan-crow, Lee Ann; Hinson, Jack A.

2012-01-01

312

Protective Effects of Statins after Embolic Focal Cerebral Ischemia in Endothelial Nitric Oxide Synthase Knockout Mice  

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Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by upregulating endogenous tissue plasminogen activator (tPA) and by enhancing clot lysis in a model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal caroti...

Asahi, Minoru; Thomas, Sunu; Yoshimura, Shin-ichi; Sumii, Toshihisa; Mori, Tatsuro; Qiu, Jianhua; Amin-hanjani, Sepideh; Huang, Paul L.; Liao, James K.; Lo, Eng H.; Moskowitz, Michael A.

2005-01-01

313

Mammalian N-acetylglutamate synthase  

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N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA-(NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirme...

Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

2004-01-01

314

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

315

Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.  

Science.gov (United States)

Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time. PMID:24849013

Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

2014-06-01

316

Structural analysis of chorismate synthase from Plasmodium falciparum: a novel target for antimalaria drug discovery.  

Science.gov (United States)

The shikimate pathway in Plasmodium falciparum provides several targets for designing novel antiparasitic agents for the treatment of malaria. Chorismate synthase (CS) is a key enzyme in the shikimate pathway which catalyzes the seventh and final step of the pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS. The structure of PfCS along with cofactor FMN was predicted by homology modeling using crystal structure of Helicobacter pylori chorismate synthase (HpCS). The quality of the model was validated using structure analysis servers and molecular dynamics. Dimeric form of PfCS was generated and the FMN binding mechanism involving movement of loop near active site has been proposed. Active site pocket has been identified and substrate 5-enolpyruvylshikimate 3-phosphate (EPSP) along with screened potent inhibitors has been docked. The study resulted in identification of putative inhibitors of PfCS with binding efficiency in nanomolar range. The selected putative inhibitors could lead to the development of anti-malarial drugs. PMID:21801743

Tapas, Satya; Kumar, Abhinav; Dhindwal, Sonali; Preeti; Kumar, Pravindra

2011-11-01

317

Nitric Oxide synthases and atrial fibrillation  

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Full Text Available 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 synthase 2 in multiple cell types in the myocardium. In certain conditions, the NOS enzymes may become uncoupled, shifting from production of nitric oxide to superoxide anion, a potent free radical and oxidant. Multiple lines of evidence suggest a role for nitric oxide synthases in the pathogenesis of atrial fibrillation. Therapeutic approaches to reduce atrial fibrillation by modulation of nitric oxide synthase activity may be beneficial, although further investigation of this strategy is needed.

CynthiaAnnCarnes

2012-04-01

318

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

1985-08-15

319

Intracellular compartmentation of CTP synthase in Drosophila.  

Science.gov (United States)

Compartmentation is essential for the localization of biological processes within a eukaryotic cell. ATP synthase localizes to organelles such as mitochondria and chloroplasts. By contrast, little is known about the subcellular distribution of CTP synthase, the critical enzyme in the production of CTP, a high-energy molecule similar to ATP. Here I describe the identification of a novel intracellular structure containing CTP synthase, termed the cytoophidium, in Drosophila cells. I find that cytoophidia are present in all major cell types in the ovary and exist in a wide range of tissues such as brain, gut, trachea, testis, accessory gland, salivary gland and lymph gland. In addition, I find CTP synthase-containing cytoophidia in other fruit fly species. The observation of compartmentation of CTP synthase now permits a broad range of questions to be addressed concerning not only the structure and function of cytoophidia but also the organization and regulation of CTP synthesis. PMID:20513629

Liu, Ji-Long

2010-05-01

320

Squalene Synthase As a Target for Chagas Disease Therapeutics  

Science.gov (United States)

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A.; Wang, Andrew H.-J.; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

2014-01-01

 
 
 
 
321

Nitric oxide synthase inhibition prevents acute QA-induced neurotoxicity  

Directory of Open Access Journals (Sweden)

Full Text Available In the present study we employed 7-NI, reportedly a selective inhibitor of neuronal nitric oxide synthase (NOS and the non-specific potent NOS inhibitor I-NAME, to investigate the possible involvement of nitric oxide (NO in quinolinic acid (QA-induced striatal toxicity in the rat. QA was administered unilaterally into the striatum of adult Wistar rats in the single dose of 150 nmol/L. The second and third group were treated with 7-NI and QA and I-NAME and QA. The control group was treated with O.9% saline solution likewise. Nitrite levels were decreased in the ipsi- and contralateral striatum, forebrain cortex and hippocampus in the group treated with NOS inhibitors (7-NI, I-NAME and QA compared to QA-treated animals. As 7-NI selectively inhibits the neuronal form of NOS, this study suggests that NO produced from a neuronal and not an epithelial source may contribute to neuronal damage in this model.

Vasiljevi? Ivana D.

2002-01-01

322

Squalene synthase as a target for chagas disease therapeutics.  

Science.gov (United States)

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. PMID:24789335

Shang, Na; Li, Qian; Ko, Tzu-Ping; Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A; Wang, Andrew H-J; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

2014-05-01

323

Mitochondrial F0F1-ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone  

Science.gov (United States)

BACKGROUND AND PURPOSE 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F0F1-ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH Kinetic analyses were performed on F0F1-ATP synthase in sub-mitochondrial particles and soluble F1-ATPase. Activity assays and immunodetection of the inhibitor protein IF1 were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS T1AM was a non-competitive inhibitor of F0F1-ATP synthase whose binding was mutually exclusive with that of the inhibitors IF1 and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF1 binding site, causing IF1 release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F0F1-ATP synthase consistent with displacement of endogenous IF1,, reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS Effects of T1AM on F0F1-ATP synthase were twofold: IF1 displacement and enzyme inhibition. By targeting F0F1-ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF1 and aurovertin binding sites are described.

Cumero, S; Fogolari, F; Domenis, R; Zucchi, R; Mavelli, I; Contessi, S

2012-01-01

324

The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases.  

Science.gov (United States)

Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme. PMID:2108169

DeWitt, D L; el-Harith, E A; Kraemer, S A; Andrews, M J; Yao, E F; Armstrong, R L; Smith, W L

1990-03-25

325

Umbelliferone Aminoalkyl Derivatives asInhibitors of Human Oxidosqualene: Lanosterol Cyclase  

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Abstract Human and murine lanosterol synthases (EC 5.4.99.7) were studied as targets of a series of umbelliferone aminoalkyl derivatives previously tested as inhibitors of oxidosqualene cyclases from other eukaryotes. Tests were carried out on cell cultures of human keratinocytes and mouse 3T3 fibroblasts incubated with radiolabeled acetate, and on homogenates prepared from yeast cells expressing human lanosterol synthase, incubated with radiolabeled oxidosqualene. In cell cultures of both hu...

Balliano, Gianni; Cravotto, Giancarlo; Viola, Franca Cecilia; Ermondi, Giuseppe; Taramino, Silvia; Tagliapietra, Silvia Maria; Oliaro Bosso, Simonetta

2009-01-01

326

Inducible nitric oxide synthase as a possible target in hypertension.  

Science.gov (United States)

Nitric oxide (NO) is an important vasodilator produced by vascular endothelium. Its enzymatic formation is derived from three different synthases: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) synthases. While relatively small amounts of NO produced by eNOS are important to cardiovascular homeostasis, high NO levels produced associated with iNOS activity may have detrimental consequences to the cardiovascular system and contribute to hypertension. In this article, we reviewed current literature and found mounting evidence indicating that increased iNOS expression and activity contribute to the pathogenesis of hypertension and its complications. Excessive amounts of NO produced by iNOS up-regulation can react with superoxide anions forming peroxynitrite, thereby promoting nitrosative stress and endothelial dysfunction. In addition, abnormal iNOS activity can up-regulate arginase activity, allowing it to compete with eNOS for L-arginine, thereby resulting in reduced NO bioavailability. This may also lead to eNOS uncoupling with enhanced production of superoxide anions instead of NO. All these alterations mediated by iNOS apparently contribute to hypertension and its complications. We also reviewed current evidence showing the effects of iNOS inhibitors on different animal models of hypertension. iNOS inhibition apparently exerts antihypertensive effects, decreases oxidative and nitrosative stress, and improves vascular function. Together, these studies highlight the possibility that iNOS is a potential pharmacological target in hypertension. PMID:24102471

Oliveira-Paula, Gustavo H; Lacchini, Riccardo; Tanus-Santos, Jose E

2014-02-01

327

Structure and Inhibition of Mouse Leukotriene C4 Synthase  

Science.gov (United States)

Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes’ steady-state kinetics. Although structural differences near the active site and along the C-terminal ?-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development.

Niegowski, Damian; Qureshi, Abdul Aziz

2014-01-01

328

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

329

A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*  

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Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

Klundt, Tim; Bocola, Marco; Lu?tge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

2009-01-01

330

Cellulose Synthase Complexes: Composition and Regulation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Live cell imaging has greatly advanced our knowledge on the molecular mechanism by which cellulose is deposited. Both the actin and microtubule cytoskeleton are involved in assuring the proper distribution, organization, and dynamics of cellulose synthase complexes (CSCs). This review is an update on the most recent progress on the characterization of the composition, regulation, and trafficking of CSCs. With the newly identified cellulose synthase interactive protein 1 (CSI1) on hand, we beg...

Lei, Lei; Li, Shundai; Gu, Ying

2012-01-01

331

Reduced activity of ATP synthase in mitochondria causes cytoplasmic male sterility in chili pepper.  

Science.gov (United States)

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the inability to produce functional pollen. The CMS-associated protein Orf507 (reported as Orf456 in previous researches) was previously identified as a candidate gene for mediating male sterility in pepper. Here, we performed yeast two-hybrid analysis to screen for interacting proteins, and found that the ATP synthase 6 kDa subunit containing a mitochondrial signal peptide (MtATP6) specifically interacted with Orf507. In addition, the two proteins were found to be interacted in vivo using bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Further functional characterization of Orf507 revealed that the encoded protein is toxic to bacterial cells. Analysis of tissue-specific expression of ATP synthase 6 kDa showed that the transcription level was much lower in anthers of the CMS line than in their wild type counterparts. In CMS plants, ATP synthase activity and content were reduced by more than half compared to that of the normal plants. Taken together, it can be concluded that reduced ATP synthase activity and ATP content might have affected pollen development in CMS plants. Here, we hypothesize that Orf507 might cause MtATP6 to be nonfunctional by changing the latter's conformation or producing an inhibitor that prevents the normal functioning of MtATP6. Thus, further functional analysis of mitochondrial Orf507 will provide insights into the mechanisms underlying CMS in plants. PMID:23274393

Li, Jinjie; Pandeya, Devendra; Jo, Yeong Deuk; Liu, Wing Yee; Kang, Byoung-Cheorl

2013-04-01

332

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

1987-01-01

333

Inhibition of inositol phosphorylceramide synthase by aureobasidin A in Candida and Aspergillus species.  

Science.gov (United States)

Inositol phosphorylceramide (IPC) synthase is an enzyme common to fungi and plants that catalyzes the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC. The reaction is a key step in fungal sphingolipid biosynthesis and the target of the antibiotics galbonolide A, aureobasidin A, and khafrefungin. As a first step toward understanding the antifungal spectrum of IPC synthase inhibitors, we examined the sensitivity of IPC synthase to aureobasidin A in membrane preparations of Candida species (Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) and Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). As expected, preparations from the five Candida species, all exquisitely susceptible to aureobasidin A (MICs, 400 pmol/min/mg of protein) sensitive to aureobasidin A (50% inhibitory concentrations [IC(50)s], 2 to 4 ng/ml). Surprisingly, preparations from the four Aspergillus species, including A. fumigatus and A. flavus, which are intrinsically resistant to aureobasidin A (MICs, >50 microgram/ml), had IPC synthase activity (specific activity, 1 to 3 pmol/min/mg of protein) also sensitive to aureobasidin A (IC(50)s, 3 to 5 ng/ml). The mammalian multidrug resistance modulators verapamil, chlorpromazine, and trifluoperazine lowered the MIC of aureobasidin A for A. fumigatus from >50 microgram/ml to 2 to 3 microgram/ml, suggesting that the resistance of this major fungal pathogen is the result of increased efflux. PMID:10681333

Zhong, W; Jeffries, M W; Georgopapadakou, N H

2000-03-01

334

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

335

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

Energy Technology Data Exchange (ETDEWEB)

A ..beta..-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 ..beta..-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 IC/sub 50/ of 0.12 ..mu..M. The enzymes HMG-CoA reductase, ..beta..-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 (/sup 14/C)acetate into sterols with an IC/sub 50/ of 6 ..mu..M, while incorporation of (/sup 3/H)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 ..mu..M L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed.

Greenspan, M.D.; Yudkovitz, J.B.; Lo, C.Y.L.; Chen, J.S.; Alberts, A.W.; Hunt, V.M.; Chang, M.N.; Yang, S.S.; Thompson, K.L.; Chiang, Y.C.P.; Chabala, J.C.

1987-11-01

336

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 (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). 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.

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

2009-06-01

337

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

Science.gov (United States)

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

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

1986-06-01

338

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.

339

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

340

Muscle NMDA receptors regulate the resting membrane potential through NO-synthase.  

Science.gov (United States)

The early postdenervation depolarization of rat diaphragm muscle fibres (8-10 mV) is substantially smaller (3 mV) when muscle strips are bathed with 1 mM L-glutamate (GLU) or N-methyl-D-aspartate (NMDA). The effects of GLU and NMDA are not seen in the presence of aminophosphonovaleric acid (APV), a blocker of NMDA-subtype of glutamate receptors, 5 mM Mg2+ (which blocks NMDA-controlled ion channels) and L-nitroarginine methylester (NAME), an inhibitor of NO-synthase. This indicates that NMDA-subtype of GLU receptors might be involved in the regulation of the membrane potential in muscle fibres, most probably through the NO-synthase system. PMID:8869279

Urazaev, A K; Magsumov, S T; Poletayev, G I; Nikolsky, E E; Vyskocil, F

1995-01-01

 
 
 
 
341

Activation and inhibition of CTP synthase from Trypanosoma brucei, the causative agent of African sleeping sickness.  

Science.gov (United States)

CTP Synthase from Trypanosoma brucei (TbCTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of antiprotozoal agents. GTP activates glutamine-dependent CTP formation catalyzed by TbCTPS at concentrations below 0.2 mM, but inhibits this activity at concentrations above 0.2 mM. TbCTPS catalyzes ammonia-dependent CTP formation, which is inhibited by purine derivatives such as GTP, guanosine, caffeine, and uric acid with IC(50) values of 460, 380, 480, and 100 ?M, respectively. These observations suggest that the purine ring may serve as a useful scaffold for the development of inhibitors of trypanosomal CTP synthase. PMID:21840216

Steeves, Craig H; Bearne, Stephen L

2011-09-15

342

Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.  

Science.gov (United States)

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases. PMID:10497176

Hörnsten, L; Su, C; Osbourn, A E; Garosi, P; Hellman, U; Wernstedt, C; Oliw, E H

1999-10-01

343

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

Science.gov (United States)

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

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

2010-06-01

344

Nuclear genetic defects of mitochondrial ATP synthase.  

Science.gov (United States)

Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis. PMID:24564666

Hejzlarová, K; Mrá?ek, T; Vrbacký, M; Kaplanová, V; Karbanová, V; N?sková, H; Pecina, P; Houšt?k, J

2014-01-01

345

Molecular cloning of prostacyclin (PGI2) synthase  

International Nuclear Information System (INIS)

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

1986-05-01

346

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass appr...

Coxon, F. P.; Helfrich, M. H.; Larijani, B.; Muzylak, M.; Dunford, J. E.; Marshall, D.; Mckinnon, A. D.; Nesbitt, S. A.; Horton, M. A.; Seabra, M. C.; Ebetino, F. H.; Rogers, M. J.

2001-01-01

347

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

2008-01-01

348

Exploration of the Active Site of Neuronal Nitric Oxide Synthase by the Design and Synthesis of Pyrrolidinomethyl 2-Aminopyridine Derivatives  

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Neuronal nitric oxide synthase (nNOS) represents an important therapeutic target for the prevention of brain injury and the treatment of various neurodegenerative disorders. A series of trans substituted amino pyrrolidinomethyl 2-aminopyridine derivatives (8–34) was designed and synthesized. A structure-activity relationship analysis led to the discovery of low nanomolar nNOS inhibitors [(±)-32 and (±)-34] with more than 1000-fold selectivity for nNOS over eNOS. Four enantiomerically pure...

2010-01-01

349

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

350

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

351

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

352

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.

353

Diversity and analysis of bacterial terpene synthases.  

Science.gov (United States)

Terpenoid compounds are generally considered to be plant or fungal metabolites, although a small number of odorous terpenoid metabolites of bacterial origin have been known for many years. Recently, extensive bacterial genome sequencing and bioinformatic analysis of deduced bacterial proteins using a profile hidden Markov model have revealed more than a hundred distinct predicted terpene synthase genes. Although some of these synthase genes might be silent in the parent microorganisms under normal laboratory culture conditions, the controlled overexpression of these genes in a versatile heterologous host has made it possible to identify the biochemical function of cryptic genes and isolate new terpenoid metabolites. PMID:22999173

Yamada, Yuuki; Cane, David E; Ikeda, Haruo

2012-01-01

354

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

355

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

356

Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis.  

Science.gov (United States)

ATP synthase was isolated from beef heart mitochondria by extraction with N,N-bis-(3-D-gluconamidopropyl)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion-exchange and gel-permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12-14 subunits and contained three tightly bound nucleotides. The co-reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light-driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (> 90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide-p-trifluormethoxyphenylhydrazone (> 95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light-driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria. PMID:8269926

Deisinger, B; Nawroth, T; Zwicker, K; Matuschka, S; John, G; Zimmer, G; Freisleben, H J

1993-12-01

357

Brainstem prolactin mRNA is enhanced in mice with suppressed neuronal nitric oxide synthase activity.  

Science.gov (United States)

Prolactin (PRL) and vasoactive intestinal polypeptide (VIP) mRNA levels were elevated in the brainstem of neuronal nitric oxide synthase (nNOS) gene knockout (KO) mice compared to the levels in nNOS control mice. In addition, PRL mRNA levels increased in the hypothalamus and the brainstem of nNOS control mice after administration of 7-nitro-indazole (7-NI), a relatively selective nNOS inhibitor. The results suggest that NO inhibits PRL. No differences in the genes measured were observed in inducible NOS KO mice. PMID:15469894

Chen, Lichao; Taishi, Ping; Duricka, Deborah; Krueger, James M

2004-10-22

358

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

359

Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earl...

1994-01-01

360

Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mice lacking neuronal nitric oxide synthase gene (ncNOS) were used to determine the enzymatic source of nitric oxide (NO) and its relationship with other putative inhibitory neurotransmitters. Inhibitory junction potentials (IJP) of circular smooth muscle of gastric fundus were studied. The IJP in the wild-type mice consists of overlapping components, the fast and slow IJPs. NOS inhibitor L-NA or VIP receptor antagonist VIP(10-28), blocks the slow IJP but not the fast IJP. The fast UP is bloc...

Mashimo, H.; He, X. D.; Huang, P. L.; Fishman, M. C.; Goyal, R. K.

1996-01-01

 
 
 
 
361

Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal?eosinophils  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor N?-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migrat...

1997-01-01

362

Glycogen synthase kinase 3? and 3? have distinct functions during cardiogenesis of zebrafish embryo  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3? (51 kDa) and GSK3? (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3? and GSK3?, making it difficult to ...

Lee Huang-Chieh; Tsai Jen-Ning; Liao Pei-Yin; Tsai Wei-Yuan; Lin Kai-Yen; Chuang Chung-Cheng; Sun Chi-Kuang; Chang Wen-Chang; Tsai Huai-Jen

2007-01-01

363

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

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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

364

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

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

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

1999-01-01

365

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

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

1992-01-01

366

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

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

Ober, Dietrich; Hartmann, Thomas

1999-01-01

367

Producing dicarboxylic acids using polyketide synthases  

Science.gov (United States)

The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

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

2013-10-29

368

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  

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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

369

14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase.  

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We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme. PMID:9359857

Sala, A; Garcia, M; Zarini, S; Rossi, J C; Folco, G; Durand, T

1997-11-15

370

The very-long-chain fatty acid synthase is inhibited by chloroacetamides.  

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The first elongation step to form very-long-chain fatty acids (VLCFAs) is catalyzed by the VLCFA-synthase. CoA-activated fatty acids react with malonyl-CoA to condense a C2-unit. As shown with recombinant enzyme this reaction is specifically inhibited by chloroacetamide herbicides. The inhibition is alleviated when the inhibitor (e.g. metazachlor) is incubated together with adequate concentrations of the substrate (e.g. oleoyl-CoA). Malonyl-CoA has no influence. However, once a chloroacetamide has been tightly bound to the synthase after an appropriate time it cannot be displaced anymore by the substrate. In contrast, oleoyl-CoA, is easily removed from the synthase by metazachlor. The irreversible binding of the chloroacetamides and their competition with the substrate explains the very low half-inhibition values of 10(-8) M and below. Chiral chloroacetamides like metolachlor or dimethenamid give identical results. However, only the (S)-enantiomers are active. PMID:15813378

Götz, Thomas; Böger, Peter

2004-01-01

371

Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.  

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Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7827039

Tang, M S; Askonas, L J; Penning, T M

1995-01-24

372

Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.  

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Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

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

2010-02-26

373

Mechanisms of action of FdUMP[10]: metabolite activation and thymidylate synthase inhibition.  

Science.gov (United States)

FdUMP[10] is a multimer of FdUMP, a suicide inhibitor of thymidylate synthase (TS), and was designed to bypass resistance to 5-fluorouracil (5FU). The aim of the study was to compare the effect of FdUMP[10] with 5FU and 5-fluoro-2-deoxyuridine (FUdR) in their efficacy to inhibit their target TS in resistant cells. Therefore cell lines FM3A/0, FM3A/TK- (deficient in thymidine kinase) and FM3A/TS- (deficient in thymidylate synthase) were used to determine TK dependency and specificity for TS inhibition. FdUMP[10] inhibited cell growth with greater potency than 5FU and FdUMP. Direct folate-based inhibitors Raltitrexed, GW1843U89 and Pemetrexed were also evaluated using these cell lines. In TK-deficient cells these folate-based inhibitors had greater potency than the fluoropyrimidines (FPs). Surprisingly, Pemetrexed even inhibited cell growth in TS-deficient cells. Incubation with nucleotidase and phosphatase inhibitors resulted in a reduction of cytotoxicity of FdUMP[10], indicating that the drug can be degraded outside the cells. In the TS in situ inhibition assay (TSIA) 24 h exposure of FM3A cells to 0.5 microM FdUMP and 0.05 microM FdUMP[10] decreased TSIA to 7 and 1% of control. Inhibition of nucleotidase and phosphatase activities reduced the effect of FdUMP[10], while the inhibitory effect was lower in cells lacking TK. FdUMP[10] can enter the cells intact, but also to some extent after dephosphorylation. In conclusion, FdUMP[10] can bypass resistance to FUdR by direct inhibition of TS. PMID:17549381

Bijnsdorp, I V; Comijn, E M; Padron, J M; Gmeiner, W H; Peters, G J

2007-07-01

374

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine.  

Science.gov (United States)

The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated from the Indian medicinal plants Pergularia pallida and Alangium lamarckii respectively. Cytotoxicity studies showed that cell growth of L. leichmannii was inhibited (IC50 = 40-45 microM) by all the three alkaloids, the concentrations > 80-90 microM resulting in complete loss of the enzyme activity. Ki values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were 10 x 10(-6) M, 9 x 10(-6) M and 7 x 10(-6) M respectively. These are typed as 'non-competitive' inhibitors of TS. All the three alkaloids inhibited (IC50 = 50 microM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n = 10), acute lymphocytic leukemia (n = 8) and metastatic solid tumours (n = 3). PMID:10844999

Rao, K N; Bhattacharya, R K; Veankatachalam, S R

1999-12-01

375

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

Science.gov (United States)

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, hymeglusin’s effects on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of inhibitor from culture media, a growth curve inflection point at 3.1 hr is observed (versus 0.7 hr for 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/solvent accessibility. The E. faecalis mvaS-hymeglusin co-crystal structure (1.95 Å) reveals virtually complete occlusion of bound inhibitor in a narrow tunnel that is largely occluded 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.

2012-01-01

376

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

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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

377

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

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In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-? (IFN-?). JAK inhibitors AG-490 and WHI-P154 decreased IFN-?-induced nuclear levels of signal transducer and activator of transcription 1? (STAT1?). JAK inhibitors ...

Sareila, Outi; Korhonen, Riku; Ka?rpa?nniemi, Outi; Nieminen, Riina; Kankaanranta, Hannu; Moilanen, Eeva

2006-01-01

378

Prostaglandin-H synthase inhibition by malonamides. Ring-opened analogues of phenylbutazone.  

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Recent reports of serious concern regarding the safe clinical use of phenylbutazone and its hydroxylated metabolite (oxyphenbutazone) as antiinflammatory agents have prompted the further investigation of ring-opened (malonamide) derivatives as potentially preferable therapeutic derivatives. Earlier reports have claimed reduced toxicity among similar derivatives. These studies reveal the relative degree of prostaglandin-H (PGH) synthase inhibitory activity among a series of malonamide derivatives. Contrary to observations in the pyrazolidinedione series, incorporation of a nonpolar butyl side chain in these malonamides was not beneficial but, rather, detrimental to enzyme-inhibitory activity. Although none of the reported nonbutylated malonamides was as potent an inhibitor of this enzyme as phenylbutazone, they all showed some inhibitory activity. PGH synthase inhibitory activity was especially pronounced in the bis(p-hydroxy anilide) derivatives, even extending to succinamide and adipamide derivatives. Of some interest is the observation that all of these p-hydroxy anilide derivatives were more potent inhibitors of this enzyme than acetaminophen. PMID:3100804

Vennerstrom, J L; Holmes, T J

1987-02-01

379

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

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Full Text Available Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS was 5802 bp long with an open reading frame of 4704 bp that e