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

Science.gov (United States)

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

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

Science.gov (United States)

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

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

2009-11-01

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

Scientific Electronic Library Online (English)

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

Patrícia Andrea, Monqueiro; Pedro Jacob, Christoffoleti.

2001-03-01

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

Directory of Open Access Journals (Sweden)

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

Ribas A. Vidal

1999-12-01

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

Scientific Electronic Library Online (English)

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

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

1999-12-01

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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

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

Energy Technology Data Exchange (ETDEWEB)

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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Functional expression of plant acetolactate synthase genes in Escherichia coli  

OpenAIRE

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

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

1989-01-01

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Acetolactate synthase mutation conferring imidazolinone-specific herbicide resistance in Amaranthus hybridus.  

Science.gov (United States)

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branched-chain amino acids in plants and is the target of several herbicides. ALS inhibitors have enjoyed popularity as herbicides due to numerous attributes, although their current adequacy in weed control programs is hampered by herbicide resistance. Most cases of ALS-inhibitor resistance have resulted from selection of an altered target site. The study herein reports on an alanine by threonine amino acid substitution at position 122 of ALS as the basis for imidazolinone-specific resistance in an A. hybridus population from Illinois. In vitro inhibition of enzymatic activity (I(50)) required 1000-fold greater concentration of imazethapyr in the resistant population compared with a susceptible control. This mutation represents the second ALS alteration associated with herbicide resistance in a natural A. hybridus population. PMID:16455361

Trucco, Federico; Hager, Aaron G; Tranel, Patrick J

2006-03-01

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

Scientific Electronic Library Online (English)

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

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

2003-08-01

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

Directory of Open Access Journals (Sweden)

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

D.L.P. Gazziero

2003-08-01

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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  

Directory of Open Access Journals (Sweden)

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

Patrícia Andrea Monquero

2003-01-01

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

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

2014-06-01

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

International Nuclear Information System (INIS)

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

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2013-02-15

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

Directory of Open Access Journals (Sweden)

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

Nanaka Kikuchi

2012-03-01

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

OpenAIRE

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

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

2013-01-01

19

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

Science.gov (United States)

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

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

2014-01-01

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

Energy Technology Data Exchange (ETDEWEB)

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

Van Dyk, D.E.; Schloss, J.V.

1987-05-01

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

International Nuclear Information System (INIS)

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

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

Directory of Open Access Journals (Sweden)

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

De Cauwer, Benny

2014-02-01

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

OpenAIRE

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

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

2013-01-01

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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.

2002-08-01

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

Science.gov (United States)

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

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

2013-04-01

27

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

OpenAIRE

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

Kingsbury, Joanne M.; Mccusker, John H.

2010-01-01

28

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

Directory of Open Access Journals (Sweden)

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

Patrick W. Geier

2013-04-01

29

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

Scientific Electronic Library Online (English)

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

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

2006-07-01

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Selectable Tolerance to Herbicides by Mutated Acetolactate Synthase Genes Integrated into the Chloroplast Genome of Tobacco1[OA  

Science.gov (United States)

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3?-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation. PMID:18515641

Shimizu, Masanori; Goto, Maki; Hanai, Moeko; Shimizu, Tsutomu; Izawa, Norihiko; Kanamoto, Hirosuke; Tomizawa, Ken-Ichi; Yokota, Akiho; Kobayashi, Hirokazu

2008-01-01

31

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

Science.gov (United States)

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

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

2013-09-01

32

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

Science.gov (United States)

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

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

2014-11-01

33

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

Scientific Electronic Library Online (English)

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

Alexandre Magno, Brighenti.

2012-06-01

34

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

Science.gov (United States)

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

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

2014-08-01

35

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

Science.gov (United States)

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

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

2013-11-01

36

Acetolactate synthase activity in Euphorbia heterophylla resistant to ALS- and protox- inhibiting herbicides / Atividade da enzima acetolactato sintase em Euphorbia heterophylla com resistência múltipla aos herbicidas inibidores da ALS e da protox  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese O objetivo deste trabalho foi determinar a atividade da enzima ALS em biótipos de leiteiro (Euphorbia heterophylla) com resistência múltipla aos inibidores da ALS e da Protox na presença e ausência dos herbicidas imazapyr, imazethapyr e nicosulfuron. Efetuou-se ensaio in vitro da enzima acetolactato [...] sintase (ALS) extraída de plantas dos biótipos Vitorino, Bom Sucesso do Sul e Medianeira (com resistência múltipla aos inibidores da ALS e da Protox) e de um biótipo suscetível, na ausência e presença dos herbicidas imazapyr, imazethapyr e nicosulfuron. Na ausência dos herbicidas, os biótipos com resistência múltipla demonstraram maior afinidade da enzima pelo substrato piruvato em comparação ao biótipo suscetível. Os herbicidas imazapyr, imazethapyr e nicosulfuron produziram reduzido efeito sobre a atividade da enzima ALS dos biótipos resistentes e, ao contrário, elevado efeito inibitório sobre a ALS do biótipo suscetível. Os fatores de resistência foram elevados, superiores a 438, 963 e 474 para os biótipos Vitorino, Bom Sucesso do Sul e Medianeira, respectivamente. A resistência observada deve-se à insensibilidade da enzima ALS aos herbicidas tanto do grupo das imidazolinonas quanto das sulfonilureias, caracterizando resistência cruzada. Abstract in english The objective of this study was to determine the activity of the enzyme acetolactate synthase in biotypes of wild poinsettia (Euphorbia heterophylla) with multiple resistance to ALS- and Protox- inhibitors in the presence and absence of imazapyr, imazethapyr and nicosulfuron. We conducted in vitro a [...] ssay of ALS enzyme extracted from plants of Vitorino, Bom Sucesso do Sul and Medianeira biotypes (with multiple resistance) and a susceptible population in the absence and presence of imazapyr, imazethapyr and nicosulfuron. In the absence of herbicides, biotypes with multiple resistance showed higher affinity for the substrate of the enzyme compared with the susceptible population. The herbicides imazapyr, imazethapyr and nicosulfuron had little effect on the enzyme activity of ALS-resistant biotypes and, conversely, high inhibitory effect on ALS of the susceptible population. Resistance factors were very high, greater than 438, 963 and 474 for Vitorino, Bom Sucesso do Sul and Medianeira biotypes, respectively. The resistance to ALS inhibitors is due to the insensitivity of ALS to herbicides of both imidazolinone and sulfonylurea groups, characterizing a cross-resistance.

E., Xavier; M.C., Oliveira; M.M., Trezzi; R.A., Vidal; F., Diesel; F.D., Pagnoncelli; E., Scalcon.

2013-12-01

37

Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.  

Science.gov (United States)

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

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

2014-07-10

38

Novel potent and selective inhibitors of inducible nitric oxide synthase.  

Science.gov (United States)

We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases. PMID:7536889

Nakane, M; Klinghofer, V; Kuk, J E; Donnelly, J L; Budzik, G P; Pollock, J S; Basha, F; Carter, G W

1995-04-01

39

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.

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

40

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

41

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

42

Inhibitors of glycogen synthase 3 kinase  

Science.gov (United States)

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

43

Enhancement of vascular targeting by inhibitors of nitric oxide synthase  

International Nuclear Information System (INIS)

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

44

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.

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

1067-10-01

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)

46

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

47

Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.  

Science.gov (United States)

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

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

2015-01-01

48

Glucosylceramide synthase inhibitors differentially affect expression of glycosphingolipids.  

Science.gov (United States)

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

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

2015-04-01

49

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.  

Science.gov (United States)

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus. PMID:20645653

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

50

Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors  

OpenAIRE

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 rem...

Fernandes, Chantal; Anjos, Jorge; Walker, Louise A.; Silva, Branca M. A.; Cortes, Lui?sa; Mota, Marta; Munro, Carol A.; Gow, Neil A. R.; Gonc?alves, Teresa

2014-01-01

51

Fatty acid synthase inhibitors isolated from Punica granatum L.  

Scientific Electronic Library Online (English)

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

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

2012-05-01

52

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

53

Biochemistry: Acetohydroxyacid Synthase  

OpenAIRE

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

54

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), Mg²? and flavin-adenine dinucleotide (FAD). AHAS is the target for several classes of commercial herbicides that include the sulfonylurea and imidazolinone families. These herbicides are potent and selective inhibitors of AHAS with Ki values that can be in the low nM range. Such compounds also exhibit low application rates as herbicides (typically ~3 g ha?¹) and have low mammalian toxicity (LD?? values typically >4g/kg), thereby highlighting their utility and effectiveness as biocidal agents. However, somewhat surprisingly given the central importance of AHAS in the metabolism of microorganisms, no inhibitors of this enzyme have been commercialized into antimicrobial agents. Here we provide an overview of the biochemical characterization of AHASs from bacterial and fungal sources, analyse the structural features of these enzymes that are criticial to catalysis andprovide the current data on AHAS inhibitors that have potential to be developed into antimicrobial therapeutics. PMID:23688082

Pue, Nason; Guddat, Luke W

2014-01-01

55

Distância genética e geográfica entre acessos de picão-preto suscetíveis e resistentes a herbicidas inibidores da acetolactato sintase / Genetic and geographic distance among beggar-ticks accesses susceptible and resistant to acetolactate sintase herbicide inhibitors  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese O objetivo deste trabalho foi avaliar o grau de similaridade genética entre acessos de picão-preto, suscetíveis e resistentes aos herbicidas inibidores da enzima acetolactato sintase (ALS) e a relação entre similaridade genética e distância geográfica desses acessos. Sementes dos acessos foram colet [...] adas no Estado do Paraná e cultivadas em casa de vegetação, na Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, em outubro de 2004. Depois da confirmação da resistência ou suscetibilidade dos acessos aos inibidores da enzima ALS, realizou-se a extração de DNA. Por meio da técnica de RAPD, foi possível avaliar a similaridade genética entre os acessos de picão-preto. Na análise conjunta dos acessos, dos 20 iniciadores utilizados, 17 apresentaram-se polimórficos, amplificando um total de 94 bandas. A similaridade genética média foi baixa e equivalente a 37%. A análise de regressão evidenciou que não há relação entre distância genética e geográfica nos acessos de picão-preto avaliados. A baixa similaridade geral entre esses acessos evidencia que a resistência aos herbicidas na região se configura pela seleção de indivíduos resistentes preexistentes na população. Abstract in english The objective of this work was to evaluate the degree of genetic similarity among beggar-ticks accesses, susceptible and resistant to acetolactate sintase (ALS) herbicide inhibitors and the relationship among the genetic similarity and geographic distance of this accesses. Beggar-ticks seeds were sa [...] mpled at Paraná state and were grown in the greenhouse at Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil, in October 2004. After the confirmation of resistance or susceptibility to ALS inhibitors herbicides, the DNA extraction was performed. Through RAPD analysis, it was possible to evaluate the genetic similarity among beggar-ticks accesses. In the whole analysis of the accesses, from 20 primers assessed, only 17 displayed polymorphism and amplified a total of 94 bands. Average genetic similarity was low (37%). Regression analysis evidenced that there is no relationship between genetic and geographic distance for the beggar-ticks accesses. Low general similarity among accesses evidences that resistance in the region is represented by selection of resistant individuals already existing in the population.

Fabiane Pinto, Lamego; Luciane Vilela, Resende; Paulo Roberto, Da-Silva; Ribas Antonio, Vidal; Anderson Luis, Nunes.

2006-06-01

56

Distância genética e geográfica entre acessos de picão-preto suscetíveis e resistentes a herbicidas inibidores da acetolactato sintase Genetic and geographic distance among beggar-ticks accesses susceptible and resistant to acetolactate sintase herbicide inhibitors  

Directory of Open Access Journals (Sweden)

Full Text Available O objetivo deste trabalho foi avaliar o grau de similaridade genética entre acessos de picão-preto, suscetíveis e resistentes aos herbicidas inibidores da enzima acetolactato sintase (ALS e a relação entre similaridade genética e distância geográfica desses acessos. Sementes dos acessos foram coletadas no Estado do Paraná e cultivadas em casa de vegetação, na Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, em outubro de 2004. Depois da confirmação da resistência ou suscetibilidade dos acessos aos inibidores da enzima ALS, realizou-se a extração de DNA. Por meio da técnica de RAPD, foi possível avaliar a similaridade genética entre os acessos de picão-preto. Na análise conjunta dos acessos, dos 20 iniciadores utilizados, 17 apresentaram-se polimórficos, amplificando um total de 94 bandas. A similaridade genética média foi baixa e equivalente a 37%. A análise de regressão evidenciou que não há relação entre distância genética e geográfica nos acessos de picão-preto avaliados. A baixa similaridade geral entre esses acessos evidencia que a resistência aos herbicidas na região se configura pela seleção de indivíduos resistentes preexistentes na população.The objective of this work was to evaluate the degree of genetic similarity among beggar-ticks accesses, susceptible and resistant to acetolactate sintase (ALS herbicide inhibitors and the relationship among the genetic similarity and geographic distance of this accesses. Beggar-ticks seeds were sampled at Paraná state and were grown in the greenhouse at Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil, in October 2004. After the confirmation of resistance or susceptibility to ALS inhibitors herbicides, the DNA extraction was performed. Through RAPD analysis, it was possible to evaluate the genetic similarity among beggar-ticks accesses. In the whole analysis of the accesses, from 20 primers assessed, only 17 displayed polymorphism and amplified a total of 94 bands. Average genetic similarity was low (37%. Regression analysis evidenced that there is no relationship between genetic and geographic distance for the beggar-ticks accesses. Low general similarity among accesses evidences that resistance in the region is represented by selection of resistant individuals already existing in the population.

Fabiane Pinto Lamego

2006-06-01

57

Nitric oxide synthase inhibitors containing the carboxamidine group or its isosteres  

Energy Technology Data Exchange (ETDEWEB)

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

Proskuryakov, Sergei Ya; Konoplyannikov, Anatoly G; Skvortzov, Valery G [Medical Radiological Research Centre, Russian Academy of Medical Sciences (Russian Federation); Mandrugin, Andrey A; Fedoseev, Vladimir M [Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)

2005-09-30

58

Nitric oxide synthase inhibitors containing the carboxamidine group or its isosteres  

International Nuclear Information System (INIS)

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

59

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

60

Thiouracil antithyroid drugs as a new class of neuronal nitric oxide synthase inhibitors.  

Science.gov (United States)

Two established antithyroid drugs, 6-propyl-2-thiouracil and 6-methyl-2-thiouracil, as well as S-methylthiouracil, are shown to be competitive inhibitors of nitric oxide synthase (NOS) (K(I) values ranging from 14 to 60 microM), with moderate selectivity for the neuronal isoform. Other thioureylene and thioamide-containing heterocyclic systems proved virtually ineffective as NOS inhibitors. Besides offering novel useful leads for inhibitor design as well as to probe the active site of neuronal NOS, the results of this study may have interesting implications in relation to the antithyroid activity of thiouracils and their possible neurological effects. PMID:11401533

Palumbo, A; d'Ischia, M

2001-04-01

61

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

OpenAIRE

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

62

Correction of Cystathionine ?-synthase Deficiency in Mice by Treatment with Proteasome Inhibitors  

OpenAIRE

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant...

Gupta, Sapna; Wang, Liqun; Anderl, Janet; Slifker, Michael J.; Kirk, Christopher; Kruger, Warren D.

2013-01-01

63

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

OpenAIRE

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

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

2009-01-01

64

Imaging Pharmacodynamics of the alpha Folate Targeted Thymidylate Synthase Inhibitor BGC 945  

OpenAIRE

The assessment of tissue-specific pharmacodynamics is desirable in the development of tumour-targeted therapies. Plasma deoxyuridine (dUrd) levels - a measure of systemic thymidylate synthase (TS) inhibition - has limited application for studying the pharmacodynamics of novel TS inhibitors targeted to the high affinity alpha-folate receptor (FR). Here we have evaluated the utility of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) for imaging the tissue pharmacodynamics of BG...

Pillai, Radhakrishna G.; Forster, Martin; Perumal, Meg; Mitchell, Fraser; Leyton, Julius; Aibgirhio, Franklin I.; Golovko, Oksana; Jackman, Ann L.; Aboagye, Eric O.

2008-01-01

65

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors  

OpenAIRE

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and recepto...

Srivastava, Vivek; Gupta, Satya Prakash; Siddiqi, Mohd Imran; Mishra, Bhartendu Nath

2010-01-01

66

Glycogen synthase kinase-3 inhibitors augment TRAIL-induced apoptotic death in human hepatoma cells.  

Science.gov (United States)

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC. PMID:18938143

Beurel, Eléonore; Blivet-Van Eggelpoël, Marie-José; Kornprobst, Michel; Moritz, Sylviane; Delelo, Roland; Paye, François; Housset, Chantal; Desbois-Mouthon, Christèle

2009-01-01

67

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

International Nuclear Information System (INIS)

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

68

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

Science.gov (United States)

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

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

2014-07-01

69

Structure and activity of NO synthase inhibitors specific to the L-arginine binding site.  

Science.gov (United States)

Synthesis of compounds containing a fragment similar to the guanidine group of L-arginine, which is a substrate of nitric oxide synthase (NOS), is the main direction in creating NOS inhibitors. The inhibitory effect of such compounds is caused not only by their competition with the substrate for the L-arginine-binding site and/or oxidizing center of the enzyme (heme) but also by interaction with peptide motifs of the enzyme that influence its dimerization, affinity for cofactors, and interaction with associated proteins. Structures, activities, and relative in vitro and in vivo specificities of various NOS inhibitors (amino acid and non-amino acid) with linear or cyclic structure and containing guanidine, amidine, or isothiuronium group are considered. These properties are mainly analyzed by comparison with effects of the inhibitors on the inducible NOS. PMID:15701046

Proskuryakov, S Ya; Konoplyannikov, A G; Skvortsov, V G; Mandrugin, A A; Fedoseev, V M

2005-01-01

70

Effects of new nitric oxide synthase inhibitors on spontaneous locomotor activity  

Directory of Open Access Journals (Sweden)

Full Text Available Introduction. New nitric oxide synthase (NOS inhibitors: 3-bromo-7-nitro- indazole (3-Br-7-NI, 1-(2-trifluoromethylphenyl imidazole (TRIM, S-methyl-L-thiocitrulline (S-Me-TC and 7-nitroindazole (7-NI reduce spontaneous locomotor activity in mice. Material and methods. In order to elucidate central effects of NOS inhibitors on locomotor activity, the influence of 7-NI on electroencephalographic (EEG power spectrum in rats was investigated. Results. 7-NI reduced the EEG power density in all frequency bands in rats, suggesting a depression of the central neuronal activity. The electrophysiologic power was most reduced in the range of 7-9 Hz of the rhythmic slow activity (theta rhythm, which is in accordance with decreased locomotor activity observed following administration of NOS inhibitors. Conclusion. The present results indicate that nitric oxide exerts an excitatory effect on central neuronal structures involved in regulation of locomotion. .

Džolji? Eleonora

2006-01-01

71

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.

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

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

74

Attenuation of human nasal airway responses to bradykinin and histamine by inhibitors of nitric oxide synthase.  

OpenAIRE

1. The effects of inhibitors of nitric oxide synthase and local anaesthetics were studied on changes in human nasal airway patency and albumin extravasation in response to bradykinin and histamine, in vivo. 2. Compared with the action of the vasoconstrictor, ephedrine, 2.5 mumol, NG-nitro-L-arginine methyl ester (L-NAME), 1 mumol alone, did not change the resting value of the minimal cross-sectional area (A min) of the human nasal airway. L-NAME, 0.1 to 10 mumol, produced a dose-related inhib...

Dear, J. W.; Ghali, S.; Foreman, J. C.

1996-01-01

75

Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase  

OpenAIRE

Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was scr...

Bulfer, Stacie L.; Mcquade, Thomas J.; Larsen, Martha J.; Trievel, Raymond C.

2010-01-01

76

In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors.  

Science.gov (United States)

Selective type II (inducible) nitric oxide synthase (NOS) inhibitors have several potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases. The ability of two novel, selective inhibitors of type II NOS, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), to inhibit type II NOS function in vivo was studied in lipopolysaccharide (LPS) treated rats. Type II NOS activity was assessed by measuring changes in plasma nitrite and nitrate concentrations ([NOx]). Both EIT and AMT elicited a dose-dependent and > 95% inhibition of the LPS-induced increase in plasma [NOx]. The ED50 values for EIT and AMT were 0.4 and 0.2 mg/kg, respectively. In addition, the administration of LPS and either NOS inhibitor resulted in a dose-dependent increase in animal mortality; neither compound was lethal when administered alone. Pretreatment with L-arginine (but not D-arginine) prevented the mortality, while not affecting the type II NOS-dependent NO production, suggesting the toxicity may be due to inhibition of one of the other NOS isoforms (endothelial or neuronal). Thus, although EIT and AMT are potent inhibitors of type II NOS function in vivo, type II NOS inhibitors of even greater selectivity may need to be developed for therapeutic applications. PMID:7585335

Tracey, W R; Nakane, M; Basha, F; Carter, G

1995-05-01

77

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

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

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

2014-09-01

78

[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

79

Structure of ACC synthase inactivated by the mechanism-based inhibitor L-vinylglycine.  

Science.gov (United States)

L-Vinylglycine (L-VG) is both a substrate for and a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to alpha-ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Angstroms resolution. The active site contains an external aldimine of the adduct of L-VG with the pyridoxal 5'-phosphate cofactor. The side chain gamma-carbon of L-VG is covalently bound to the epsilon-amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase. Biochemistry 39, 2436-2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate. PMID:15848188

Capitani, Guido; Tschopp, Markus; Eliot, Andrew C; Kirsch, Jack F; Grütter, Markus G

2005-04-25

80

ELIGLUSTAT TARTRATE: Glucosylceramide Synthase Inhibitor Treatment of Type 1 Gaucher Disease.  

Science.gov (United States)

Eliglustat tartrate (Genz-112638) is a novel, orally administered agent currently in development for the treatment of lysosomal storage disorders, including type 1 Gaucher disease and Fabry disease. This glucosylceramide analogue acts as an inhibitor of glucosylceramide synthase, a Golgi complex enzyme that catalyzes the formation of glucosylceramide from ceramide and UDP-glucose and is the first step in the formation of glucocerebroside-based glycosphingolipids. Pre-clinical pharmacological studies demonstrate that the agent has a high therapeutic index, excellent oral bioavailability and limited toxicity. Phase I studies in healthy volunteers revealed limited toxicity with an excellent pharmacodynamic response, as measured by decreased plasma glucosylceramide concentrations. Phase II studies in patients with type 1 Gaucher disease have demonstrated promising clinical responses, as measured by decreases in spleen size, improvement in hemoglobin concentrations and increased platelet counts. Two randomized phase III trials testing the efficacy and safety of eliglustat tartrate are currently in progress. PMID:22563139

Shayman, J A

2010-08-01

81

Evaluation of deoxyhypusine synthase inhibitors targeting BCR-ABL positive leukemias.  

Science.gov (United States)

Effective inhibition of BCR-ABL tyrosine kinase activity with Imatinib represents a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, more than 30 % of patients with CML in chronic phase do not respond adequately to Imatinib and the drug seems not to affect the quiescent pool of BCR-ABL positive leukemic stem and progenitor cells. Therefore, despite encouraging clinical results, Imatinib can still not be considered a curative treatment option in CML. We recently reported downregulation of eukaryotic initiation factor 5A (eIF5A) in Imatinib treated K562 cells. Furthermore, the inhibition of eIF5A by siRNA in combination with Imatinib has been shown to exert synergistic cytotoxic effects on BCR-ABL positive cell lines. Based on the structure of known deoxyhypusine synthase (DHS) inhibitors such as CNI-1493, a drug design approach was applied to develop potential compounds targeting DHS. Here we report the biological evaluation of selected novel (DHSI-15) as compared to established (CNI-1493, deoxyspergualin) DHS inhibitors. We show that upon the compounds tested, DHSI-15 and deoxyspergualin exert strongest antiproliferative effects on BCR-ABL cells including Imatinib resistant mutants. However, this effect did not seem to be restricted to BCR-ABL positive cell lines or primary cells. Both compounds are able to induce apoptosis/necrosis during long term incubation of BCR-ABL positive BA/F3 derivates. Pharmacological synergism can be observed for deoxyspergualin and Imatinib, but not for DHSI-15 and Imatinib. Finally we show that deoxyspergualin is able to inhibit proliferation of CD34+ progenitor cells from CML patients. We conclude that inhibition of deoxyhypusine synthase (DHS) can be supportive for the anti-proliferative treatment of leukemia and merits further investigation including other cancers. PMID:22415796

Ziegler, Patrick; Chahoud, Tuhama; Wilhelm, Thomas; Pällman, Nora; Braig, Melanie; Wiehle, Valeska; Ziegler, Susanne; Schröder, Marcus; Meier, Chris; Kolodzik, Adrian; Rarey, Matthias; Panse, Jens; Hauber, Joachim; Balabanov, Stefan; Brümmendorf, Tim H

2012-12-01

82

Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells  

Science.gov (United States)

Canonical Wnt/?-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months. PMID:22745733

Noble, Brenda L.; Mendez, Daniel C.; Caseley, Paul S.; Peterson, Sarah C.; Routledge, Tyler J.; Patel, Nilay V.

2012-01-01

83

Correction of cystathionine ?-synthase deficiency in mice by treatment with proteasome inhibitors.  

Science.gov (United States)

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant human CBS enzyme (either p.I278T or p.S466L) that has less than 5% of normal liver CBS activity, resulting in a 10-30-fold elevation in plasma homocysteine levels. We show that treatment of these mice with two different proteasome inhibitors can induce liver Hsp70, Hsp40, and Hsp27, increase levels of active CBS, and lower plasma homocysteine levels to within the normal range. However, response rates varied, with 100% (8/8) of the p.S466L animals showing correction, but only 38% (10/26) of the p.I278T animals. In total, our data show that treatment with proteostasis modulators can restore significant enzymatic activity to mutant misfolded CBS enzymes and suggests that they may be useful in treating certain types of genetic diseases caused by missense mutations. PMID:23592311

Gupta, Sapna; Wang, Liqun; Anderl, Janet; Slifker, Michael J; Kirk, Christopher; Kruger, Warren D

2013-08-01

84

Nitric oxide synthase inhibitors reduce sarcomere addition in rat skeletal muscle.  

Science.gov (United States)

1. Mechanical stimuli are thought to modulate the number of sarcomeres in series (sarcomere number) in skeletal muscle fibres. However, the mechanisms by which muscle cells transduce mechanical signals into serial sarcomere addition have not been explored. In this study, we test the hypothesis that nitric oxide positively modulates sarcomere addition. 2. The soleus muscle was cast-immobilized in a shortened position in 3-week-old female Wistar rats. After 4 weeks, the casts were removed, creating a period of rapid sarcomere addition. During the remobilization period, nitric oxide synthase (NOS) inhibitors or substrate were administered. 3. Rats treated with the non-isoform-specific NOS inhibitor L-nitro-arginine methyl ester during 3 weeks of remobilization had smaller soleus sarcomere numbers than control rats. Rats treated with 1-(2-trifluoromethyl-phenyl)-imidazole, which has greater specificity for the neuronal isoform than for the endothelial isoform of NOS, also had smaller soleus sarcomere numbers than control rats. These results suggest that inhibition of the neuronal isoform of NOS reduces sarcomere addition during remobilization. 4. Rats treated with L-arginine, the substrate for NOS, during 1 week of remobilization had soleus sarcomere numbers for the immobilized-remobilized muscle which were closer to that for the contralateral, non-immobilized muscle than did rats that were not treated with L-arginine. 5. These results support the hypothesis that nitric oxide derived from the neuronal isoform of NOS positively modulates sarcomere addition. PMID:10432349

Koh, T J; Tidball, J G

1999-08-15

85

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

Directory of Open Access Journals (Sweden)

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

Wilson Abrao Saad

2006-01-01

86

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

87

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

Directory of Open Access Journals (Sweden)

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

Irene Mavelli

2012-02-01

88

Trophoblastic Neoplasms Express Fatty Acid Synthase, Which May Be a Therapeutic Target via Its Inhibitor C93  

OpenAIRE

Fatty acid synthase (FASN) is an emerging tumor-associated marker and a promising antitumor therapeutic target. In this study, we analyzed the expression of FASN in normal and molar placentas, as well as gestational trophoblastic neoplasia, and assessed the effects of a new FASN inhibitor, C93, on cellular proliferation and apoptosis in choriocarcinoma cells. Using a FASN-specific monoclonal antibody, we found that FASN immunoreactivity was detected in the cytotrophoblast and intermediate (ex...

Ueda, Stefanie M.; Mao, Tsui-lien; Kuhajda, Francis P.; Vasoontara, Chanont; Giuntoli, Robert L.; Bristow, Robert E.; Kurman, Robert J.; Shih, Ie-ming

2009-01-01

89

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.  

OpenAIRE

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the sub...

Salvemini, D.; Manning, P. T.; Zweifel, B. S.; Seibert, K.; Connor, J.; Currie, M. G.; Needleman, P.; Masferrer, J. L.

1995-01-01

90

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

91

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

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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.

C.A.A. Perotti

1999-11-01

92

Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.  

Science.gov (United States)

The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC?? of 0.7 ?g/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell. PMID:23237384

Lee, Yu-Ting; Cui, Chang-Jun; Chow, Eve W L; Pue, Nason; Lonhienne, Thierry; Wang, Jian-Guo; Fraser, James A; Guddat, Luke W

2013-01-10

93

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

Science.gov (United States)

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

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

2012-02-01

94

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

Science.gov (United States)

Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of free fatty acids, is up-regulated in many cancers. FASN is essential for cancer cell survival and contributes to drug resistance and poor prognosis. However, it is not expressed in most nonlipogenic normal tissues. Thus, FASN is a desirable target for drug discovery. Although different FASN inhibitors have been identified, none has successfully moved into clinical use. In this study, using in silico screening of an FDA-approved drug database, we identified proton pump inhibitors (PPIs) as effective inhibitors of the thioesterase activity of human FASN. Further investigation showed that PPIs inhibited proliferation and induced apoptosis of cancer cells. Supplementation of palmitate, the end product of FASN catalysis, rescued cancer cells from PPI-induced cell death. These findings provide new evidence for the mechanism by which this FDA-approved class of compounds may be acting on cancer cells. PMID:25513712

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

2015-01-22

95

L-arginine chlorination results in the formation of a nonselective nitric-oxide synthase inhibitor.  

Science.gov (United States)

Reduced nitric oxide (NO) bioavailability and impaired vascular function are the key pathological characteristics of inflammatory diseases such as atherosclerosis. We have recently found that leukocyte-derived hypochlorous acid is able to react with the nitric-oxide synthase (NOS) substrate L-arginine to produce chlorinated L-arginine (cl-L-Arg). Interestingly, cl-L-Arg potently inhibits the formation of NO metabolites in cultured endothelial cells. It is unknown whether cl-L-Arg has a direct inhibitory effect on endothelial NOS (eNOS). In addition, the effect of cl-L-Arg on the other NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), is also unknown. Therefore, we designed the current study to test the effects of cl-L-Arg on eNOS, nNOS, and iNOS. Using recombinant NOS, we found that cl-L-Arg had a direct inhibitory effect on the activity of NOS. The effect of cl-L-Arg on NOS activity is nonselective, as all three NOS isoforms were inhibited with a similar IC(50). We further determined the effect of cl-L-Arg on the three NOS isoforms at the tissue level. The results demonstrated that cl-L-Arg potently inhibited all three NOS isoform-mediated vessel reactivities, as well as the NOS signaling molecule cGMP. Cl-L-Arg might serve as a novel endogenous NOS inhibitor and an important mediator for vascular dysfunction under inflammatory conditions such as atherosclerosis. Blocking cl-L-Arg formation may be a new therapeutic approach to cardiovascular diseases. PMID:16717106

Yang, Jian; Ji, Ruirui; Cheng, Yunhui; Sun, Ju-Zhong; Jennings, Lisa K; Zhang, Chunxiang

2006-09-01

96

Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation  

Energy Technology Data Exchange (ETDEWEB)

Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

Zhang, Y.; Cao, R; Yin, F; Hudock, M; Guo, R; Song, Y; No, J; Bergan, K; Leon, A; et al,

2009-01-01

97

Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation  

Science.gov (United States)

Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anti-cancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth, how cell activity can be predicted based on enzyme inhibition data, and, using x-ray diffraction, solid state NMR and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS. PMID:19309137

Zhang, Yonghui; Cao, Rong; Yin, Fenglin; Hudock, Michael P.; Guo, Rey-Ting; Krysiak, Kilannin; Mukherjee, Sujoy; Gao, Yi-Gui; Robinson, Howard; Song, Yongcheng; No, Joo Hwan; Bergan, Kyle; Leon, Annette; Cass, Lauren; Goddard, Amanda; Chang, Ting-Kai; Lin, Fu-Yang; Van Beek, Ermond; Papapoulos, Socrates; Wang, Andrew H.-J.; Kubo, Tadahiko; Ochi, Mitsuo; Mukkamala, Dushyant; Oldfield, Eric

2009-01-01

98

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

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

Bahmanzadeh M.

2008-03-01

99

Design, synthesis, and evaluation of new type of L-amino acids containing pyridine moiety as nitric oxide synthase inhibitor.  

Science.gov (United States)

New amino acids 7-12 were designed and synthesized as candidate inhibitors of human nitric oxide synthase (NOS). The 2-aminopyridine-containing l-amino acids 8 had potent inhibitory activity toward all of the human NOS isozymes. However, the regioisomers 9 and 10, and 2-methylpyridine-containing compound 11 had much lower inhibitory activity. Human NOS isozymes were also inhibited by 7, which lacks an amino group on the pyridine moiety. A computational docking study was carried out to investigate the mechanism of the inhibitory effect. PMID:16466923

Ijuin, Ryosuke; Umezawa, Naoki; Higuchi, Tsunehiko

2006-05-15

100

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

International Nuclear Information System (INIS)

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

101

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

International Nuclear Information System (INIS)

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

102

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

Science.gov (United States)

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

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

2001-01-01

103

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

104

Differential effects of glycogen synthase kinase 3 (GSK3) inhibition by lithium or selective inhibitors in the central nervous system.  

Science.gov (United States)

Glycogen synthase kinase (GSK3) is a constitutively active serine-threonine kinase associated to neurological and psychiatric disorders. GSK3 inhibition is considered a mediator of the efficacy of the mood-stabiliser lithium. This study aimed at comparing the central nervous system effect of lithium with the selective GSK3 inhibitors AZ1080 and compound A in biochemical, cellular, and behavioural tests. Collapsin response mediator protein 2 is a neuron-specific GSK3 substrate. Lithium, AZ1080, and compound A inhibited its phosphorylation in rat primary neurons with different pIC50. After systemic treatments with lithium or GSK3 inhibitors to assess specific functional responses, phosphorylation was unchanged in adult rat brain, while it was strongly inhibited by GSK3 inhibitors in pups, differently from lithium. Lithium may exert neurotrophic effect by increasing brain-derived neurotrophic factor (BDNF) levels: in the present experimental conditions, lithium exerted opposite effects on plasma BDNF levels compared to GSK3 inhibitors, suggesting this effect might not be necessarily mediated by GSK3 inhibition alone. While plasma thyroid-stimulating hormone and luteinising hormone were not affected by lithium, they were decreased by selective inhibitors. GH and prolactin displayed similar responses towards reduction. Follicle-stimulating hormone levels were not altered by treatments, whereas melatonin was specifically increased by AZ1080. Lithium impaired mouse spontaneous locomotion and decreased amphetamine-induced hyper-locomotion. AZ1080 had no effects on locomotion, while compound A reduced spontaneous locomotor activity without effects on amphetamine-induced hyper-locomotion. The present results indicate that a broad correlation between the effects of lithium and selective GSK3 inhibitors could not be devised, suggesting alternative mechanisms, whereas overlapping results could be obtained in specific assays. PMID:23793101

Caberlotto, Laura; Carboni, Lucia; Zanderigo, Floriana; Andreetta, Filippo; Andreoli, Michela; Gentile, Gabriella; Razzoli, Maria

2013-10-01

105

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

106

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

OpenAIRE

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

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

1980-01-01

107

Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3? inhibitor, promotes prostate cancer growth in soft tissue and bone  

OpenAIRE

Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3?) inhibits Wnt signaling through inducing ?-catenin degradation. Therefore, AR79, an inhibitor of GSK3?, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tu...

Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; Mccauley, Laurie K.; Keller, Evan T.

2013-01-01

108

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

Science.gov (United States)

The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. PMID:25760598

Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

2015-03-01

109

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

Science.gov (United States)

The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5?-methylthio­adenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethyl­aniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. PMID:25760598

Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

2015-01-01

110

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

Science.gov (United States)

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

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

2011-01-01

111

Endogenous nitric oxide synthase inhibitors in the biology of disease: markers, mediators and regulators?  

OpenAIRE

The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of al...

Caplin, Ben; Leiper, James

2012-01-01

112

Substituted Pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis Thymidylate Synthase Inhibitors  

OpenAIRE

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 individuals1, 2. 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 classic...

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

2013-01-01

113

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

International Nuclear Information System (INIS)

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

114

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

Directory of Open Access Journals (Sweden)

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

115

Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I  

Science.gov (United States)

Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity. PMID:21775138

Sayahi, Halimah; Zimhony, Oren; Jacobs, William R.; Shekhtman, Alexander; Welch, John T.

2015-01-01

116

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

Directory of Open Access Journals (Sweden)

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

Sheela Sharma

2002-01-01

117

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

OpenAIRE

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

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

2014-01-01

118

Marine natural products as inhibitors of cystathionine beta-synthase activity.  

Science.gov (United States)

A library consisting of characterized marine natural products as well as synthetic derivatives was screened for compounds capable of inhibiting the production of hydrogen sulfide (H2S) by cystathionine beta-synthase (CBS). Eight hits were validated and shown to inhibit CBS activity with IC50 values ranging from 83 to 187?M. The majority of hits came from a series of synthetic polyandrocarpamine derivatives. In addition, a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions is reported. PMID:25666819

Thorson, Megan K; Van Wagoner, Ryan M; Harper, Mary Kay; Ireland, Chris M; Majtan, Tomas; Kraus, Jan P; Barrios, Amy M

2015-03-01

119

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

Science.gov (United States)

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 accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3? SB216763 or SB415286, or with GSK-3?-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3? inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3?, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3? inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3?, MDM2 and p53. PMID:21738215

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

2012-03-01

120

Thienopyrimidine bisphosphonate (ThPBP) inhibitors of the human farnesyl pyrophosphate synthase: optimization and characterization of the mode of inhibition.  

Science.gov (United States)

Human farnesyl pyrophosphate synthase (hFPPS) controls the post-translational prenylation of small GTPase proteins that are essential for cell signaling, cell proliferation, and osteoclast-mediated bone resorption. Inhibition of hFPPS is a clinically validated mechanism for the treatment of lytic bone diseases, including osteoporosis and cancer related bone metastases. A new series of thienopyrimidine-based bisphosphonates (ThP-BPs) were identified that inhibit hFPPS with low nanomolar potency. Crystallographic evidence revealed binding of ThP-BP inhibitors in the allylic subpocket of hFPPS. Simultaneous binding of inorganic pyrophosphate in the IPP subpocket leads to conformational closing of the active site cavity. The ThP-BP analogues are significantly less hydrophilic yet exhibit higher affinity for the bone mineral hydroxyapatite than the current N-BP drug risedronic acid. The antiproliferation properties of a potent ThB-BP analogue was assessed in a multiple myeloma cell line and found to be equipotent to the best current N-BP drugs. Consequently, these compounds represent a new structural class of hFPPS inhibitors and a novel scaffold for the development of human therapeutics. PMID:23998921

Leung, Chun Yuen; Park, Jaeok; De Schutter, Joris W; Sebag, Michael; Berghuis, Albert M; Tsantrizos, Youla S

2013-10-24

121

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

Science.gov (United States)

A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach led to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by Surface Plasmon Resonance (SPR) experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo. PMID:25437621

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

2015-01-27

122

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.

2005-03-01

123

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

International Nuclear Information System (INIS)

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

124

Effects of L-NAME, a non-specific nitric oxide synthase inhibitor, on AlCl3-induced toxicity in the rat forebrain cortex  

OpenAIRE

The present experiments were done to determine the effectiveness of a non-specific nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on oxidative stress parameters induced by aluminium chloride (AlCl3) intrahippocampal injections in Wistar rats. Animals were sacrificed 3 h and 30 d after treatments, heads were immediately frozen in liquid nitrogen and forebrain cortices were removed. Crude mitochondrial fraction preparations of forebrain cortices were used for the bio...

Stevanovic?, Ivana D.; Jovanovic?, Marina D.; Jelenkovic?, Ankica; C?olic?, Miodrag; Stojanovic?, Ivana; Ninkovic?, Milica

2009-01-01

125

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

OpenAIRE

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

Boultadakis, Antonios; Pitsikas, Nikolaos

2010-01-01

126

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

OpenAIRE

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...

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

2011-01-01

127

HMG-CoA Reductase Inhibitor Improves Endothelial Dysfunction in Spontaneous Hypertensive Rats Via Down-regulation of Caveolin-1 and Activation of Endothelial Nitric Oxide Synthase  

OpenAIRE

Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a ...

Suh, Jung-won; Choi, Dong-ju; Chang, Hyuk-jae; Cho, Young-seok; Youn, Tae-jin; Chae, In-ho; Kim, Kwang-il; Kim, Cheol-ho; Kim, Hyo-soo; Oh, Buyng-hee; Park, Young-bae

2009-01-01

128

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

OpenAIRE

Neuronal nitric oxide synthase (nNOS) plays an important role in neurotransmission and smooth muscle relaxation. Selective inhibition of nNOS over its other isozymes is highly desirable for the treatment of neurodegenerative diseases to avoid undesirable effects. In this study, we present a workflow for the identification and prioritization of compounds as potentially selective human nNOS inhibitors. Three-dimensional pharmacophore models were constructed based on a set of known nNOS inhib...

Guanhong Xu; Yue Chen; Kun Shen; Xiuzhen Wang; Fei Li; Yan He

2014-01-01

129

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

OpenAIRE

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

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

2008-01-01

130

A Fatal Combination: A Thymidylate Synthase Inhibitor with DNA Damaging Activity  

Science.gov (United States)

2?-deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2?-deoxythymidine 5?-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 ?M EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations. PMID:25671308

Ligasová, Anna; Strunin, Dmytro; Friedecký, David; Adam, Tomáš; Koberna, Karel

2015-01-01

131

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2002-01-01

132

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase  

Science.gov (United States)

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

Snoeberger, Ning-Shiuan Nicole

133

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

134

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

Science.gov (United States)

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

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

2013-01-01

135

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

Directory of Open Access Journals (Sweden)

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

Leigh James

2006-11-01

136

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

Science.gov (United States)

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

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

2012-01-01

137

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

138

Synthesis of potent inhibitors of ?-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents.  

Science.gov (United States)

Mycobacterium tuberculosis FabH, an essential enzyme in the mycolic acid biosynthetic pathway, is an attractive target for novel anti-tubercolosis agents. Structure-based design and synthesis of 1-(4-carboxybutyl)-4-(4-(substituted benzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid derivatives 7a-h, a subset of eight potential FabH inhibitors, is described in this paper. The Vilsmeier-Haack reaction was employed as a key step. The structures of all the newly synthesized compounds were identified by IR, ¹H-NMR, ¹³C-NMR, ESI-MS and HRMS. The alamarBlue™ microassay was employed to evaluate the compounds 7a-h against Mycobacterium tuberculosis H??Rv. The results demonstrate that the compound 7d possesses good in vitro antimycobacterial activity against Mycobacterium tuberculosis H??Rv (Minimum Inhibitory Concentration value [MIC], 12.5 µg/mL).These compounds may prove useful in the discovery and development of new anti-tuberculosis drugs. PMID:22534662

Liu, Yan; Zhong, Wu; Li, Rui-Juan; Li, Song

2012-01-01

139

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

Directory of Open Access Journals (Sweden)

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

Song Li

2012-04-01

140

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

Directory of Open Access Journals (Sweden)

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

Guanhong Xu

2014-05-01

141

Inhibitor-?B kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells.  

Science.gov (United States)

Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-?B kinase-? (IKK?)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKK? on Hsp90. Interestingly, IKK? binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKK? to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKK?. The pathophysiological relevance of the IKK?-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2(Akita) in vivo model. Our study further defines the preferential involvement of ?- vs. ?-isoforms of Hsp90 in the IKK?-eNOS-Hsp90 interaction, even though both Hsp90? and Hsp90? stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKK? within the cell system that regulates NO production, but they also confirm that the IKK?-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

Natarajan, Mohan; Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L; Mohan, Sumathy

2015-04-15

142

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

143

The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.  

Science.gov (United States)

Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats. PMID:25636602

Gocmez, Semil Selcen; Yazir, Yusufhan; Sahin, Deniz; Karadenizli, Sabriye; Utkan, Tijen

2015-04-01

144

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

Science.gov (United States)

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

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

2012-01-01

145

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

OpenAIRE

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

146

CG0009, a Novel Glycogen Synthase Kinase 3 Inhibitor, Induces Cell Death through Cyclin D1 Depletion in Breast Cancer Cells  

OpenAIRE

Glycogen synthase kinase 3?/? (GSK3?/?) is a constitutively active serine/threonine kinase involved in multiple physiological processes, such as protein synthesis, stem cell maintenance and apoptosis, and acts as a key suppressor of the Wnt-?-catenin pathway. In the present study, we examined the therapeutic potential of a novel GSK3 inhibitor, CG0009, in the breast cancer cell lines, BT549, HS578T, MDA-MB-231, NCI/ADR-RES, T47D, MCF7 and MDA-MB-435, from the NCI-60 cancer cell line pane...

Kim, Hyun Mi; Kim, Choung-soo; Lee, Je-hwan; Jang, Se Jin; Hwang, Jung Jin; Ro, Seonggu; Choi, Jene

2013-01-01

147

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

OpenAIRE

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

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

2012-01-01

148

Efficacy of caspofungin, a 1,3-?-D-glucan synthase inhibitor, on Pneumocystis carinii pneumonia in rats.  

Science.gov (United States)

Pneumocystis carinii pneumonia (PcP) is a common and potentially fatal opportunistic infection in immunosuppressed patients, and the standard trimethoprim-sulfamethoxazole (TMP-SMZ) treatment has serious side effects. The cell wall of the causative fungal pathogen is enriched in 1-3-?-D-glucan, providing an alternative therapeutic target. We directly compared the efficacy of the 1,3-?-D-glucan synthase inhibitor caspofungin to TMP-SMZ for promoting survival and reducing lung cyst number during the early phase of treatment in a rat model of PcP. Rats were immunosuppressed using dexamethasone for 8 weeks and PcP infection confirmed in test animals by lung print smear. The remaining rats were randomly divided into three control groups, a baseline group and two observed for 7 or 14 days, two caspofungin groups treated intravenously for 7 or 14 days (1 mg/kg/d), and 2 TMP-SMZ positive control groups treated by oral gavage for 7 or 14 days (300 mg/kg/d). Mortality was markedly reduced by both caspofungin and TMP-SMZ after 14 days (caspofungin: 20.0%, TMP-SMZ: 13.3%, Control: 40.0%). Body weight gain in caspofungin-treated rats after 7 (3.04 ± 3.54%) and 14 (4.27 ± 2.79%) days was similar to that in TMP-SMZ-treated rats (3.35 ± 1.88% and 5.85 ± 2.78%, respectively), whereas untreated controls showed weight loss. Lung weight to body weight ratio, and mean cyst number per 50 microscopic fields were significantly lower (all P caspofungin-treated rats than untreated controls at both 7 and 14 days, and similar to those in the TMP-SMZ-treated rats (all P > 0.05 vs. caspofungin). Caspofungin exhibited similar efficacy to TMP-SMZ for enhancing survival and reducing lung edema and cyst load in a rat model of PcP, suggesting potential clinical utility against PcP. PMID:25288652

Sun, Peipei; Tong, Zhaohui

2014-11-01

149

Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography-tandem mass spectrometry.  

Science.gov (United States)

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography-tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z' value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis. PMID:24959941

Yurek, David; Yu, Lan; Schrementi, James; Bell, Michael G; McGee, James; Kowala, Mark; Kuo, Ming-shang; Wang, Jian

2014-10-01

150

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

International Nuclear Information System (INIS)

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

151

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2006-09-15

152

An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.  

Science.gov (United States)

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 potential dual inhibitors of human Thymidylate synthase (hTS) and human dihydrofolate reductase (hDHFR). These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs. PMID:23577115

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

2013-01-01

153

The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes.  

Science.gov (United States)

We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [(3)H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [(3)H]2-amino-4-picoline bound saturably and with high affinity to human NOSs. Affinity constants (K(D) values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS isoforms (iNOS, nNOS, eNOS). Binding of [(3)H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity. L-NIL [L-N(6)-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective while L-NA (N(G)-nitro-L-arginine) and L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate assay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzyme inhibition and the inhibition of [(3)H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms. PMID:11040050

Boer, R; Ulrich, W R; Klein, T; Mirau, B; Haas, S; Baur, I

2000-11-01

154

Small-Molecule Inhibitor of Glycogen Synthase Kinase 3? 6-Bromoindirubin-3-oxime Inhibits Hematopoietic Regeneration in Stem Cell Recipient Mice.  

Science.gov (United States)

Small-molecule inhibitors of glycogen synthase kinase 3? (GSK3?) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3? inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3? inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant. PMID:25329250

Shen, Sylvie; Xu, Ning; Klamer, Guy; Ko, Kap-Hyoun; Khoo, Melissa; Ma, David; Moore, John; O'Brien, Tracey A; Dolnikov, Alla

2015-03-15

155

Discovery of a Novel Class of Orally Active Antifungal ?-1,3-d-Glucan Synthase Inhibitors?  

OpenAIRE

The echinocandins are a class of semisynthetic natural products that target ?-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. ...

Walker, Scott S.; Xu, Yiming; Triantafyllou, Ilias; Waldman, Michelle F.; Mendrick, Cara; Brown, Nathaniel; Mann, Paul; Chau, Andrew; Patel, Reena; Bauman, Nicholas; Norris, Christine; Antonacci, Barry; Gurnani, Maya; Cacciapuoti, Anthony; Mcnicholas, Paul M.

2011-01-01

156

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

157

A leukotriene C4 synthase inhibitor with the backbone of 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid.  

Science.gov (United States)

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner. PMID:23378248

Ago, Hideo; Okimoto, Noriaki; Kanaoka, Yoshihide; Morimoto, Gentaro; Ukita, Yoko; Saino, Hiromichi; Taiji, Makoto; Miyano, Masashi

2013-05-01

158

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

159

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

Science.gov (United States)

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

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

2012-04-01

160

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

Science.gov (United States)

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

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

2015-01-01

161

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2013-10-15

162

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

OpenAIRE

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

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

2001-01-01

163

2,4-Diamino-5-methyl-6-substituted Arylthio-furo[2,3-d]pyrimidines as Novel Classical and Nonclassical Antifolates as Potential Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors1a,b  

OpenAIRE

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6–16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog ...

Gangjee, Aleem; Jain, Hiteshkumar D.; Phan, Jaclyn; Guo, Xin; Queener, Sherry F.; Kisliuk, Roy L.

2009-01-01

164

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

Directory of Open Access Journals (Sweden)

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

Adam Curtis

1992-01-01

165

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.

166

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

Science.gov (United States)

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

167

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

Science.gov (United States)

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 antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation. PMID:7537518

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

1995-05-01

168

Effect of S-methylisothiourea, an inducible nitric oxide synthase inhibitor, in joint pain and pathology in surgically induced model of osteoarthritis.  

Science.gov (United States)

The aim of the present study was to evaluate in vivo modulatory effect of S-methylisothiourea (SMT), a preferential inhibitor of inducible nitric oxide synthase (iNOS) on pain and pathology in the surgical model of osteoarthritis (OA) in rats. The OA was produced by the anterior cruciate ligament transection (ACLT) and medial meniscectomy (MMx) of right knee. SMT was administered 1 day prior to the production of OA and continued up to day 42 postoperation. Mechanical hyperalgesia, thermal hyperalgesia, tail flick latency after repeated flexion and extension of OA knee and knee diameter of right knee were determined at weekly intervals. Serum levels of IL-1?, TNF-? and nitrite concentration were determined at the end of the experiment. Glycosaminoglycan (GAG) content, collagen content and histopathological evaluation of articular cartilage were also determined at the end of the experiment. SMT reduced mechanical hyperalgesia and the serum levels of IL-1?, TNF-? and nitrite. Further, SMT reduced the loss of GAG from articular cartilage. Microscopically, SMT reduced the severity of the cartilage lesion. The results indicate the effectiveness of SMT in attenuating the pain and pathology of experimental OA phase by reducing the production of nitric oxide and interleukin-1? and tumor necrosis factor-?, which are known to play a major role in the pathophysiology of OA. PMID:25111192

Balaganur, Venkanna; Pathak, Nitya Nand; Lingaraju, Madhu C; More, Amar Sunil; Latief, Najeeb; Kumari, Rashmi Rekha; Kumar, Dinesh; Tandan, Surendra K

2014-01-01

169

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.

170

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

Science.gov (United States)

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

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

2012-08-01

171

21 CFR 173.115 - Alpha-acetolactate decarboxylase (?-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...

2010-04-01

172

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.

173

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

Scientific Electronic Library Online (English)

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

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

1047-10-01

174

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2012-12-15

175

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

Directory of Open Access Journals (Sweden)

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

176

Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats.  

Science.gov (United States)

1. A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically-instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 micro g kg(-1) h(-1)) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4-8 after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well-maintained over the following 8 h of LPS infusion. 2. By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta. (43.3 +/- 7.8, 28.8 +/- 3.3, 50.8 +/- 7.2, 3.04 +/- 0.29, 3.76 +/- 0.94 pmol min(-1) mg(-1) protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 +/- 1.23 to 29.44 +/- 7.08 micromol l(-1)), and further by 6 h (228.10 +/- 29.20 micromol l(-1)), but were less 24 h after onset of LPS infusion (74.96 +/- 11.34 micromol l(-1)). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8-24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3. Pretreatment with NG-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1) bolus, 30 mg kg(-1) h(-1) infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6-8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation. PMID:8640339

Gardiner, S M; Kemp, P A; March, J E; Bennett, T

1995-10-01

177

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

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

Suborov Evgeny V

2012-06-01

178

Thiopyranol[2,3,4-c,d]indoles as inhibitors of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase.  

Science.gov (United States)

The attachment of an arylacetic or benzoic acid moiety to the thiopyrano[2,3,4-c,d]indole nucleus results in compounds which are highly potent and selective 5-lipoxygenase (5-LO) inhibitors. These compounds are structurally simpler than previous compounds of similar potency in that they contain a single chiral center. From the data presented, 2-[[1-(3-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]- 4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]-phenylacetic acid, 14b, was shown to inhibit 5-hydroperoxyeicosatetraenoic acid (5-HPETE) production by human 5-LO (IC50 of 18 nM). The acid 14b is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or human leukocyte leukotriene A4 (LTA4) hydrolase (IC50 > 20 microM). In addition, 14b was inactive in a 5-lipoxygenase-activating protein (FLAP) binding assay at 10 microM. In vivo studies showed that 14b is bioavailable in rat and functionally active in the hyperreactive rat model of antigen-induced dyspnea (74% inhibition at 0.5 mk/kg po; 2 h pretreatment). In the conscious squirrel monkey model of asthma, 14b showed excellent functional activity at 0.1 mg/kg against antigen-induced bronchoconstriction (94% inhibition of the increase in RL and 100% inhibition in the decrease in Cdyn; n = 4). Resolution of this compound gave (-)-14b, the most potent enantiomer (IC50 = 10 nM in the human 5-LO assay), which was shown to possess the S configuration at the chiral center by X-ray crystallographic analysis of an intermediate. Subsequent studies on the aryl thiopyrano[2,3,4-c,d]indole series of inhibitors led to the discovery of potent dual inhibitors of both FLAP and 5-LO, the most potent of which is 2-[[1-(4-chlorobenzyl)-4-methyl-6-(quinolin-2-ylmethoxy)-4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]phenylacetic acid, 19. Acid 19 has an IC50 of 100 nM for the inhibition of 5-HPETE production by human 5-LO and is active in a FLAP binding assay with an IC50 of 32 nM. Furthermore, thiopyrano[2,3,4-c,d]indoles such as 1 and 14b are capable of inhibiting the LTC4 synthase reaction in a dose dependent manner (IC50s of 11 and 16 microM, respectively, compared to that of LTC2 at 1.2 microM) in contrast to other, structurally distinct 5-LO inhibitors. It has also been observed that the thiopyrano[2,3,4-c,d]indole class of compounds strongly promotes the translocation of 5-LO from the cytosol to a membrane fraction in the presence or absence of the ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7473582

Hutchinson, J H; Charleson, S; Evans, J F; Falgueyret, J P; Hoogsteen, K; Jones, T R; Kargman, S; Macdonald, D; McFarlane, C S; Nicholson, D W

1995-10-27

179

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

Science.gov (United States)

Echinocandins inhibit the synthesis of ?-1,3-d-glucan in Candida and are the first-line therapy in numerous clinical settings. Their use is limited by poor oral bioavailability, and they are available only as intravenous therapies. Derivatives of enfumafungin are novel orally bioavailable glucan synthase inhibitors. We performed an in vivo pharmacodynamic (PD) evaluation with a novel enfumafungin derivative, SCY-078 (formerly MK-3118), in a well-established neutropenic murine model of invasive candidiasis against C. albicans, C. glabrata, and C. parapsilosis. The SCY-078 MICs varied 8-fold. Oral doses of 3.125 to 200 mg/kg SCY-078 salt in sterile water produced peak levels of 0.04 to 2.66 ?g/ml, elimination half-lives of 5.8 to 8.5 h, areas under the concentration-time curve from 0 to 24 h (AUC0-24 h) of 0.61 to 41.10 ?g · h/ml, and AUC from 0 to infinity (AUC0-?) values of 0.68 to 40.31 ?g · h/ml. The pharmacokinetics (PK) were approximately linear over the dose range studied. Maximum response (Emax) and PK/PD target identification studies were performed with 4 C. albicans, 4 C. glabrata, and 3 C. parapsilosis isolates. The PD index AUC/MIC was explored by using total (tAUC) and free (fAUC) drug concentrations. The maximum responses were 4.0, 4.0, and 4.3 log10 CFU/kidney reductions for C. albicans, C. glabrata, and C. parapsilosis, respectively. The AUC/MIC was a robust predictor of efficacy (R(2), 0.53 to 0.91). The 24-h PD targets were a static dose of 63.5 mg/kg, a tAUC/MIC of 500, and an fAUC/MIC of 1.0 for C. albicans; a static dose of 58.4 mg/kg, a tAUC/MIC of 315, and an fAUC/MIC of 0.63 for C. glabrata; and a static dose of 84.4 mg/kg, a tAUC/MIC of 198, and an fAUC/MIC of 0.40 for C. parapsilosis. The mean fAUC/MIC values associated with a 1-log kill endpoint against these species were 1.42, 1.26, and 0.91 for C. albicans, C. glabrata, and C. parapsilosis, respectively. The static and 1-log kill endpoints were measured relative to the burden at the start of therapy. The static and 1-log kill doses, as well as the total and free drug AUC/MIC PD targets, were not statistically different between species but were numerically lower than those observed for echinocandins. SCY-078 is a promising novel oral glucan synthase inhibitor against Candida species, and further investigation is warranted. PMID:25512406

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

2015-02-01

180

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

Directory of Open Access Journals (Sweden)

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

181

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

182

The selective prostaglandin endoperoxide synthase-2 inhibitor, NS-398, reduces prostaglandin production and ovulation in vivo and in vitro in the rat.  

Science.gov (United States)

Two isoforms of prostaglandin G/H synthase, PGS-1 and PGS-2, catalyze the formation of prostaglandins (PG). Nonselective PGS inhibitors, e.g., indomethacin, reduce the number of ovulations and PG levels in many animal models. This study evaluated the effects of the selective PGS-2 inhibitor NS-398, compared to indomethacin, on ovulation number and on PG and steroid production both in vivo and in vitro in the rat. NS-398 reduced the synthesis of PGE2 in isolated, LH-stimulated preovulatory follicles incubated in vitro. The inhibition by NS-398 was similar to that of indomethacin. Maximal inhibition was noted from 0.1 microM. Neither progesterone nor cAMP production was affected by NS-398 or indomethacin. The effect of in vivo administration of NS-398 (1, 3, or 10 mg/kg BW, s. c.) to proestrous rats 1 h after the injection of an ovulatory dose of hCG was monitored in follicles extirpated 10 h after hCG. These follicles were incubated in vitro, and NS-398 dose-dependently reduced PGE2 production. The synthesis of cAMP and progesterone was not altered. In separate experiments, the same doses of NS-398 were injected to determine their effect on ovulation in vivo. The number of ovulations was decreased by the highest dose of NS-398. In the in vitro ovarian perfusion model, NS-398 (10 microM) reduced the number of ovulations initiated by LH and isobutylmethylxanthine. Lower doses of NS-398 (0.1 and 1 microM) were less effective. The production of prostanoids (PGE2, PGF2alpha, and 6-keto-PGF1alpha) was reduced in a dose-dependent manner by NS-398. The secretion of steroids was not affected. This study demonstrates that selective inhibition of PGS-2 by NS-398 reduces LH/hCG-stimulated production of prostanoids and the number of ovulations both in vivo and in vitro. These results provide direct evidence to strengthen the role of the inducible, granulosa cell-expressed PGS-2 as one of the key regulators in the ovulatory process and also document that the elevated and perhaps sustained levels of PG are obligatory for ovulation. PMID:9780312

Mikuni, M; Pall, M; Peterson, C M; Peterson, C A; Hellberg, P; Brännström, M; Richards, J S; Hedin, L

1998-11-01

183

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

OpenAIRE

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

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

1986-01-01

184

Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone  

OpenAIRE

The action of taxifolin on the angiotensin-converting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (N?-nitro-l-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-l-histidyl-l-leucine, and the ROS/RNS production was measured by oxidation of dichlorodihydrofluorescein. It w...

Arutyunyan, Tamara V.; Korystova, Antonina F.; Kublik, Ludmila N.; Levitman, Maria Kh; Shaposhnikova, Vera V.; Korystov, Yuri N.

2012-01-01

185

Effect of NO synthase inhibitor 2-amino-5,6-dihydro-4H-1,3-thiazine on endotoxin-induced changes in hemodynamic parameters and respiration in rats.  

Science.gov (United States)

We studied the effect of NO synthase inhibitor 2-amino-5,6-dihydro-4H-1,3-thiazine (2-ADT) on the cardiovascular system in rats with endotoxic shock. Blood pressure, heart rate, and respiratory rate were recorded. E. coli lipopolysaccharide decreased blood pressure and heart rate. 2-ADT in a dose of 5 mg/kg normalized these hemodynamic parameters. The normalizing effect of 2-ADT decreased with increasing the dose of this preparation. 2-ADT in high doses (10, 20, and 30 mg/kg) and repeated injections of the preparation caused death of experimental animals. PMID:15665955

Proskuryakov, S Ya; Filimonova, M V; Verkhovskii, Yu G; Konoplyannikov, A G; Mandrugin, A A; Fedoseev, V M; Skvortsov, V G

2004-10-01

186

A selective thromboxane A2 (TXA2) synthase inhibitor, ozagrel, attenuates lung injury and decreases monocyte chemoattractant protein-1 and interleukin-8 mRNA expression in oleic acid-induced lung injury in guinea pigs.  

Science.gov (United States)

This study examined the effect of ozagrel, a thromboxane A(2) synthase inhibitor, on the accumulation of leucocytes and chemokine mRNA expression in lungs experimentally injured using oleic acid (OA). OA injection into guinea pigs rapidly increased thromboxane A(2) generation and subsequently increased total protein concentration and the numbers of macrophages and neutrophils in bronchoalveolar lavage fluid and increased monocyte chemoattractant protein-1 and interleukin-8 mRNA expression in the whole lung. Administration of ozagrel prevented these changes associated with OA injection. Ozagrel is a promising drug candidate for preventing acute lung injury. PMID:19783866

Ishitsuka, Yoichi; Moriuchi, Hiroshi; Isohama, Yoichiro; Tokunaga, Hidehiro; Hatamoto, Keita; Kurita, Sumika; Irikura, Mitsuru; Iyama, Ken-ichi; Irie, Tetsumi

2009-10-01

187

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

OpenAIRE

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

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

2011-01-01

188

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors.  

Science.gov (United States)

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC(50) values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis. PMID:15985365

Müller-Vieira, Ursula; Angotti, Marc; Hartmann, Rolf W

2005-08-01

189

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

International Nuclear Information System (INIS)

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

190

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

International Nuclear Information System (INIS)

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

191

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

OpenAIRE

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

Ferrero, Aldo; Vidotto, Francesco; Busi, Roberto

2006-01-01

192

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors.  

Science.gov (United States)

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR. PMID:20056546

Gangjee, Aleem; Jain, Hiteshkumar D; Phan, Jaclyn; Guo, Xin; Queener, Sherry F; Kisliuk, Roy L

2010-01-15

193

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

Energy Technology Data Exchange (ETDEWEB)

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

Roeda, Dirk; Crouzel, Christian; Brouillet, Emmanuel; Valette, Heric

1996-05-01

194

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis*  

OpenAIRE

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus re...

Yamaji, Toshiyuki; Nishikawa, Kiyotaka; Hanada, Kentaro

2010-01-01

195

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

Czech Academy of Sciences Publication Activity Database

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

Broží?ková, Carole; Otáhal, Jakub

2013-01-01

196

Cyclooxygenase-independent induction of p21WAF-1/cip1, apoptosis and differentiation by L-745,337, a selective PGH synthase-2 inhibitor, and salicylate in HT-29 cells.  

Science.gov (United States)

In order to dissect out cyclooxygenase-dependent from cyclooxygenase-independent mechanisms in the antiproliferative effects of selective prostaglandin H synthase (PGHS)-2 inhibitors, we compared the effects of L-745,337 (a highly selective PGHS-2 inhibitor) with sodium salicylate (a weak PGHS inhibitor) on prostanoid production, induction of the cyclin-dependent kinase inhibitor p21WAF-1/cip1, mutant p53 (m273-p53) levels, apoptosis and differentiation in human colon adenocarcinoma HT-29 cells. L-745,337 dose-dependently suppressed the cyclooxygenase activity of HT-29 cells (IC50: 0.24 microM). Four-day treatment with L-745,337 caused a concentration-dependent inhibition of cell growth (IC50: 0.9 mM) associated with the induction of p21WAF-1/cip1 and an increase in the proportion of apoptotic nuclei (EC50: 0.1 and 0.34 mM, respectively) while reducing the levels of m273-p53 (IC50: 0.2 mM). Sodium salicylate, at the concentration of 10 mM that did not affect prostanoid formation, caused a 60% reduction of cell growth associated with a 3-fold induction of p21WAF-1/cip1 and a 60% increase in the proportion of apoptotic nuclei. Ultrastructural analysis showed that L-745,337 (0.5 mM) and sodium salicylate (10 mM) caused the induction of a differentiated phenotype. We conclude that high concentrations of L-745,337 and sodium salicylate inhibit colon cancer cell growth by a mechanism unrelated to cyclooxygenase inhibition that may involve p53-independent induction of the tumor suppressor p21WAF-1/cip1. PMID:14634277

Santini, G; Sciulli, M G; Marinacci, R; Fusco, O; Spoletini, L; Pace, A; Ricciardulli, A; Natoli, C; Procopio, A; Maclouf, J; Patrignani, P

1999-06-01

197

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

OpenAIRE

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

198

A leukotriene C4 synthase inhibitor with the backbone of 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid  

OpenAIRE

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-...

Ago, Hideo; Okimoto, Noriaki; Kanaoka, Yoshihide; Morimoto, Gentaro; Ukita, Yoko; Saino, Hiromichi; Taiji, Makoto; Miyano, Masashi

2013-01-01

199

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

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

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

2014-06-27

200

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

OpenAIRE

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

Vials, A.; Burnstock, G.

1992-01-01

201

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

Science.gov (United States)

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

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

2012-04-11

202

Role of the anterior region of the third ventricle in the cardiovascular responses produced by systemic injection of a nitric oxide synthase inhibitor  

Science.gov (United States)

This study examined whether a prior electrolytic lesion of the tissue surrounding the anteroventral third ventricle (AV3V) would affect the increase in mean arterial blood pressure (MAP) and the fall in heart rate (HR) produced by systemic injection of the nitric oxide synthesis (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 25 micromol/kg, i.v.) in conscious rats. L-NAME produced a smaller increase in MAP in AV3V-lesion than in sham-lesion rats (+19+/-3 vs. +40+/-3 mmHg, respectively; PL-NAME produced similar falls in HR in the AV3V-lesion and sham-lesion rats (-103+/-15 vs. -97+/-8 bpm, respectively; PL-NAME-induced pressor response is dependent upon the integrity of the AV3V region, whereas the L-NAME-induced bradycardia is not. Copyright 1999 Elsevier Science B. V.

Lewis, S. J.; Whalen, E. J.; Beltz, T. G.; Johnson, A. K.

1999-01-01

203

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

Science.gov (United States)

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

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

2013-05-01

204

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

Science.gov (United States)

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

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

2015-02-15

205

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

Science.gov (United States)

The rationale of this dose matching/dose escalating study was to compare a panel of flavonoids—luteolin, resveratrol, and quercetin—against the metabolite flux-controlling properties of a synthetic targeted fatty acid synthase inhibitor drug C75 on multiple macromolecule synthesis pathways in pancreatic tumor cells using [1,2-13C2]-d-glucose as the single precursor metabolic tracer. MIA PaCa-2 pancreatic adenocarcinoma cells were cultured for 48 h in the presence of 0.1% DMSO (control), or 50 or 100 ?M of each test compound, while intracellular glycogen, RNA ribose, palmitate and cholesterol as well as extra cellular 13CO2, lactate and glutamate production patterns were measured using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). The use of 50% [1,2-13C2]-d-glucose as tracer resulted in an average of 24 excess 13CO2 molecules for each 1,000 CO2 molecule in the culture media, which was decreased by 29 and 33% (P < 0.01) with 100 ?M C75 and luteolin treatments, respectively. Extracellular tracer glucose-derived 13C-labeled lactate fractions (?m) were between 45.52 and 47.49% in all cultures with a molar ratio of 2.47% M + 1/?m lactate produced indirectly by direct oxidation of glucose in the pentose cycle in control cultures; treatment with 100 ?M C75 and luteolin decreased this figure to 1.80 and 1.67%. The tracer glucose-derived 13C labeled fraction (?m) of ribonucleotide ribose was 34.73% in controls, which was decreased to 20.58 and 8.45% with C75, 16.15 and 6.86% with luteolin, 27.66 and 19.25% with resveratrol, and 30.09 and 25.67% with quercetin, respectively. Luteolin effectively decreased nucleotide precursor synthesis pentose cycle flux primarily via the oxidative branch, where we observed a 41.74% flux (M + 1/?m) in control cells, in comparison with only a 37.19%, 32.74%, or a 26.57%, 25.47% M + 1/?m flux (P < 0.001) after 50 or 100 ?M C75 or luteolin treatment. Intracellular de novo fatty acid palmitate (C16:0) synthesis was severely and equally blocked by C75 and luteolin treatments indicated by the 5.49% (control), 2.29 or 2.47% (C75) and 2.21 or 2.73% (luteolin) tracer glucose-derived 13C-labeled fractions, respectively. On the other hand there was a significant 192 and 159% (P < 0.001), and a 103 and 117% (P < 0.01) increase in tracer glucose-derived cholesterol after C75 or luteolin treatment. Only resveratrol and quercetin at 100 ?M inhibited tracer glucose-derived glycogen labeling (?m) and turnover by 34.8 and 23.8%, respectively. The flavonoid luteolin possesses equal efficacy to inhibit fatty acid palmitate de novo synthesis as well as nucleotide RNA ribose turnover via the oxidative branch of the pentose cycle in comparison with the targeted fatty acid synthase inhibitor synthetic compound C75. Luteolin is also effective in stringently controlling glucose entry and anaplerosis in the TCA cycle, while it promotes less glucose flux towards cholesterol synthesis than that of C75. In contrast, quercetin and resveratrol inhibit glycogen synthesis and turnover as their underlying mechanism of controlling tumor cell proliferation. Therefore the flavonoid luteolin controls fatty and nucleic acid syntheses as well as energy production with pharmacological strength, which can be explored as a non-toxic natural treatment modality for pancreatic cancer. PMID:22754424

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

2011-01-01

206

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  

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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

207

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

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

2006-04-01

208

Nitric Oxide Synthase Inhibitors as Antidepressants  

OpenAIRE

Affective and anxiety disorders are widely distributed disorders with severe social and economic effects. Evidence is emphatic that effective treatment helps to restore function and quality of life. Due to the action of most modern antidepressant drugs, serotonergic mechanisms have traditionally been suggested to play major roles in the pathophysiology of mood and stress-related disorders. However, a few clinical and several pre-clinical studies, strongly suggest involvement of the nitric oxi...

Vallo Volke; Gregers Wegener

2010-01-01

209

Construction of a brewer's yeast having alpha-acetolactate decarboxylase gene from Acetobacter aceti ssp. xylinum integrated in the genome.  

Science.gov (United States)

alpha-Acetolactate decarboxylase (ALDC) gene from Acetobacter aceti ssp. xylinum has several possible initiation codons in the N-terminus. To determine the initiation codon of the ALDC giving the highest expression levels, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was linked just upstream of each possible initiation codon. The ALDC whose translation starts 130 bp downstream from the first ATG codon had the highest activity in yeast cells. When expression levels of the ALDC gene were compared using three strong yeast promoters of glycolytic genes, alcohol dehydrogenase I (ADC1), phosphoglycerate kinase (PGK) and GPD, the GPD promoter was the strongest. The ALDC gene was integrated in a ribosomal RNA gene of a brewer's yeast by co-transformation with an expression plasmid of G418-resistance gene. The laboratory-scale growth test confirmed that the total diacetyl concentration was reduced in wort. PMID:7764564

Yamano, S; Kondo, K; Tanaka, J; Inoue, T

1994-02-14

210

Cloning and expression of the gene encoding alpha-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast.  

Science.gov (United States)

Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding alpha-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione. PMID:7764563

Yamano, S; Tanaka, J; Inoue, T

1994-02-14

211

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

Science.gov (United States)

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

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

2015-04-01

212

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

OpenAIRE

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

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

1988-01-01

213

Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells.  

Science.gov (United States)

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases. PMID:7530001

Nakane, M; Pollock, J S; Klinghofer, V; Basha, F; Marsden, P A; Hokari, A; Ogura, T; Esumi, H; Carter, G W

1995-01-17

214

Inibidores seletivos de prostaglandina endoperóxido sintase-2 (PGHS-2): nova estratégia para o tratamento da inflamação Selective inhibitors of prostaglandin endoperoxide synthase-2 (PGHS-2): new target to the treatment for inflammatory diseases  

OpenAIRE

Prostaglandins (PG's), produced from arachidonic acid metabolism, are potent mediators of inflammation. Nonsteroidal anti-inflammatory (NSAIDs) exert their effects by inhibition of prostaglandin endoperoxide synthase (PGHS) enzyme, which catalyses the first committed step in arachidonic acid metabolism. Two isoforms of PGHS are known: PGHS-1, constitutively expressed in most tissues, and is responsible for physiological production of PG's. The second isoform, PGHS-2, is induced by cytokines, ...

Adriana dos Santos Lages; Nelilma Correia Romeiro; Carlos Alberto Manssour Fraga; Eliezer Jesus Barreiro

1998-01-01

215

Higher plant cellulose synthases  

OpenAIRE

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

Richmond, Todd

2000-01-01

216

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

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

1710-17-01

217

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

218

Pharmacological characterization of N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea (BM-573), a novel thromboxane A2 receptor antagonist and thromboxane synthase inhibitor in a rat model of arterial thrombosis and its effects on bleeding time.  

Science.gov (United States)

The present study was undertaken to characterize the antiplatelet and antithrombotic effects of BM-573 [N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea], an original combined thromboxane receptor antagonist and thromboxane synthase inhibitor in rats, and to determine its effects on mice bleeding time. Intraperitoneal injection of a single dose of 5 mg/kg BM-573 to rats inhibited U-46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F(2))-induced washed platelet aggregation 30 min and 1, 2, and 4 h after drug administration with a maximum antiplatelet effect observed after 1 and 2 h. In a rat model of thrombosis induced by ferric chloride application on the abdominal aorta, BM-573 significantly reduced the thrombus weight by 92.53, 80.20, 64.75, and 18.21% at doses of 5, 2, 0.5, and 0.2 mg/kg, respectively. Time to occlusion of abdominal aorta in the BM-573-treated group (41.50 +/- 5.21 min) was significantly prolonged compared with the vehicle-treated rats (16.16 +/- 0.79 min). Like furegrelate, seratrodast, and acetylsalicylic acid, BM-573 did not affect the tail bleeding time induced by tail transection in mice compared with vehicle-treated mice. Moreover, BM-573, a close derivative of the loop diuretic torasemide, failed to induce a significant increase in diuresis in rat and did not produce a decrease in blood glucose concentration as observed with the sulfonylurea glibenclamide. In conclusion, we have demonstrated that the nitrobenzenic sulfonylurea BM-573, an original combined thromboxane receptor antagonist and thromboxane synthase inhibitor, is a potent antithrombotic agent that does not affect bleeding time. Moreover, BM-573 lost the diuretic property of torasemide and has no impact on glycemia. PMID:14742735

Dogné, Jean-Michel; Hanson, Julien; de Leval, Xavier; Kolh, Philippe; Tchana-Sato, Vincent; de Leval, Laurence; Rolin, Stéphanie; Ghuysen, Alexandre; Segers, Patrick; Lambermont, Bernard; Masereel, Bernard; Pirotte, Bernard

2004-05-01

219

ATP Synthase Inhibition of Mycobacterium avium Is Not Bactericidal?  

Science.gov (United States)

The efficacy of ATP synthase inhibitor TMC207 was assessed in early and late Mycobacterium avium infections in mice. In contrast to what was earlier observed for M. tuberculosis, a bacteriostatic effect was obtained. In vitro, the minimal bactericidal concentration (MBC)/MIC ratio was very high. The MBC was more relevant for assessment of pharmacokinetic/pharmacodynamic relationships than the MIC. PMID:19738016

Lounis, Nacer; Gevers, Tom; Van Den Berg, Joke; Vranckx, Luc; Andries, Koen

2009-01-01

220

GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS  

OpenAIRE

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

HagitEldar-Finkelman; AnaMartinez

2011-01-01

221

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

222

Susceptibility of Plasmodium falciparum to a combination of thymidine and ICI D1694, a quinazoline antifolate directed at thymidylate synthase.  

OpenAIRE

Unlike mammalian cells, malarial parasites lack the enzymes to salvage preformed pyrimidines. For this reason, a combination of a thymidylate synthase inhibitor and the nucleoside thymidine should provide selective antimalarial activity even in the absence of any known active site differences between malarial and mammalian thymidylate synthases. To test this hypothesis, we evaluated the in vitro antimalarial activity of ICI D1694, a quinazoline antifolate that inhibits thymidylate synthase in...

Rathod, P. K.; Reshmi, S.

1994-01-01

223

Geranyl diphosphate synthase from mint  

Energy Technology Data Exchange (ETDEWEB)

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

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

1999-01-01

224

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

225

Tapentadol and nitric oxide synthase systems.  

Science.gov (United States)

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

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

2015-04-01

226

Inibidores seletivos de prostaglandina endoperóxido sintase-2 (PGHS-2: nova estratégia para o tratamento da inflamação Selective inhibitors of prostaglandin endoperoxide synthase-2 (PGHS-2: new target to the treatment for inflammatory diseases  

Directory of Open Access Journals (Sweden)

Full Text Available Prostaglandins (PG's, produced from arachidonic acid metabolism, are potent mediators of inflammation. Nonsteroidal anti-inflammatory (NSAIDs exert their effects by inhibition of prostaglandin endoperoxide synthase (PGHS enzyme, which catalyses the first committed step in arachidonic acid metabolism. Two isoforms of PGHS are known: PGHS-1, constitutively expressed in most tissues, and is responsible for physiological production of PG's. The second isoform, PGHS-2, is induced by cytokines, mitogens and endotoxins in inflammatory cells, and appears to be responsible for the elevated production of PG's during inflammation. With the recent discovery of the inducible PGHS (PGHS-2, the medicinal chemist now possesses a novel target for designing therapeutic agents that could provide suitable anti-inflammatory activity without the ulcerogenic and renal side effects associated with currently available NSAIDs, all of which inhibit both PGHS-1 and PGHS-2.

Adriana dos Santos Lages

1998-11-01

227

Inibidores seletivos de prostaglandina endoperóxido sintase-2 (PGHS-2): nova estratégia para o tratamento da inflamação / Selective inhibitors of prostaglandin endoperoxide synthase-2 (PGHS-2): new target to the treatment for inflammatory diseases  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese [...] Abstract in english Prostaglandins (PG's), produced from arachidonic acid metabolism, are potent mediators of inflammation. Nonsteroidal anti-inflammatory (NSAIDs) exert their effects by inhibition of prostaglandin endoperoxide synthase (PGHS) enzyme, which catalyses the first committed step in arachidonic acid metabol [...] ism. Two isoforms of PGHS are known: PGHS-1, constitutively expressed in most tissues, and is responsible for physiological production of PG's. The second isoform, PGHS-2, is induced by cytokines, mitogens and endotoxins in inflammatory cells, and appears to be responsible for the elevated production of PG's during inflammation. With the recent discovery of the inducible PGHS (PGHS-2), the medicinal chemist now possesses a novel target for designing therapeutic agents that could provide suitable anti-inflammatory activity without the ulcerogenic and renal side effects associated with currently available NSAIDs, all of which inhibit both PGHS-1 and PGHS-2.

Adriana dos Santos, Lages; Nelilma Correia, Romeiro; Carlos Alberto Manssour, Fraga; Eliezer Jesus, Barreiro.

1998-11-01

228

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

229

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2009-08-17

230

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

OpenAIRE

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5?-deoxy-5?-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSA...

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

2011-01-01

231

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

OpenAIRE

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

232

Monoterpene synthases from common sage (Salvia officinalis)  

Science.gov (United States)

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

1999-01-01

233

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.

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

2001-04-01

234

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

Directory of Open Access Journals (Sweden)

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

235

S-methylmethionine is both a substrate and an inactivator of 1-aminocyclopropane-1-carboxylate synthase.  

Science.gov (United States)

S-methyl-L-methionine (SMM) is ubiquitous in the tissues of flowering plants, but its precise function remains unknown. It is both a substrate and an inhibitor of the pyridoxal 5(')-phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase, due to its structural similarity to the natural substrate of this enzyme, S-adenosyl-L-methionine. In the reaction with ACC synthase, SMM can either be transaminated to yield 4-dimethylsulfonium-2-oxobutyrate; converted to alpha-ketobutyrate, ammonia, and dimethylsulfide; or inactivate the enzyme covalently after elimination of dimethylsulfide. These results suggest a previously unrecognized role for SMM in the regulation of ACC synthase activity in plants. PMID:14678788

Ko, SaeHee; Eliot, Andrew C; Kirsch, Jack F

2004-01-01

236

Novel Pyridyl Substituted 4,5-Dihydro-[1,2,4]triazolo[4,3-a]quinolines as Potent and Selective Aldosterone Synthase Inhibitors with Improved in Vitro Metabolic Stability.  

Science.gov (United States)

CYP11B2 inhibition is a promising treatment for diseases caused by excessive aldosterone. To improve the metabolic stability in human liver miscrosomes of previously reported CYP11B2 inhibitors, modifications were performed via a combination of ligand- and structure-based drug design approaches, leading to pyridyl 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolones. Compound 26 not only exhibited a much longer half-life (t1/2 ? 120 min), but also sustained inhibitory potency (IC50 = 4.2 nM) and selectivity over CYP11B1 (SF = 422), CYP17, CYP19, and a panel of hepatic CYP enzymes. PMID:25711516

Hu, Qingzhong; Yin, Lina; Ali, Amjad; Cooke, Andrew J; Bennett, Jonathan; Ratcliffe, Paul; Lo, Michael Man-Chu; Metzger, Edward; Hoyt, Scott; Hartmann, Rolf W

2015-03-12

237

Design, synthesis and biological evaluation of N-alkyl or aryl substituted isoindigo derivatives as potential dual cyclin-dependent kinase 2 (CDK2)/glycogen synthase kinase 3? (GSK-3?) phosphorylation inhibitors.  

Science.gov (United States)

A series of N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and selective anti-proliferative activity against K562 cells (IC50 7.8 ?M) and induced the apoptosis of K562 cells in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3? (Ser9), ?-catenin and c-myc proteins, up-regulated the expression of GSK-3?, consequently, suppressed Wnt/?-catenin signaling pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3? was simulated using molecular docking tools. All of these studies gave a better understanding to the molecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3? (Ser9) phosphorylation inhibitors applied in cancer chemotherapy. PMID:25151579

Zhao, Ping; Li, Yanzhong; Gao, Guangwei; Wang, Shuai; Yan, Yun; Zhan, Xiaoping; Liu, Zenglu; Mao, Zhenmin; Chen, Shaoxiong; Wang, Liqun

2014-10-30

238

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

OpenAIRE

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

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

2009-01-01

239

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

OpenAIRE

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

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

1994-01-01

240

Structure of a cephalosporin synthase.  

OpenAIRE

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

Valega?rd, K.; Scheltinga, Ac; Lloyd, Md; Hara, T.; Ramaswamy, S.; Perrakis, A.; Thompson, A.; Lee, Hj; Baldwin, Je; Schofield, CJ; Hajdu, J.; Andersson, I.

1998-01-01

241

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2011-11-17

242

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

OpenAIRE

Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40?Å.

Atkinson, Sarah C.; Dogovski, Con; Dobson, Renwick C. J.; Perugini, Matthew A.

2012-01-01

243

Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues.  

OpenAIRE

1. Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH-groups. 2. The NO synthase inhibitor, NG-nitro-L-arginine...

Panagiotidis, G.; Akesson, B.; Rydell, E. L.; Lundquist, I.

1995-01-01

244

Cyclophilin D Modulates Mitochondrial F0F1-ATP Synthase by Interacting with the Lateral Stalk of the Complex*  

OpenAIRE

Blue native gel electrophoresis purification and immunoprecipitation of F0F1-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F1 sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were obse...

Giorgio, Valentina; Bisetto, Elena; Soriano, Maria Eugenia; Dabbeni-sala, Federica; Basso, Emy; Petronilli, Valeria; Forte, Michael A.; Bernardi, Paolo; Lippe, Giovanna

2009-01-01

245

Convergent evolution of coenzyme M biosynthesis in the Methanosarcinales: cysteate synthase evolved from an ancestral threonine synthase.  

Science.gov (United States)

The euryarchaeon Methanosarcina acetivorans has no homologues of the first three enzymes that produce the essential methanogenic coenzyme M (2-mercaptoethanesulfonate) in Methanocaldococcus jannaschii. A single M. acetivorans gene was heterologously expressed to produce a functional sulfopyruvate decarboxylase protein, the fourth canonical enzyme in this biosynthetic pathway. An adjacent gene, at locus MA3297, encodes one of the organism's two threonine synthase homologues. When both paralogues from this organism were expressed in an Escherichia coli threonine synthase mutant, the MA1610 gene complemented the thrC mutation, whereas the MA3297 gene did not. Both PLP (pyridoxal 5'-phosphate)-dependent proteins were heterologously expressed and purified, but only the MA1610 protein catalysed the canonical threonine synthase reaction. The MA3297 protein specifically catalysed a new beta-replacement reaction that converted L-phosphoserine and sulfite into L-cysteate and inorganic phosphate. This oxygen-independent mode of sulfonate biosynthesis exploits the facile nucleophilic addition of sulfite to an alpha,beta-unsaturated intermediate (PLP-bound dehydroalanine). An amino acid sequence comparison indicates that cysteate synthase evolved from an ancestral threonine synthase through gene duplication, and the remodelling of active site loop regions by amino acid insertion and substitutions. The cysteate product can be converted into sulfopyruvate by an aspartate aminotransferase enzyme, establishing a new convergent pathway for coenzyme M biosynthesis that appears to function in members of the orders Methanosarcinales and Methanomicrobiales. These differences in coenzyme M biosynthesis afford the opportunity to develop methanogen inhibitors that discriminate between the classes of methanogenic archaea. PMID:19761441

Graham, David E; Taylor, Stephanie M; Wolf, Rachel Z; Namboori, Seema C

2009-12-15

246

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

247

Heterocyclic inhibitors of glycogen synthase kinase GSK-3  

OpenAIRE

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

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

2006-01-01

248

Biphenyl synthase, a novel type III polyketide synthase.  

Science.gov (United States)

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

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

2007-05-01

249

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

Directory of Open Access Journals (Sweden)

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

250

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

Scientific Electronic Library Online (English)

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

P.J., Christoffoleti.

2001-04-01

251

Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo  

DEFF Research Database (Denmark)

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

Volke, V; Wegener, Gregers

1999-01-01

252

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

Science.gov (United States)

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

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

2013-04-01

253

Glycogen synthase kinase 3: more than a namesake.  

Science.gov (United States)

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target. PMID:19366350

Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

2009-03-01

254

Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.  

Science.gov (United States)

1. The synthesis of nitrite and citrulline from L-arginine by immune-stimulated rat alveolar macrophages and the modulation of this synthesis were studied. 2,4-Diamino-6-hydroxypyrimidine (DAHP), 6R-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-sepiapterin were potent inhibitors of the recombinant interferon-gamma induced production of nitrogen oxides in intact cultured cells with I50 values for BH4 and L-sepiapterin of approximately 10 microM. They were equally effective in inhibiting the induced production of citrulline. This inhibitory effect was concentration-dependent for all three modulators investigated. 2. The inhibitory effects were not dependent on incubation times of either 24 or 48 h, on the immune-stimulus used (lipopolysaccharide, interferon-gamma), or whether these stimuli were added during or after the induction period. 3. Pterin-6-carboxylic acid (PCA), which cannot be converted into BH4, and methotrexate (MTX), which inhibits dihydrofolatereductase but not de novo biosynthesis of BH4, did not change the production of nitrite. 4. The data indicate that DAHP, an inhibitor of the de novo biosynthesis of the co-factor BH4, blocks the nitric oxide synthase activity in intact cells. Since the pterins BH4 and L-sepiapterin blocked the L-arginine dependent production of nitrite and citrulline, the activity of nitric oxide synthase in phagocytic cells may be regulated by metabolic endproducts of the de novo biosynthesis of BH4. PMID:1281717

Jorens, P. G.; van Overveld, F. J.; Bult, H.; Vermeire, P. A.; Herman, A. G.

1992-01-01

255

Attenuation by prolonged nitric oxide synthase inhibition of the enhancement of fibrinolysis caused by environmental stress in the rat.  

OpenAIRE

1. Nitric oxide (NO) suppresses platelet aggregation and plasminogen activator inhibitor (PAI) release from platelets, playing physiological and/or pathological roles in the haemostatic system. We investigated the effect of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, on the disseminated intravascular coagulation (DIC)-like phenomena in rats under environmental stress, induced by prolonged fluctuation in air temperature, known as SART (specific alternation of rhythm in...

Kawabata, A.; Hata, T.

1996-01-01

256

The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase.  

Science.gov (United States)

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere. PMID:2893789

Mukohata, Y; Yoshida, M

1987-10-01

257

Assay of Deoxyhypusine Synthase Activity  

OpenAIRE

Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (

Wolff, Edith C.; Lee, Seung Bum; Park, Myung Hee

2011-01-01

258

Identification of avian wax synthases  

OpenAIRE

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

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

2012-01-01

259

Deoxyribonuclease inhibitors.  

Science.gov (United States)

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

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

2014-12-17

260

A functional cellulose synthase from ascidian epidermis  

OpenAIRE

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

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

2004-01-01

261

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

OpenAIRE

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

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

2011-01-01

262

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

Directory of Open Access Journals (Sweden)

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

Ketabchi Farzaneh

2012-01-01

263

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

OpenAIRE

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

264

Artesunate potentiates antibiotics by inactivating heme-harbouring bacterial nitric oxide synthase and catalase  

OpenAIRE

Abstract Background A current challenge of coping with bacterial infection is that bacterial pathogens are becoming less susceptible to or more tolerant of commonly used antibiotics. It is urgent to work out a practical solution to combat the multidrug resistant bacterial pathogens. Findings Oxidative stress-acclimatized bacteria thrive in rifampicin by generating antibiotic-detoxifying nitric oxide (NO), which can be repressed by artesunate or an inhibitor of nitric oxide synthase (NOS). Sup...

Guo Xiao-Xia; Zeng Li-Xiang; Yang Xue-Qin; Wu Pei; Xiao Na; Zeng Qing-Ping; Zhang Ping-Zu; Qiu Frank

2011-01-01

265

Cerulenin Blockade of Fatty Acid Synthase Reverses Hepatic Steatosis in ob/ob Mice  

OpenAIRE

Fatty liver or hepatic steatosis is a common health problem associated with abnormal liver function and increased susceptibility to ischemia/reperfusion injury. The objective of this study was to investigate the effect of the fatty acid synthase inhibitor cerulenin on hepatic function in steatotic ob/ob mice. Different dosages of cerulenin were administered intraperitoneally to ob/ob mice for 2 to 7 days. Body weight, serum AST/ALT, hepatic energy state, and gene expression patterns in ob/ob ...

Cheng, Gang; Palanisamy, Arun P.; Evans, Zachary P.; Sutter, Alton G.; Jin, Lan; Singh, Inderjit; May, Harold; Schmidt, Michael G.; Chavin, Kenneth D.

2013-01-01

266

Development of a Pterin-based Fluorescent Probe for Screening Dihydropteroate Synthase  

OpenAIRE

Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin sub-site rather than to the adjacent p-amino benzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate th...

Zhao, Ying; Hammoudeh, Dalia; Lin, Wenwei; Das, Sourav; Yun, Mi-kyung; Li, Zhenmei; Griffith, Elizabeth; Chen, Taosheng; White, Stephen W.; Lee, Richard E.

2011-01-01

267

Producing biofuels using polyketide synthases  

Science.gov (United States)

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

268

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

269

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

270

Chitin synthases from Saprolegnia are involved in tip growth and represent a potential target for anti-oomycete drugs.  

Science.gov (United States)

Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control. PMID:20865175

Guerriero, Gea; Avino, Mariano; Zhou, Qi; Fugelstad, Johanna; Clergeot, Pierre-Henri; Bulone, Vincent

2010-01-01

271

Hepatic stellate cells promote hepatocyte engraftment in rat liver after prostaglandin-endoperoxide synthase inhibition  

Science.gov (United States)

Background & Aims Hepatic inflammation occurs immediately after cells are transplanted to the liver, but the mechanisms that underlie this process are not fully defined. We examined cyclooxygenase pathways that mediate hepatic inflammation through synthesis of prostaglandins (PG), prostacyclins, thromboxanes and other prostanoids following transplantation of hepatocytes. Methods We transplanted F344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient F344 rats. Changes in cyclooxygenase pathways were analyzed and specific pathways were blocked pharmacologically; the effects on cell engraftment and native liver cells were determined. Results Transplantation of hepatocytes induced hepatic expression of prostaglandin-endoperoxide synthases 1 and 2, which catalyze production of prostaglandin H2, as well as the downstream factor thromboxane synthase, which produces thromboxane A2 (a regulator of vascular and platelet responses in inflammation). Transplanted hepatocytes were in proximity with liver cells that express prostaglandin-endoperoxide synthases. The number of engrafted hepatocytes increased in rats given naproxen or celecoxib before transplantation, but not in rats given furegrelate (an inhibitor of thromboxane synthase) or clopodigrel (an anti-platelet drug). Naproxen and celecoxib did not prevent hepatic ischemia or activation of neutrophils, Kupffer cells or inflammatory cytokines, but they did induce hepatic stellate cells to express cytoprotective genes, vascular endothelial growth factor and hepatocyte growth factor, and matrix-type metalloproteinases and tissue inhibitor of metalloproteinase-1, which regulate hepatic remodeling. Conclusions Activation of cyclooxygenase pathways interferes with engraftment of transplanted hepatocytes in the liver. Pharmacological blockade of prostaglandin-endoperoxide synthases stimulated hepatic stellate cells and improved cell engraftment. PMID:19303017

Enami, Yuta; Bandi, Sriram; Kapoor, Sorabh; Krohn, Natan; Joseph, Brigid; Gupta, Sanjeev

2009-01-01

272

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex.  

Science.gov (United States)

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

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

2014-06-19

273

Asymmetric dimethylarginine in adults with cystathionine ?-synthase deficiency.  

Science.gov (United States)

In hyperhomocysteinemia (HHcy), an independent risk factor for cardiovascular diseases, endothelial dysfunction due to reduced bioavailability of nitric oxide is a consistent finding. However, the underlying mechanisms remain unknown. Increased levels of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with HHcy, and may contribute, at least in part, for the homocysteine-induced endothelial dysfunction, but whether cystathionine ?-synthase (CBS) deficiency is associated with increased ADMA has hardly been investigated. To address this question, we measured total homocysteine (tHcy), ADMA and symmetric dimethylarginine (SDMA) in plasma of 22 adult CBS deficient patients, using established HPLC techniques. Results showed that in CBS deficient patients with elevated levels of tHcy (median (total range): 33 (14-237) ?mol/L), both ADMA and SDMA levels were normal. Moreover, tHcy and ADMA concentrations were not correlated (r(s)=0.017, p=0.94). Our results favor the hypothesis that the negative vascular effects of HHcy have an ADMA-independent etiology. PMID:22484094

Rocha, Monica S; Teerlink, Tom; Janssen, Mirian C H; Kluijtmans, Leo A J; Smulders, Yvo; Jakobs, Cornelis; Tavares de Almeida, Isabel; Rivera, Isabel; Castro, Rita; Blom, Henk J

2012-06-01

274

Constitutive nitric oxide synthase is present in normal human keratinocytes.  

Science.gov (United States)

Normal human keratinocytes in culture exhibit a nitric oxide synthase (NOS) activity ranging from 50 to 150 pmol/min/mg of protein. The enzyme is cytosolic and requires the presence of calcium, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide. Calmodulin antagonists (trifluoperazine and calmidazolium) inhibit the enzyme activity. We show that NG-nitro-L-arginine inhibits NOS more potently than NG-monomethyl-L-arginine and that L-canavanine is a weak inhibitor. NOS was partially purified using a 2',5'-ADP Sepharose affinity column eluted with NADPH. A partially purified fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and Western blotting. A protein with an apparent molecular weight of 152 kDa cross-reacted with monoclonal antibodies raised against the neuronal constitutive isoform of NOS. The enzyme had a Vmax of 7.3 nmol/min/mg of protein and a Km for L-arginine of 22.3 microM. These results indicate that normal human keratinocytes contain a constitutive nitric oxide synthase related to NOS I. PMID:8648171

Baudouin, J E; Tachon, P

1996-03-01

275

Inhibition of nitric oxide synthase does not prevent the induction of long-term potentiation in vivo.  

OpenAIRE

Nitric oxide (NO), a putative intercellular messenger in the CNS, may be involved in certain forms of synaptic plasticity and learning. This article reports a series of experiments investigating whether an inhibitor of NO synthase, N omega-nitro-L-arginine methyl ester (L-NAME), affects long-term potentiation (LTP) in vivo, as the results of recent in vitro experiments would predict. L-NAME, given as an acute injection at a dose sufficient to inhibit hippocampal NO synthase (> 90%), had no ef...

Bannerman, Dm; Chapman, Pf; Kelly, Pa; Butcher, Sp; Morris, Rg

1994-01-01

276

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

Science.gov (United States)

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

Paliyath, G.; Poovaiah, B. W.

1988-01-01

277

Potential role of inducible nitric oxide synthase in the sleep-wake states occurrence in old rats.  

Science.gov (United States)

Extensive evidences now suggest that an association between inducible nitric oxide synthase and oxidative stress takes place during aging. Since the part played by inducible nitric oxide synthase in the sleep impairments associated with aging still remains unexplored, we compared its involvement in old rats (20-24 months) versus adult ones (3-5 months) using polygraphic, biochemical, voltammetric and immunohistochemical techniques. The experiments were conducted either in basal condition or after a systemic injection of selected inducible nitric oxide synthase inhibitors. We found that 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (10 mg/kg, i.p.) or aminoguanidine (400 mg/kg, i.p.) was capable to suppress rapid-eye-movement sleep and induce a delayed enhancement in slow-wave sleep in old rats. These effects did not occur in adult animals. Within the frontal cortex, the laterodorsal tegmentum and dorsal raphe nuclei, the basal inducible nitric oxide synthase activity was 85-200% higher in old rats than in adult ones. In contrast, the neuronal nitric oxide synthase activity did not vary in both groups. 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine administration significantly reduced inducible nitric oxide synthase activity (70-80% according to the brain areas) independently of age, but significantly decreased the cortical nitric oxide release in old rats. Finally, in frontal cortex and dorsal raphe immunohistochemical analysis showed inducible nitric oxide synthase-positive cells again only in old animals. These data support the idea that nitric oxide produced by inducible nitric oxide synthase plays a role in the triggering and maintenance of rapid-eye-movement sleep during aging. PMID:16112470

Clément, P; Sarda, N; Cespuglio, R; Gharib, A

2005-01-01

278

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

279

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

280

Genetics Home Reference: GM3 synthase deficiency  

Science.gov (United States)

... a shortage of GM3 synthase and changes in skin pigmentation is also unknown. Read more about the ST3GAL5 ... involuntary ; mitochondria ; nervous system ; pigmentation ; recessive ; respiratory ; seizure ; skin pigmentation ; status epilepticus ; syndrome You may find definitions for ...

281

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase.  

Science.gov (United States)

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5'-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glu171 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1-37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway. PMID:25462832

Fan, Shanghua; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhou, Lin; Zhou, Jie; Chen, Tao; Chen, Guanjun; Zhang, Xian-En; Bi, Lijun

2015-01-01

282

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

Science.gov (United States)

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

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

2015-04-30

283

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

Science.gov (United States)

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

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

2015-06-01

284

Aminoguanidine selectively inhibits inducible nitric oxide synthase.  

OpenAIRE

1. Endotoxin induces nitric oxide synthase in vascular tissue, including rat main pulmonary artery. Currently available agents that cause inhibition of nitric oxide synthase are relatively non-selective between the constitutive and inducible forms of the enzyme. 2. Aminoguanidine caused a dose-dependent increase in phenylephrine-induced tension in intact and endothelium-denuded pulmonary artery rings from endotoxin-treated rats, but had no effect on sham-treated controls. 3. Contraction cause...

Griffiths, M. J.; Messent, M.; Macallister, R. J.; Evans, T. W.

1993-01-01

285

Nitric Oxide synthases and atrial fibrillation  

OpenAIRE

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

CynthiaAnnCarnes; ArunSridhar; SandorGyorke

2012-01-01

286

Novel Polyketide Synthase from Nectria haematococca  

OpenAIRE

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

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

2004-01-01

287

Deoxyhypusine synthase promotes differentiation and proliferation of T helper type 1 (Th1) cells in autoimmune diabetes.  

Science.gov (United States)

In type 1 diabetes, cytokines arising from immune cells cause islet ? cell dysfunction even before overt hyperglycemia. Deoxyhypusine synthase catalyzes the crucial hypusine modification of the factor eIF5A, which promotes the translation of a subset of mRNAs involved in cytokine responses. Here, we tested the hypothesis that deoxyhypusine synthase and, secondarily, hypusinated eIF5A contribute to the pathogenesis of type 1 diabetes using the non-obese diabetic (NOD) mouse model. Pre-diabetic NOD mice that received injections of the deoxyhypusine inhibitor N1-guanyl-1,7-diaminoheptane (GC7) demonstrated significantly improved glucose tolerance, more robust insulin secretion, and reduced insulitis compared with control animals. Analysis of tissues from treated mice revealed selective reductions in diabetogenic T helper type 1 (Th1) cells in the pancreatic lymph nodes, a primary site of antigen presentation. Isolated mouse CD90.2(+) splenocytes stimulated in vitro with anti-CD3/anti-CD28 and IL-2 to mimic autoimmune T cell activation exhibited proliferation and differentiation of CD4(+) T cell subsets (Th1, Th17, and Treg), but those treated with the deoxyhypusine synthase inhibitor GC7 showed a dose-dependent block in T cell proliferation with selective reduction in Th1 cells, similar to that observed in NOD mice. Inhibition of deoxyhypusine synthase blocked post-transcriptional expression of CD25, the high affinity IL-2 receptor ? chain. Our results suggest a previously unrecognized role for deoxyhypusine synthase in promoting T cell proliferation and differentiation via regulation of CD25. Inhibition of deoxyhypusine synthase may provide a strategy for reducing diabetogenic Th1 cells and preserving ? cell function in type 1 diabetes. PMID:24196968

Colvin, Stephanie C; Maier, Bernhard; Morris, David L; Tersey, Sarah A; Mirmira, Raghavendra G

2013-12-20

288

Deoxyhypusine Synthase Promotes Differentiation and Proliferation of T Helper Type 1 (Th1) Cells in Autoimmune Diabetes*  

Science.gov (United States)

In type 1 diabetes, cytokines arising from immune cells cause islet ? cell dysfunction even before overt hyperglycemia. Deoxyhypusine synthase catalyzes the crucial hypusine modification of the factor eIF5A, which promotes the translation of a subset of mRNAs involved in cytokine responses. Here, we tested the hypothesis that deoxyhypusine synthase and, secondarily, hypusinated eIF5A contribute to the pathogenesis of type 1 diabetes using the non-obese diabetic (NOD) mouse model. Pre-diabetic NOD mice that received injections of the deoxyhypusine inhibitor N1-guanyl-1,7-diaminoheptane (GC7) demonstrated significantly improved glucose tolerance, more robust insulin secretion, and reduced insulitis compared with control animals. Analysis of tissues from treated mice revealed selective reductions in diabetogenic T helper type 1 (Th1) cells in the pancreatic lymph nodes, a primary site of antigen presentation. Isolated mouse CD90.2+ splenocytes stimulated in vitro with anti-CD3/anti-CD28 and IL-2 to mimic autoimmune T cell activation exhibited proliferation and differentiation of CD4+ T cell subsets (Th1, Th17, and Treg), but those treated with the deoxyhypusine synthase inhibitor GC7 showed a dose-dependent block in T cell proliferation with selective reduction in Th1 cells, similar to that observed in NOD mice. Inhibition of deoxyhypusine synthase blocked post-transcriptional expression of CD25, the high affinity IL-2 receptor ? chain. Our results suggest a previously unrecognized role for deoxyhypusine synthase in promoting T cell proliferation and differentiation via regulation of CD25. Inhibition of deoxyhypusine synthase may provide a strategy for reducing diabetogenic Th1 cells and preserving ? cell function in type 1 diabetes. PMID:24196968

Colvin, Stephanie C.; Maier, Bernhard; Morris, David L.; Tersey, Sarah A.; Mirmira, Raghavendra G.

2013-01-01

289

Glycogen synthase kinase-3? is involved in C-reactive protein-induced endothelial cell activation.  

Science.gov (United States)

C-reactive protein (CRP) is a significant contributor to atherosclerosis and a powerful predictor of cardiovascular risk. The role of CRP in endothelial cell (EC) activation has been extensively investigated, but the underlying mechanisms have not been fully elucidated. The effect of glycogen synthase kinase-3? (GSK-3?) on CRP-induced EC activation was evaluated in this study. We observed that CRP decreased endothelial nitric oxide synthase (eNOS) activity during EC activation. CRP also activated GSK-3? by dephosphorylating its Ser9 level and reducing ?-catenin protein expression in a time-dependent manner. We also found that the GSK-3? inhibitors TDZD-8 and SB415286 partially restored eNOS activity and suppressed the release of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 from ECs. These data provide new evidence for the involvement of GSK-3? in EC activation. PMID:24228880

Liu, Shao-Jun; Liu, Wei-Hua; Zhong, Yun; Liu, Shi-Ming

2013-08-01

290

Identification of avian wax synthases  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.

Biester Eva-Maria

2012-02-01

291

Assay of deoxyhypusine synthase activity.  

Science.gov (United States)

Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-(3)H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [(3)H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [(3)H]deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [(3)H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter. PMID:21318875

Wolff, Edith C; Lee, Seung Bum; Park, Myung Hee

2011-01-01

292

Terpene synthases are widely distributed in bacteria.  

Science.gov (United States)

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

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

2015-01-20

293

Inhibition of nitric oxide synthase desensitizes retinal ganglion cells to light by diminishing their excitatory synaptic currents under light adaptation  

OpenAIRE

The effect of inhibiting nitric oxide synthase (NOS) on the visual responses of mouse retinal ganglion cells (RGCs) was studied under light adaptation by using patch-clamp recordings. The results demonstrated that NOS inhibitor, L-NAME, reduced the sensitivity of RGCs to light under light adaptation at different ambient light conditions. These observations were seen in all cells that recordings were made from. L-NAME diminished the excitatory synaptic currents (EPSCs), rather than increasing ...

Nemargut, Joseph P.; Wang, Guo-yong

2009-01-01

294

Possible Role of the Glycogen Synthase Kinase-3 Signaling Pathway in Trimethyltin-Induced Hippocampal Neurodegeneration in Mice  

OpenAIRE

Trimethyltin (TMT) is an organotin compound with potent neurotoxic effects characterized by neuronal destruction in selective regions, including the hippocampus. Glycogen synthase kinase-3 (GSK-3) regulates many cellular processes, and is implicated in several neurodegenerative disorders. In this study, we evaluated the therapeutic effect of lithium, a selective GSK-3 inhibitor, on the hippocampus of adult C57BL/6 mice with TMT treatment (2.6 mg/kg, intraperitoneal [i.p.]) and on cultured hip...

Kim, Juhwan; Yang, Miyoung; Kim, Sung-ho; Kim, Jong-choon; Wang, Hongbing; Shin, Taekyun; Moon, Changjong

2013-01-01

295

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

OpenAIRE

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

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

2013-01-01

296

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

297

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

298

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

Scientific Electronic Library Online (English)

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

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

2013-12-01

299

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

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

2013-06-01

300

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

Science.gov (United States)

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 migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10?8 M) and leukotriene B4 (LTB4, 10?8 M). l-NAME (but not d-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P 1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by l-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism. PMID:9391161

Zanardo, Renata C. O.; Costa, Edmar; Ferreira, Heloisa H. A.; Antunes, Edson; Martins, Antonio R.; Murad, Ferid; De Nucci, Gilberto

1997-01-01

301

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

Science.gov (United States)

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nomega-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 migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-8) M) and leukotriene B4 (LTB4, 10(-8) M). L-NAME (but not D-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P 1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0. 05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by L-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism. PMID:9391161

Zanardo, R C; Costa, E; Ferreira, H H; Antunes, E; Martins, A R; Murad, F; De Nucci, G

1997-12-01

302

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.  

OpenAIRE

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker re...

Eberhardt, S.; Korn, S.; Lottspeich, F.; Bacher, A.

1997-01-01

303

STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE  

OpenAIRE

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to polyesters class and is composed of hydroxy fatty acids. PHB is produced by microorganisms apparently in response to conditions of physiological stress. PHB synthases are the key enzymes of PHB biosynthesis. The PHB synthases obtained from Chromobacterium violaceum, belongs to the class I PHA synthases. Due to the limited structural information of PHB synthase, its functional properties including catalysis are u...

Femlin Blessia, T.; Jeya Sundara Sharmila, D.; Mohd Razip Samian; Hasni Arsad; Nur Fifyana Jamil

2012-01-01

304

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

OpenAIRE

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

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

1995-01-01

305

Determinants of aortic cyclic guanosine monophosphate in hypertension induced by chronic inhibition of nitric oxide synthase.  

OpenAIRE

Nitric oxide (NO) and atrial natriuretic factor (ANF) cause vascular relaxation by generating cyclic guanosine monophosphate (cGMP) via activation of the soluble and particulate guanylate cyclases, respectively. The chronic effects of NG-nitro-L-arginine methyl ester (L-NAME), an L-arginine antagonist and NO synthase inhibitor, on the blood pressure and plasma and aortic cGMP levels of rats were tested. Wistar rats (n = 10 per group) were given doses of L-NAME (0, 1, 5, 10, 20, 50, and 100 mg...

Arnal, J. F.; Warin, L.; Michel, J. B.

1992-01-01

306

The affinity purification and characterization of ATP synthase complexes from mitochondria  

OpenAIRE

The mitochondrial F1-ATPase inhibitor protein, IF1, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the ?-helical inhibitory region of the bound IF1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the ?-subunit in the enzyme's rotor. The intricacy of fo...

Runswick, Michael J.; Bason, John V.; Montgomery, Martin G.; Robinson, Graham C.; Fearnley, Ian M.; Walker, John E.

2013-01-01

307

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

International Nuclear Information System (INIS)

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

308

Nitric oxide synthase mediates the apelin-induced improvement of myocardial postischemic metabolic and functional recovery  

OpenAIRE

The adipocytokine apelin is capable to reduce myocardial ischemia/reperfusion injury in rodents. Cardioprotective activity of apelin may be attributed to upregulation of endothelial nitric oxide synthase (eNOS). This study was designed to examine metabolic and functional effects of a synthesized 12 C-terminal residue of apelin (A-12) and NG-nitro-L-arginine methyl ester (L-NAME), a non-selective eNOS inhibitor, in isolated working rat hearts subjected to global ischemia...

Pelogeykina, Yulia A.; Pisarenko, Oleg I.; Shulzhenko, Valentin S.; Studneva, Irina M.

2012-01-01

309

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

Directory of Open Access Journals (Sweden)

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

P.J. Christoffoleti

1994-08-01

310

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Experimentos foram instalados em condições de casa-de-vegetação com o objetivo de comparar a capacidade competitiva de biotipos resistentes e suscetíveis aos herbicidas inibidores da enzima acetolactase synthase da planta daninha kochia (Kochia scoparia L. Schard) comparada com trigo. Os resultados [...] de diversos experimentos, utilizando a metodologia chamada de substitutiva, indicaram que o trigo foi o competidor dominante, e em média uma planta de trigo reduziu o crescimento da planta de kochia resistente igual ao efeito de 4,8 plantas de kochia resistente ou 5,4 plantas de kochia suscetível. A competição chamada de intraespecífíca foi mais importante que a competição interespecífica para o trigo, porém o inverso foi verdadeiro para os biotípos resistentes e susceptíveis de kochia. Os resultados do índice de diferenciação ecológica indicaram que trigo e qualquer um dos dois biotípos de kochia estudados foram limitados apenas parcialmente pelos mesmos recursos de crescimento. No entanto, o crescimento dos biotípos resistentes e susceptíveis de kochia foram limitados pelos mesmos fatores de crescimento. Abstract in english Greenhouse experiments were conducted to compare the competitive ability of sulfonylurea resistant and susceptible kochia (Kochia scoparia L. Schard) compared to wheat. The results of several replacement series experiments indicate that wheat was the dominant competitor, and an average of one wheat [...] plant reduced resistant kochia yield per plant equal to the effect of 4.8 resistant kochia or 5.4 susceptible kochia plants. Intraspeciflc competition was more important than interspecific competition for wheat, whereas the reverse was true for the resistant and susceptible kochia. The results of the niche differentiation index (NDI) indicate that wheat and either resistant or susceptible kochia are only partly limited by the same resources. The resistant and susceptible kochia, however, are limited by the same resources.

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

1994-08-01

311

SYSTEMIC BUT NOT CENTRAL NERVOUS SYSTEM NITRIC OXIDE SYNTHASE INHIBITION EXACERBATES THE HYPERTENSIVE EFFECTS OF CHRONIC MELANOCORTIN-3/4 RECEPTOR ACTIVATION  

OpenAIRE

We examined whether systemic or central nervous system (CNS) inhibition of nitric oxide (NO) synthase exacerbates the cardiovascular responses of chronic CNS melanocortin 3/4 receptor (MC3/4R) activation. Sprague-Dawley rats implanted with telemetry probes, venous catheters and intracerebroventricular (ICV) cannulae were divided in 3 groups. After control measurements, the NO synthase inhibitor L-NAME was infused (10 ?g/kg/min, IV) for 17 days and starting on day 7 of L-NAME infusion the MC3...

Do Carmo, Jussara M.; Bassi, Mirian; Da Silva, Alexandre A.; Hall, John E.

2011-01-01

312

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

OpenAIRE

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

313

Cloning of parsley flavone synthase I.  

Science.gov (United States)

A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin. PMID:11524111

Martens, S; Forkmann, G; Matern, U; Lukacin, R

2001-09-01

314

Nuclear genetic defects of mitochondrial ATP synthase.  

Czech Academy of Sciences Publication Activity Database

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

Houšt?k, Josef; Kmoch, S.; Mayr, J. A.; Sperl, W.; Zeman, J.

315

Producing dicarboxylic acids using polyketide synthases  

Energy Technology Data Exchange (ETDEWEB)

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

316

Acceleration of oxidative stress-induced endothelial cell death by nitric oxide synthase dysfunction accompanied with decrease in tetrahydrobiopterin content.  

Science.gov (United States)

The purpose of this study was to examine whether nitric oxide (NO) synthase dysfunction accompanied with decrease in tetrahydrobiopterin (BH4) content increases H2O2-induced endothelial cell death. Endothelial cell death was measured by the release of intracellular lactate dehydrogenase (LDH). Intracellular BH4 content was changed by pretreatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, or pretreatment with sepiapterin, a substrate for the salvage pathway of BH4 synthesis, and the intracellular content was measured by high performance liquid chromatography equipped with a fluorescence detector. Moreover, production of superoxide was detected by a chemiluminescence technique using MCLA, a Cypridina luciferin analogue, for the superoxide-sensitive probe. Pretreatment with DAHP (10 mM) for 24 h decreased intracellular BH4 content to 14% and increased H2O2-induced cell death. The toxic effect of DAHP was reduced by co-pretreatment with sepiapterin (100 microM) or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NO synthase, but not by N(G)-methyl-L-arginine (L-NMA, 1 mM), the other inhibitor of NO synthase. Moreover, production of superoxide in endothelial cells induced by Ca2+-ionophore ionomycin (1 microM) increased by the pretreatment with DAHP, and the increase in superoxide production was blocked by L-NAME (1 mM) but not L-NMA (1 mM). Co-pretreatment with sepiapterin decreased the production of superoxide. These findings suggested that dysfunction of NO synthase with a decrease in BH4 content in endothelial cells produced superoxide instead of NO and increased the oxidative stress-induced endothelial cell death. PMID:9252248

Ishii, M; Shimizu, S; Yamamoto, T; Momose, K; Kuroiwa, Y

1997-01-01

317

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

Science.gov (United States)

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

318

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?  

Science.gov (United States)

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function. PMID:14550568

Barry, Fiona A; Graham, Gwenda J; Fry, Michael J; Gibbins, Jonathan M

2003-10-01

319

The affinity purification and characterization of ATP synthase complexes from mitochondria.  

Science.gov (United States)

The mitochondrial F?-ATPase inhibitor protein, IF?, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the ?-helical inhibitory region of the bound IF? occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the ?-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF? with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF? with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region. PMID:23407638

Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

2013-02-01

320

Inhibiting glycogen synthase kinase 3beta in sepsis.  

Science.gov (United States)

The serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in the regulation of many cell functions, but its role in the regulation of the inflammatory response is unknown. Here we investigate the effects of GSK-3beta inhibition on organ injury/dysfunction caused by endotoxaemia or severe inflammation in the rat. Rats received either intravenous Escherichia coli lipopolysaccharide (LPS) (6 mg/kg) or LPS (1mg/kg) plus Staphylococcus aureus peptidoglycan (PepG) (0.3mg/kg) or their vehicle (saline). The GSK-3p1 inhibitors TDZD-8, SB415286 (both 1mg/kg, i.v.), and SB216763 (0.6 mg/kg i.v.), or vehicle (10% dimethyl sulfoxide) were administered 30 min before LPS or LPS/PepG. Both endotoxaemia and co-administration of LPS/PepG resulted in multiple organ injury and dysfunction. The GSK-3beta inhibitors attenuated the organ injury/dysfunction caused by LPS or LPS/PepG. GSK-3beta inhibition reduced the Ser536 phosphorylation of nuclear factor (NF)-kappaB subunit p65 and the mRNA expression of NF-kappaB-dependent pro-inflammatory mediators, but had no effect on the NF-kappaB/DNA binding activity in the lung. GSK-3beta inhibition reduced the increase in NF-kappaB p65 activity caused by interleukin (IL)1 in human e mbryonic kidney cells in vitro. We propose that GSK-3beta inhibition may be useful in the therapy of sepsis, shock and other diseases associated with local or systemic inflammation. PMID:17380792

Dugo, Laura; Collin, Marika; Allen, David A; Patel, Nimesh S A; Bauer, Inge; Mervaala, Eero; Louhelainen, Marjut; Foster, Simon J; Yaqoob, Muhammad M; Thiemermann, Christoph

2007-01-01

321

Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c.  

Science.gov (United States)

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism. PMID:15466874

Kim, Kang Ho; Song, Min Jeong; Yoo, Eung Jae; Choe, Sung Sik; Park, Sang Dai; Kim, Jae Bum

2004-12-10

322

Tandem acyl carrier protein domains in polyunsaturated fatty acid synthases.  

Science.gov (United States)

Polyunsaturated fatty acids (PUFAs) can be biosynthesized via aerobic pathways that rely on combinations of desaturases and elongases to convert saturated fatty acids to PUFAs or anaerobic pathways that exploit polyketide synthase (PKS)-like enzymes known as PUFA synthases for de novo synthesis from acyl CoA precursors. In contrast to most fatty acid synthases (FASs) and PKSs that contain a single acyl carrier protein (ACP) domain for each cycle of fatty acid or polyketide chain elongation, all PUFA synthases known to date contain tandem ACPs (ranging from five to nine). The roles and engineering potential of such tandem ACPs in PUFA synthases remain largely unknown, although the growing demand for PUFAs and decline of current sources dictate that a greater understanding of these PUFA synthases is not only warranted, but urgently needed. This chapter describes methods and protocols developed to dissect the role and underlying biochemistry of each of the PfaA-ACPs in the Shewanella japonica PUFA synthase for eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) biosynthesis. These studies have set the stage to interrogate the roles of the other domains and subunits of the Pfa PUFA synthase in EPA and DPA biosynthesis. Applications of the methods and protocols described here to other PUFA synthases are therefore envisioned to help close the knowledge gap currently limiting microbial production of PUFAs via PUFA synthase engineering and heterologous expression. PMID:19362636

Jiang, Hui; Rajski, Scott R; Shen, Ben

2009-01-01

323

[Advances in sesquiterpene synthases cyclases of Artemisia annua].  

Science.gov (United States)

Artemisinin,a new and a very potent antimalarial drug, is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin, but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date, each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate, and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, beta-caryophyllene synthase, (E)-beta-farnesene synthase, germacrene A synthase, as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua. PMID:18257222

Shen, Hai-Yan; Li, Zhen-Qiu; Wang, Hong; Ma, Lan-Qing; Liu, Ben-Ye; Yan, Fang; Li, Guo-Feng; Ye, He-Chun

2007-11-01

324

Marked Increase in Nitric Oxide Synthase mRNA in Rat Dorsal Root Ganglia after Peripheral Axotomy: In situ Hybridization and Functional Studies  

Science.gov (United States)

Using in situ hybridization, we studied nitric oxide (NO) synthase (EC 1.14.23.-) mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. The effect of the NO synthase inhibitor N^?-nitro-L-arginine methyl ester on the nociceptive flexor reflex was also studied in axotomized rats. Nerve section induced a dramatic increase in number of NO synthase mRNA-positive cells in the ipsilateral dorsal root ganglia. In some of these cells the peptides galanin and/or vasoactive intestinal polypeptide and/or neuropeptide Y were also strongly up-regulated. Intravenous administration of nitro-L-arginine methyl ester blocked spinal hyperexcitability at much lower dosages in axotomized than in normal animals. The results suggest involvement of NO in the function of lumbar sensory neurons, especially after axotomy, perhaps preferentially at peripheral sites.

Verge, Valerie M. K.; Xu, Zhang; Xu, Xiao-Jun; Wiesenfeld-Hallin, Zsuzsanna; Hokfelt, Tomas

1992-12-01

325

Nitric oxide synthase in the pineal gland  

OpenAIRE

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

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

1996-01-01

326

Tertiary model of a plant cellulose synthase  

OpenAIRE

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

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

2013-01-01

327

Crystal Structure of the Human Prostacyclin Synthase  

OpenAIRE

Prostacyclin synthase (PGIS) catalyzes an isomerization of prostaglandin H2 to prostacyclin, a potent mediator of vasodilation and anti-platelet aggregation. Here, we report the crystal structure of human PGIS at 2.15 Å resolution, which represents the first three-dimensional structure of a class III cytochrome P450. While notable sequence divergence has been recognized between PGIS and other P450s, PGIS exhibits the typical triangular prism-shaped P450 fold with only moderate structural dif...

Chiang, Chia-wang; Yeh, Hui-chun; Wang, Lee-ho; Chan, Nei-li

2006-01-01

328

Deoxyhypusine synthase haploinsufficiency attenuates acute cytokine signaling  

OpenAIRE

Deoxyhypusine synthase (DHS) catalyzes the post-translational formation of the amino acid hypusine. Hypusine is unique to the eukaryotic translational initiation factor 5A (eIF5A), and is required for its functions in mRNA shuttling, translational elongation and stress granule formation. In recent studies, we showed that DHS promotes cytokine and ER stress signaling in the islet ? cell and thereby contributes to its dysfunction in the setting of diabetes mellitus. Here, we review the evidenc...

Templin, Andrew T.; Maier, Bernhard; Nishiki, Yurika; Tersey, Sarah A.; Mirmira, Raghavendra G.

2011-01-01

329

Nitric oxide synthase in cardiac sarcoplasmic reticulum  

OpenAIRE

NO? is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO? produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of l-arginine to l-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO? was generated from the vesicles and detec...

Xu, Kai Y.; Huso, David L.; Dawson, Ted M.; Bredt, David S.; Becker, Lewis C.

1999-01-01

330

Nitric Oxide Synthases in Heart Failure  

OpenAIRE

Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in unde...

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

2013-01-01

331

Gating NO Release from Nitric Oxide Synthase  

OpenAIRE

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

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

2011-01-01

332

Acute nitric oxide synthase inhibition and endothelin-1-dependent arterial pressure elevation  

Directory of Open Access Journals (Sweden)

Full Text Available Key evidence that endogenous nitric oxide (NO inhibits the continuous, endothelin (ET-1-mediated drive to elevate arterial pressure includes demonstrations that ET-1 mediates a significant component of the pressure elevated by acute exposure to NO synthase (NOS inhibitors. This review examines the characteristics of this pressure elevation in order to elucidate potential mechanisms associated with the negative regulation of ET-1 by NO and, thereby, provide potential insight into the vascular pathophysiology underlying NO dysregulation. We surmise that the magnitude of the ET-1-dependent component of the NOS inhibitor-elevated pressure is 1 independent of underlying arterial pressure and other pressor pathways activated by the NOS inhibitors and 2 dependent on relatively higher NOS inhibitor dose, release of stored and de novo synthesized ET-1, and ETA receptor-mediated increased vascular resistance. Major implications of these conclusions include: 1 the marked variation of the ET-1-dependent component, i.e., from 0-100% of the pressure elevation, reflects the NO-ET-1 regulatory pathway. Thus, NOS inhibitor-mediated, ET-1-dependent pressure elevation in vascular pathophysiologies is an indicator of the level of compromised/enhanced function of this pathway; 2 NO is a more potent inhibitor of ET-1-mediated elevated arterial pressure than other pressor pathways, due in part to inhibition of intravascular pressure-independent release of ET-1. Thus, the ET-1-dependent component of pressure elevation in vascular pathophysiologies associated with NO dysregulation is of greater magnitude at higher levels of compromised NO.

RobertRapoport

2014-04-01

333

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

Directory of Open Access Journals (Sweden)

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

Üçeyler Nurcan

2010-03-01

334

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

International Nuclear Information System (INIS)

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

335

Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same  

Science.gov (United States)

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

Croteau, Rodney Bruce (Pullman, WA); Burke, Charles Cullen (Moscow, ID)

2008-06-24

336

Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation  

OpenAIRE

Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein ...

Hooks, David O.; Mark Venning-Slater; Jinping Du; Rehm, Bernd H. A.

2014-01-01

337

Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer  

OpenAIRE

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

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

2010-01-01

338

Nitric oxide synthase in ferret brain: localization and characterization.  

OpenAIRE

1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it w...

Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Fo?rstermann, U.; Murad, F.

1992-01-01

339

Divinyl ether synthase gene and protein, and uses thereof  

Science.gov (United States)

The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

Howe, Gregg A. (East Lansing, MI); Itoh, Aya (Tsuruoka, JP)

2011-09-13

340

Fatty Acid Synthase Is a Key Target in Multiple Essential Tumor Functions of Prostate Cancer: Uptake of Radiolabeled Acetate as a Predictor of the Targeted Therapy Outcome  

OpenAIRE

Fatty acid synthase (FASN) expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In ...

Yoshii, Yukie; Furukawa, Takako; Oyama, Nobuyuki; Hasegawa, Yoko; Kiyono, Yasushi; Nishii, Ryuichi; Waki, Atsuo; Tsuji, Atsushi B.; Sogawa, Chizuru; Wakizaka, Hidekatsu; Fukumura, Toshimitsu; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Lewis, Jason S.; Saga, Tsuneo

2013-01-01

341

Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis.  

OpenAIRE

BACKGROUND: Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear. AIMS: We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advan...

Antoniades, C.; Shirodaria, C.; Leeson, P.; Antonopoulos, A.; Warrick, N.; Van-assche, T.; Cunnington, C.; Tousoulis, D.; Pillai, R.; Ratnatunga, C.; Stefanadis, C.; Channon, Km

2009-01-01

342

The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory  

OpenAIRE

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically as...

Pogoryelov, Denys; Yu, Jinshu; Meier, Thomas; Vonck, Janet; Dimroth, Peter; Muller, Daniel J.

2005-01-01

343

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

International Nuclear Information System (INIS)

1,2-anhydroglucitol-6P, a known inhibitor of glucose-6P isomerase, behaved as a fructose-6P site-directed irreversible inhibitor of bacterial glucosamine-6P synthase. The lack of reproducibility of the aldolase-mediated condensation of dihydroxyacetone phosphate and glycidaldehyde followed by borohydride reduction previously described prompted us to develop a chemical route to this compounds and its radiolabelled counterpart. The compound was synthesized in 13 steps from D-arabinose with a 6% overall yield. Tritium introduction was performed at step 11 (3 ? 4) allowing isolation of the title compound of high specific radioactivity. (author)

344

Isolation and functional characterization of a ?-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.  

Science.gov (United States)

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-?-caryophyllene (LaCARS) and a ?-cadinol synthase (LaCADS). ?-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum ?-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum ?-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of ?-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-?-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender. PMID:24078339

Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

2014-01-01

345

Putative role of nitric oxide synthase isoforms in the changes of nitric oxide concentration in rat brain cortex and cerebellum following sevoflurane and isoflurane anaesthesia.  

Science.gov (United States)

We have previously observed an increase in nitric oxide (NO) content in rat brain cortex following halothane, sevoflurane or isoflurane anaesthesia. This study was undertaken in order to determine whether isoform-specific nitric oxide synthase (NOS) inhibitors and inducers could modify these increases in NO contents. Rats were subjected to isoflurane and sevoflurane anaesthesia with concomitant administration of neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitro-indazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or lipopolysaccharide. NO concentration in different organs was measured by electron paramagnetic resonance (EPR) spectroscopy. 7-NI significantly decreased NO concentration in cerebellum but not in brain cortex, whereas AMT decreased NO in all the organs studied. Anaesthesia significantly increased NO concentration in brain cortex and decreased that in cerebellum. AMT abolished the NO increase in brain cortex. Anaesthesia enhanced the drastic increase in NO concentration in brain cortex after intraventricular lipopolysaccharide administration. Isoflurane was found to inhibit recombinant nNOS and iNOS activities at high concentrations (EC50=20 mM). Our data suggest a putative role for iNOS in the increase in NO levels produced by isoflurane and sevoflurane, whereas nNOS activity is probably inhibited during anaesthesia. PMID:15862801

Sjakste, Nikolajs; Sjakste, Jelizaveta; Boucher, Jean-Luc; Baumane, Larisa; Sjakste, Tatjana; Dzintare, Maija; Meirena, Dainuvite; Sharipova, Jelena; Kalvinsh, Ivars

2005-04-25

346

Inactivation of cystathionine ?-synthase with peroxynitrite  

OpenAIRE

Cystathionine ?-synthase (CBS) is a homocysteine metabolizing enzyme that contains pyridoxal phosphate (PLP) and a six-coordinate heme cofactor of unknown function. CBS was inactivated by peroxynitrite, the product of nitric oxide and superoxide radicals. The IC50 was ~150 ?M for 5 ?M ferric CBS. Stopped-flow kinetics and competition experiments showed a direct reaction with a second-order rate constant of (2.4–5.0) × 104 M?1 s?1 (pH 7.4, 37 °C). The radicals derived from peroxynit...

Celano, Laura; Gil, Magdalena; Carballal, Sebastia?n; Dura?n, Rosario; Denicola, Ana; Banerjee, Ruma; Alvarez, Beatriz

2009-01-01

347

Nitric oxide synthases: regulation and function  

OpenAIRE

Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH4). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and p...

Fo?rstermann, Ulrich; Sessa, William C.

2011-01-01

348

Research progress on neurobiology of neuronal nitric oxide synthase.  

Science.gov (United States)

Neuronal nitric oxide synthase (nNOS) is mainly expressed in neurons, to some extent in astrocytes and neuronal stem cells. The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS, including nNOS-?, nNOS-?, nNOS-µ, nNOS-? and nNOS-2. Monomer of nNOS is inactive, and dimer is the active form. Dimerization requires tetrahydrobiopterin (BH4), heme and L-arginine binding. Regulation of nNOS expression relies largely on cAMP response element-binding protein (CREB) activity, and nNOS activity is regulated by heat shock protein 90 (HSP90)/HSP70, calmodulin (CaM), phosphorylation and dephosphorylation at Ser847 and Ser1412, and the protein inhibitor of nNOS (PIN). There are primarily 9 nNOS-interacting proteins, including post-synaptic density protein 95 (PSD95), clathrin assembly lymphoid leukemia (CALM), calcium/calmodulin-dependent protein kinase II alpha (CAMKIIA), Disks large homolog 4 (DLG4), DLG2, 6-phosphofructokinase, muscle type (PFK-M), carboxy-terminal PDZ ligand of nNOS (CAPON) protein, syntrophin and dynein light chain (LC). Among them, PSD95, CAPON and PFK-M are important nNOS adapter proteins in neurons. The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death. nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states, and negatively regulates neurogenesis under physiological and pathological conditions. PMID:21270901

Luo, Chun-Xia; Zhu, Dong-Ya

2011-02-01

349

Energy landscapes and catalysis in nitric-oxide synthase.  

Science.gov (United States)

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape. PMID:24610812

Sobolewska-Stawiarz, Anna; Leferink, Nicole G H; Fisher, Karl; Heyes, Derren J; Hay, Sam; Rigby, Stephen E J; Scrutton, Nigel S

2014-04-25

350

Inducible nitric oxide synthase haplotype associated with migraine and aura.  

Science.gov (United States)

Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive NO amounts possibly contributing to migraine are derived from increased expression and activity of inducible NO synthase (iNOS). We tested the hypothesis that two functional, clinically relevant iNOS genetic polymorphisms (C(-1026)A-rs2779249 and G2087A-rs2297518) are associated with migraine with or without aura. We studied 142 healthy women without migraine (control group) and 200 women with migraine divided into two groups: 148 with migraine without aura (MWA) and 52 with aura (MA). Genotypes were determined by real-time polymerase chain reaction using the Taqman(®) allele discrimination assays. The PHASE 2.1 software was used to estimate the haplotypes. The A allele for the G2087A polymorphism was more commonly found in the MA group than in the MWA group (28 vs. 18%; P 0.05). The haplotype combining both A alleles for the two polymorphisms was more commonly found in the MA group than in the control group or in the MWA group (19 vs. 10 or 8%; P = 0.0245 or 0.0027, respectively). Our findings indicate that the G2087A and the C(-1026)A polymorphism in the iNOS gene affect the susceptibility to migraine with aura when their effects are combined within haplotypes, whereas the G2087A affects the susceptibility to aura in migraine patients. These finding may have therapeutic implications when examining the effects of selective iNOS inhibitors. PMID:22234503

de O S Mansur, Thiago; Gonçalves, Flavia M; Martins-Oliveira, Alisson; Speciali, Jose G; Dach, Fabiola; Lacchini, Riccardo; Tanus-Santos, Jose E

2012-05-01

351

p63 promotes cell survival through fatty acid synthase.  

Science.gov (United States)

There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects. PMID:19517019

Sabbisetti, Venkata; Di Napoli, Arianna; Seeley, Apryle; Amato, Angela M; O'Regan, Esther; Ghebremichael, Musie; Loda, Massimo; Signoretti, Sabina

2009-01-01

352

Kinetic and chemical mechanisms of homocitrate synthase from Thermus thermophilus.  

Science.gov (United States)

The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg(2+) or Mn(2+) capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with ?-ketoglutarate (?-Kg) binding prior to acetyl-CoA (AcCoA) with Mn(2+), whereas it is steady state random with Mg(2+), suggesting a difference in the competence of the E·Mn·?-Kg·AcCoA and E·Mg·?-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d(3)) in the presence of Mg(2+) is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg(2+) or Mn(2+) as the divalent metal ion, whereas V/K(?-Kg) (with Mn(2+)) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as ?-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS. PMID:21733842

Kumar, Vidya Prasanna; West, Ann H; Cook, Paul F

2011-08-19

353

The effect of nitric oxide synthase inhibition on the plasma fibrinolytic system in septic shock in rats.  

OpenAIRE

1. We have investigated the effect of pretreatment of rats with nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on the E. coli lipopolysaccharide (LPS)-induced changes in the plasma fibrinolytic system, platelet count, fibrinogen level, as well as in gross and microscopic pathophysiological changes indicative of disseminated intravascular coagulation (DIC) in rats. 2. E. coli LPS (6 mg kg-1, i.p.) produced a decrease in the levels of plasma fibrinogen and a dro...

Korbut, R.; Warner, T. D.; Gryglewski, R. J.; Vane, J. R.

1994-01-01

354

Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase.  

OpenAIRE

1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Br...

Constantin-teodosiu, D.; Greenhaff, P. L.; Gardiner, S. M.; Randall, M. D.; March, J. E.; Bennett, T.

1995-01-01

355

Inhibition of Nitric Oxide Synthase by L-NAME Promotes Cisplatin-Induced Nephrotoxicity in Male Rats  

OpenAIRE

Objective. Nitric oxide (NO) has numerous important functions in the kidney. The role of NO in cisplatin (CP)-induced nephrotoxicity is not completely understood. This study was designed to determine the role of NO synthase inhibitor (L-NAME) on the severity of CP-induced nephrotoxicity in rats. Methods. Sixty four male (M) and female (F) Wistar rats were randomly divided into eight groups. The sham groups (group 1, male, n = 6 and group 2, female, n = 6) received saline. Groups 3 (male, n = ...

Fatemeh Moslemi; Mehdi Nematbakhsh; Fatemeh Eshraghi-Jazi; Ardeshir Talebi; Hamid Nasri; Farzaneh Ashrafi; Maryam Moeini; Azam Mansouri; Zahra Pezeshki

2013-01-01

356

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

OpenAIRE

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

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

2014-01-01

357

Reduction in Embryonic Malformations and Alleviation of Endoplasmic Reticulum Stress by Nitric Oxide Synthase Inhibition in Diabetic Embryopathy  

OpenAIRE

Maternal diabetes-induced neural tube defects (NTDs) are associated with increased programmed cell death (apoptosis) in the neuroepithelium, which is related to intracellular nitrosative stress. To alleviate nitrosative stress, diabetic pregnant mice were fed via gavage an inhibitor of nitric oxide (NO) synthase (NOS) 2, L-N6-(1-iminoethyl)-lysine (L-NIL; 80 mg/kg), once a day from embryonic (E) day 7.5 to 9.5 during early stages of neurulation. The treatment significantly reduced NTD rate in...

Zhao, Zhiyong; Eckert, Richard L.; Reece, E. Albert

2012-01-01

358

STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE  

Directory of Open Access Journals (Sweden)

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

T. Femlin Blessia

2012-02-01

359

Inhibition of Glycogen Synthase Kinase 3 (GSK3) Increases the Cytotoxicity of Enzastaurin  

Science.gov (United States)

Cutaneous T cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our lab has previously demonstrated that the Protein Kinase C (PKC) ? inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL where it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complimentary signaling pathways and identified Glycogen Synthase Kinase 3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated anenhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in up-regulation of ? catenin total protein and ? catenin-mediated transcription. Inhibition of ? catenin-mediated transcription or shRNA knockdown of ? catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of ? catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome. PMID:21471986

Rovedo, Mark; Krett, Nancy L; Rosen, Steven T

2011-01-01

360

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

Directory of Open Access Journals (Sweden)

Full Text Available Recent advances in understanding the pathophysiological mechanisms contributing to Fragile X Syndrome (FXS have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP. FMRP may have several functions, but it is most well-established as an RNA-binding protein that regulates translation, and it is by this mechanism that FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3 may contribute to the therapeutic treatment of FXS is reviewed here. In the mouse model of FXS, which lacks FMRP expression (FX mice, GSK3 is hyperactive in several brain regions. Furthermore, significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in FXS patients also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS.

RichardScottJope

2011-11-01

361

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase  

Science.gov (United States)

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma.

Asuncion Valenzuela, Malyn M; Castro, Imilce; Gonda, Amber; Diaz Osterman, Carlos J; Jutzy, Jessica M; Aspe, Jonathan R; Khan, Salma; Neidigh, Jonathan W; Wall, Nathan R

2015-01-01

362

Torque generation mechanism of ATP synthase  

Science.gov (United States)

ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

2010-03-01

363

Deoxyhypusine synthase haploinsufficiency attenuates acute cytokine signaling.  

Science.gov (United States)

Deoxyhypusine synthase (DHS) catalyzes the post-translational formation of the amino acid hypusine. Hypusine is unique to the eukaryotic translational initiation factor 5A (eIF5A), and is required for its functions in mRNA shuttling, translational elongation, and stress granule formation. In recent studies, we showed that DHS promotes cytokine and ER stress signaling in the islet ? cell and thereby contributes to its dysfunction in the setting of diabetes mellitus. Here, we review the evidence supporting a role for DHS (and hypusinated eIF5A) in cellular stress responses, and provide new data on the phenotype of DHS knockout mice. We show that homozygous knockout mice are embryonic lethal, but heterozygous knockout mice appear normal with no evidence of growth or metabolic deficiencies. Mouse embryonic fibroblasts from heterozygous knockout mice attenuate acute cytokine signaling, as evidenced by reduced production of inducible nitric oxide synthase, but show no statistically significant defects in proliferation or cell cycle progression. Our data are discussed with respect to the utility of sub-maximal inhibition of DHS in the setting of inflammatory states, such as diabetes mellitus. PMID:21389784

Templin, Andrew T; Maier, Bernhard; Nishiki, Yurika; Tersey, Sarah A; Mirmira, Raghavendra G

2011-04-01

364

Uncovering the structures of modular polyketide synthases.  

Science.gov (United States)

Covering: up to 2014The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions. PMID:25310997

Weissman, Kira J

2015-02-26

365

Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity  

DEFF Research Database (Denmark)

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers.

Hempel, Casper; Kohnke, Hannes

2014-01-01

366

Changes in pulmonary vascular tone during exercise. Effects of nitric oxide (NO) synthase inhibition, L-arginine infusion, and NO inhalation.  

Science.gov (United States)

Nitric oxide (NO) is a potent endogenous vasodilator. Its role in the normal and stressed pulmonary circulation is unclear. To better understand the importance of endogenous NO in normal physiological responses, we studied the effects of altered NO availability on the change in pulmonary vascular tone that accompanies exercise. In paired studies we measured blood flow and pressures in the pulmonary circulation at rest and during treadmill exercise at a speed of 4 mph with and without (a) N omega-nitro-L-arginine, 20 mg/kg intravenously, a selective inhibitor of NO synthase; (b) L-arginine, 200 mg/kg intravenously, substrate for NO synthase; (c) combination of the inhibitor and substrate; and (d) inhalation of NO > 30 ppm, to determine if endogenous release of NO elicits maximal vasodilation. In addition, we sought to determine the site of NO effect in the pulmonary circulation by preconstriction with either U-44619 or hypoxia (fraction of inspired O2 = 0.12) using a distal wedged pulmonary catheter technique. NO synthase inhibition raised pulmonary vascular tone equally at rest and exercise. L-Arginine reversed the effects of NO synthase inhibition but had no independent effect. NO inhalation did not reduce pulmonary vascular tone at rest or enhance the usual reduction in pulmonary vascular resistance with exercise. The effect of NO synthase inhibition was in pulmonary vessels upstream from small veins, suggesting that endogenous NO dilates primarily small arteries and veins at rest. We conclude that, in sheep, endogenous NO has a basal vasodilator function that persists during, but is not enhanced by, exercise. PMID:7527429

Koizumi, T; Gupta, R; Banerjee, M; Newman, J H

1994-12-01

367

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

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Full Text Available Backgrounds: The blocking of nitric oxide synthase (NOS activity may reason vasoconstriction with formation of reactive oxygen species. Propolis has biological and pharmacological properties, such as antioxidant. The aim of this study was to examine the antioxidant effects of propolis which natural product on biochemical parameters in brain and lung tissues of acute nitric oxide synthase inhibited rats by N?-nitro-L-arginine methyl ester (L-NAME. Methods: Rats have been received L-NAME (40 mg/kg, intraperitoneally, NOS inhibitor for 15 days to produce hypertension and propolis (200mg/kg, by gavage the lastest 5 of 15 days. Results: There were the increase (PPP Conclusion: The application of L-NAME to the Wistar rats resulted in well developed oxidative stress. Also, propolis may influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of hypertensive diseases and oxidative stress. Keywords: Catalase, L-NAME, Malondialdehyde, Oxidative stress, Propolis, Rat

Oguz Cakir

2013-06-01

368

Quantitation of glycogen synthase kinase-3 sensitive proteins in neuronal membrane rafts.  

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We report a quantitative proteomic study to investigate the changes induced in membrane rafts by the inhibition of glycogen synthase kinase-3. Sensitive quantitation of membrane raft proteins using isobaric tagging chemistries was enabled by a novel hybrid proteomic method to isolate low-microgram (10-30 microg) membrane raft protein preparations as unresolved bands in a low-density acrylamide gel. Samples were in-gel digested, differentially tagged and combined for 2-D LC and quantitative MS. Analysis of hippocampal membrane preparations using this approach resulted in a sixfold increase in sensitivity and a threefold increase in the number of quantifiable proteins compared with parallel processing using a traditional in-solution method. Quantitative analysis of membrane raft preparations from a human neuronal cell line treated with glycogen synthase kinase-3 inhibitors SB415286 or lithium chloride, that have been reported to modulate processing of the Alzheimer amyloid precursor protein, identified several protein changes. These included decreases in lamin B1 and lamin B receptor, as well as increases in several endosome regulating rab proteins, rab5, rab7 and rab11 that have been implicated in processing of the amyloid precursor protein in Alzheimer's disease. PMID:19526546

Thompson, Andrew J; Williamson, Ritchie; Schofield, Emma; Stephenson, John; Hanger, Diane; Anderton, Brian

2009-06-01

369

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation.  

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Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases. PMID:16956297

Sanchez, Verónica Muriel; Crespo, Alejandro; Gutkind, J Silvio; Turjanski, Adrián Gustavo

2006-09-14

370

Leukaemia inhibitory factor stimulates proliferation of olfactory neuronal progenitors via inducible nitric oxide synthase.  

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Neurogenesis continues in the adult brain and in the adult olfactory epithelium. The cytokine, leukaemia inhibitory factor and nitric oxide are both known to stimulate neuronal progenitor cell proliferation in the olfactory epithelium after injury. Our aim here was to determine whether these observations are independent, specifically, whether leukaemia inhibitory factor triggers neural precursor proliferation via the inducible nitric oxide synthase pathway. We evaluated the effects of leukaemia inhibitory factor on inducible form of nitric oxide synthase (iNOS) expression, and cell proliferation in olfactory epithelial cell cultures and olfactory neurosphere-derived cells. Leukaemia inhibitory factor induced expression of iNOS and increased cell proliferation. An iNOS inhibitor and an anti-leukaemia inhibitory factor receptor blocking antibody inhibited leukaemia inhibitory factor-induced cell proliferation, an effect that was reversed by a NO donor. Altogether, the results strongly suggest that leukaemia inhibitory factor induces iNOS expression, increasing nitric oxide levels, to stimulate proliferation of olfactory neural precursor cells. This finding sheds light on neuronal regeneration occurring after injury of the olfactory epithelium. PMID:23024784

Lopez-Arenas, Estefania; Mackay-Sim, Alan; Bacigalupo, Juan; Sulz, Lorena

2012-01-01

371

Cerulenin blockade of fatty acid synthase reverses hepatic steatosis in ob/ob mice.  

Science.gov (United States)

Fatty liver or hepatic steatosis is a common health problem associated with abnormal liver function and increased susceptibility to ischemia/reperfusion injury. The objective of this study was to investigate the effect of the fatty acid synthase inhibitor cerulenin on hepatic function in steatotic ob/ob mice. Different dosages of cerulenin were administered intraperitoneally to ob/ob mice for 2 to 7 days. Body weight, serum AST/ALT, hepatic energy state, and gene expression patterns in ob/ob mice were examined. We found that cerulenin treatment markedly improved hepatic function in ob/ob mice. Serum AST/ALT levels were significantly decreased and hepatic ATP levels increased in treated obese mice compared to obese controls, accompanied by fat depletion in the hepatocyte. Expression of peroxisome proliferator-activated receptors ? and ? and uncoupling protein 2 were suppressed with cerulenin treatment and paralleled changes in AST/ALT levels. Hepatic glutathione content were increased in some cases and apoptotic activity in the steatotic livers was minimally changed with cerulenin treatment. In conclusion, these results demonstrate that fatty acid synthase blockade constitutes a novel therapeutic strategy for altering hepatic steatosis at non-stressed states in obese livers. PMID:24086674

Cheng, Gang; Palanisamy, Arun P; Evans, Zachary P; Sutter, Alton G; Jin, Lan; Singh, Inderjit; May, Harold; Schmidt, Michael G; Chavin, Kenneth D

2013-01-01

372

Fgk3 glycogen synthase kinase is important for development, pathogenesis, and stress responses in Fusarium graminearum.  

Science.gov (United States)

Wheat scab caused by Fusarium graminearum is an important disease. In a previous study, the FGK3 glycogen synthase kinase gene orthologous to mammalian GSK3 was identified as an important virulence factor. Although GSK3 orthologs are well-conserved, none of them have been functionally characterized in fungal pathogens. In this study, we further characterized the roles of FGK3 gene. The ?fgk3 mutant had pleiotropic defects in growth rate, conidium morphology, germination, and perithecium formation. It was non-pathogenic in infection assays and blocked in DON production. Glycogen accumulation was increased in the ?fgk3 mutant, confirming the inhibitory role of Fgk3 on glycogen synthase. In FGK3-GFP transformants, GFP signals mainly localized to the cytoplasm in conidia but to the cytoplasm and nucleus in hyphae. Moreover, the expression level of FGK3 increased in response to cold, H2O2, and SDS stresses. In the ?fgk3 mutant, cold, heat, and salt stresses failed to induce the expression of the stress response-related genes FgGRE2, FgGPD1, FgCTT1, and FgMSN2. In the presence of 80?mM LiCl, a GSK3 kinase inhibitor, the wild type displayed similar defects to the ?fgk3 mutant. Overall, our results indicate that FGK3 is important for growth, conidiogenesis, DON production, pathogenicity, and stress responses in F. graminearum. PMID:25703795

Qin, Jun; Wang, Guanghui; Jiang, Cong; Xu, Jin-Rong; Wang, Chenfang

2015-01-01

373

Inducible nitric oxide synthase inhibits oxygen consumption in collateral-dependent myocardium.  

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Following coronary artery occlusion growth of collateral vessels can provide an effective blood supply to the dependent myocardium. The ischemia, which results in growth of collateral vessels, recruits an inflammatory response with expression of cytokines and growth factors, upregulation of endothelial nitric oxide (NO) synthase (eNOS) in vascular endothelial cells, and expression of inducible nitric oxide synthase (iNOS) in both vessels and cardiac myocytes. Because NO is a potent collateral vessel dilator, this study examined whether NO derived from iNOS or constitutive NOS regulates myocardial blood flow (MBF) in the collateral region. Nonselective NOS inhibition with N(G)-nitro-l-arginine (LNA) caused vasoconstriction with a significant decrease in MBF to the collateral region during exercise. In contrast, the highly selective iNOS inhibitor 1400W caused a 21 ± 5% increase of MBF in the collateral region. This increase in MBF following selective iNOS blockade was proportionate to an increase in myocardial O2 consumption (MVo2). The results suggest that NO produced by iNOS inhibits MVo2 in the collateralized region, so that the increase in MBF following iNOS blockade was the result of metabolic vasodilation secondary to an increase in MVo2. Thus the coordinated expression of iNOS to restrain MVo2 and eNOS to maintain collateral vasodilation act to optimize the O2 supply-demand relationship and protect the collateralized myocardium from ischemia. PMID:24322607

Chen, Yingjie; Zhang, Ping; Li, Jingxin; Xu, Xin; Bache, Robert J

2014-02-01

374

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia.  

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Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K(ATP) channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia. PMID:11744335

Lacza, Z; Puskar, M; Figueroa, J P; Zhang, J; Rajapakse, N; Busija, D W

2001-12-15

375

Crystallization and preliminary X-ray diffraction study of the farnesyl diphosphate synthase from Trypanosoma brucei.  

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Farnesyl diphosphate synthase (FPPS) catalyses the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate and is an RNAi-validated drug target in Trypanosoma brucei, the causative agent of African sleeping sickness. A T. brucei FPPS (390 amino acids) has been expressed in Escherichia coli and the recombinant protein has been crystallized in the absence and presence of the bisphosphonate inhibitor minodronate. Diffraction data were collected at 100 K using synchrotron radiation from both crystal types. Crystals obtained in the absence of minodronate belong to space group I222, with unit-cell parameters a = 61.43, b = 118.12, c = 120.04 A, while crystals grown in the presence of minodronate belong to space group C2, with unit-cell parameters a = 131.98, b = 118.10, c = 63.25 A, beta = 112.48 degrees. An initial model of the drug-free protein has been built using a homology model with the molecular-replacement method and refined to 3.3 A resolution. It shows mostly helical structure and resembles the structure of avian farnesyl diphosphate synthase, but with the addition of two loop regions. PMID:15388934

Mao, Junhong; Gao, Yi-Gui; Odeh, Sarah; Robinson, Howard; Montalvetti, Andrea; Docampo, Roberto; Oldfield, Eric

2004-10-01

376

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

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As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

2011-09-28

377

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

Science.gov (United States)

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

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

2002-04-01

378

Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target  

DEFF Research Database (Denmark)

To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.

Yang, Zhiping; Huang, Yuh-Chin T

2014-01-01

379

Helicobacter pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3?.  

Science.gov (United States)

Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Helicobacter pylori (H. pylori) infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking. Here we explored the effect of H. pylori infection on tau phosphorylation. The results showed that H. pylori filtrate induced significant tau hyperphosphorylation at several AD-related tau phosphorylation sites, such as Thr205, Thr231, and Ser404, both in mouse neuroblastoma N2a cells and rat brains with activation of glycogen synthase kinase-3? (GSK-3?). Application of GSK-3 inhibitors efficiently attenuated the H. pylori-induced tau hyperphosphorylation. Our data provide evidence supporting the role of H. pylori infection in AD-like tau pathology, suggesting that H. pylori eradication may be beneficial in the prevention of tauopathy. PMID:25079798

Wang, Xiu-Lian; Zeng, Ji; Yang, Yang; Xiong, Yan; Zhang, Zhi-Hua; Qiu, Mei; Yan, Xiong; Sun, Xu-Ying; Tuo, Qing-Zhang; Liu, Rong; Wang, Jian-Zhi

2015-01-01

380

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A  

International Nuclear Information System (INIS)

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

381

Instability of N-acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices.  

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Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides. It inhibits ceramide synthase, which is a proposed underlying mechanism responsible for the myriad of toxic endpoints observed. We previously reported that N-acetylation of FB1 prevents ceramide synthase inhibition, but cautioned that impure preparations of FA1 can contain a contaminant with the ability to inhibit ceramide synthase. We now report that FA1 spontaneously rearranges to O-acetylated analogs. These rearrangement products are putative inhibitors of ceramide synthase. Rat liver slices exposed to impure FA1 containing O-acetylated FB1 had sphinganine/sphingosine (Sa:So) ratios of 1.15-1.64. Control slices had Sa:So ratios of 0.07-0.24. Clean-up to remove the O-acetylated FB1 yielded purified FA1, which produced Sa:So ratios in liver slices of 0.08-0.18. After storage for approximately 1 year as either a dry powder in a desiccator, or as a dried film at 4 degrees C, the purified FA1 again contained O-acetylated FB1, and was capable of ceramide synthase inhibition. FA1 was most stable in neutral solution, but in acidic solution the equilibrium shifted towards the O-acetylated forms. FA1 in solid form also rearranged, but more slowly than in acid solution. As FA1 is considerably less cytotoxic than FB1, these results provide additional support for the conclusion that a primary amino group is necessary for both ceramide synthase inhibition and toxicity. PMID:11527566

Norred, W P; Riley, R T; Meredith, F I; Poling, S M; Plattner, R D

2001-11-01

382

Feature selection and linear/nonlinear regression methods for the accurate prediction of glycogen synthase kinase-3beta inhibitory activities.  

Science.gov (United States)

Few variables were selected from a pool of calculated Dragon descriptors through three different feature selection methods, namely genetic algorithm (GA), successive projections algorithm (SPA), and fuzzy rough set ant colony optimization (fuzzy rough set ACO). Each set of selected descriptors was regressed against the bioactivities of a series of glycogen synthase kinase-3beta (GSK-3beta) inhibitors, through linear and nonlinear regression methods, namely multiple linear regression (MLR), artificial neural network (ANN), and support vector machines (SVM). The fuzzy rough set ACO/SVM-based model gave the best estimation/prediction results, demonstrating the nonlinear nature of this analysis and suggesting fuzzy rough set ACO, first introduced in chemistry here, as an improved variable selection method in QSAR for the class of GSK-3beta inhibitors. PMID:19338295

Goodarzi, Mohammad; Freitas, Matheus P; Jensen, Richard

2009-04-01

383

Characterisation of the tryptophan synthase alpha subunit in maize  

Directory of Open Access Journals (Sweden)

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

Gierl Alfons

2008-04-01

384

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

OpenAIRE

Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative s