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1

Use of acetolactate synthase inhibitors on cultivated plants  

UK PubMed Central (United Kingdom)

The present invention relates to a method for increasing the plant health in a cultivated plant comprising the application of an with an acetolactate synthase inhibitor to the cultivated plant, parts of such plants, plant propagation materials, or at the locus of growth of the cultivated plant.

GEWEHR MARKUS; GLADWIN ROBERT JOHN; LOGEMANN JUERGEN; PUENTE PILAR; STUIVER MAARTEN HENDRIK; VOESTE DIRK

2

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

UK PubMed Central (United Kingdom)

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

Richie DL; Thompson KV; Studer C; Prindle VC; Aust T; Riedl R; Estoppey D; Tao J; Sexton JA; Zabawa T; Drumm J; Cotesta S; Eichenberger J; Schuierer S; Hartmann N; Movva NR; Tallarico JA; Ryder NS; Hoepfner D

2013-05-01

3

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

Science.gov (United States)

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

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

2013-03-11

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Resistance to acetolactate synthase inhibitors and quinclorac in a biotype of false cleavers (Galium spurium).  

UK PubMed Central (United Kingdom)

A false cleavers population that survived treatment with triasulfuron/bromoxynil in 1996 was identified in central Alberta, Canada, in a field that had been treated with acetolactate synthase (ALS) inhibitors in 3 of the previous 6 yr. In greenhouse studies, this biotype was highly resistant to the ALS inhibitors triasulfuron, thifensulfuron/tribenuron, and sulfometuron and moderately resistant to imazethapyr; GR50 values were > 16, > 5, > 1.0, and 9.9, respectively. In addition, cross-resistance was identified to the auxin-type herbicide quinclorac (GR50 value > 6.7) but not to fluroxypyr (GR50 value 1) or MCPA/mecoprop/dicamba. Quinclorac had not been used previously in this field. Analysis of ALS extracted from the resistant biotype and a susceptible biotype from a nearby location indicated that resistance to ALS inhibitors was due to an altered target site with reduced sensitivity to a broad range of ALS inhibitors. The ALS I50 values for triasulfuron, metsulfuron, chlorsulfuron, thifensulfuron, and imazethapyr were 36, 34, 92, 96, and 14 times higher, respectively, for the resistant compared to the susceptible biotype. The mechanism of resistance to quinclorac is unknown. This is the first report of high-level herbicide resistance in this weed species.

Hall LM; Stromme KM; Horsman GP; Devine MD

1998-07-01

5

Resistance to Acetolactate Synthase-Inhibiting Herbicides  

UK PubMed Central (United Kingdom)

Nucleotide sequences are disclosed that may be used to impart herbicide resistance to green plants. The sources of novel herbicide resistance were originally isolated in mutant Coreopsis plants. Green plants transformed with these sequences are resistant to herbicides that normally inhibit acetolactate synthase (ALS), particularly imidazolinone and sulfonylurea herbicides.

OARD JAMES H; ZHANG NENGYI; SANDERS DEARL E

6

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 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), enzima que catalisa a reação seguinte do acetolactato na cadeia de biossíntese dos aminoácidos valina, leucina e isoleucina, provocando assim, o acúmulo de acetolacta (more) to, 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 catalyzes 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 pyruv (more) ate 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.

Monqueiro, Patrícia Andrea; Christoffoleti, Pedro Jacob

2001-03-01

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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  

Directory of Open Access Journals (Sweden)

Full Text Available Foi avaliada a atividade da acetolactato sintase (ALS), em plantas resistentes e suscetíveis de B. pilosa e A. quitensis após a aplicação de herbicidas inibidores da ALS. O método baseia-se na utilização do ácido ciclopropanodicarboxílico (CPCA) para inibir a cetoácido reductoisomerase (KARI), enzima 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.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 catalyzes 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

2001-01-01

8

The oxygenase reaction of acetolactate synthase detected by chemiluminescence.  

Science.gov (United States)

In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen-consuming side reaction. Partially purified acetolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn2+ and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalse or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthesis. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species. PMID:7957905

Durner, J; Gailus, V; Böger, P

1994-10-31

9

The oxygenase reaction of acetolactate synthase detected by chemiluminescence.  

UK PubMed Central (United Kingdom)

In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen-consuming side reaction. Partially purified acetolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn2+ and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalse or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthesis. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species.

Durner J; Gailus V; Böger P

1994-10-01

10

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

UK PubMed Central (United Kingdom)

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

Duhoux A; Délye C

2013-01-01

11

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; Aldo Merotto Jr.

1999-01-01

12

Selection of transgenic rice plants using a herbicide tolerant form of the acetolactate synthase gene.  

UK PubMed Central (United Kingdom)

Acetolactate synthase (ALS) is an enzyme in the biosynthetic pathway for branched-chain amino acids, and bispyribac-sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of ALS activity. However, it appears that a mutated form of rice ALS [OsmALS (W548L/S627I)] confers resistance to BS. We succeeded in using OsmALS with native OsALS promoter and terminator region for a selection marker of rice transformation. Because this selection marker cassette is originally from the rice endogenous genome, it can be expected to be publicly acceptable.

Endo M; Shimizu T; Toki S

2012-01-01

13

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase.  

Science.gov (United States)

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP. PMID:11118309

Chong, C K; Choi, J D

2000-12-20

14

Roles of conserved methionine residues in tobacco acetolactate synthase.  

Science.gov (United States)

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site. PMID:12821153

Tien Le, Dung; Yoon, Moon-Young; Kim, Young Tae; Choi, Jung-Do

2003-07-11

15

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; C.E.C. Prete; M. Sumiya

2003-01-01

16

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 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 daninhas através de herbicidas, com diferentes mecanismos de ação, sobre plantas de Bidens subalternans, foi conduzido o presente trabalho, que envolveu um experimento (more) 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 herbicid (more) es, 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.

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

2003-08-01

17

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 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 províncias de Córdoba e Tucumã (Argentina), selecionaram biótipos resistentes (R) destas plantas daninhas. Esta pesquisa foi desenvolvida para estudar o manejo, crescimento, (more) 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, 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 biotype (more) s. 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.

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

2003-01-01

18

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; Pedro Jacob Christoffoleti; Helaine Carrer

2003-01-01

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A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase.  

Science.gov (United States)

Acetolactate synthase (ALS) inhibitors are among the most commonly used herbicides. They fall into four distinct families of compounds: sulfonylureas, imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We have investigated the molecular basis of imidazolinone tolerance of two field isolates of cocklebur (Xanthium sp.) from Mississippi and Missouri. In both cases, tolerance was conferred by a form of ALS that was less sensitive to inhibitors than the wild type. The insensitivity pattern of the Mississippi isolate was similar to that of a commercial mutant of corn generated in the laboratory: ICI 8532 IT. Sequencing revealed that the same residue (Ala57-->Thr) was mutated in both Mississippi cocklebur and ICI 8532 IT corn. ALS from the Missouri isolate was highly insensitive to all the ALS herbicide families, similar in this respect to another commercial corn mutant: Pioneer 3180 IR corn. Sequencing of ALS from both plants revealed a common mutation that changed Trp552 to Leu. The sensitive cocklebur ALS cDNA, fused with a glutathione S-transferase, was functionally expressed in Escherichia coli. The recombinant protein had enzymatic properties similar to those of the plant enzyme. All the possible point mutations affecting Trp552 were investigated by site-directed mutagenesis. Only the Trp-->Leu mutation yielded an active enzyme. This mutation conferred a dramatically reduced sensitivity toward representatives of all four chemical families, demonstrating its role in herbicide tolerance. This study indicates that mutations conferring herbicide tolerance, obtained in an artificial environment, also occur in nature, where the selection pressure is much lower. Thus, this study validates the use of laboratory models to predict mutations that may develop in natural populations. PMID:7615543

Bernasconi, P; Woodworth, A R; Rosen, B A; Subramanian, M V; Siehl, D L

1995-07-21

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Weed control with selected herbicides in acetolactate synthase-resistant sorghum  

UK PubMed Central (United Kingdom)

The recent development of grain sorghum hybrids with resistance to acetolactate synthase (ALS)-inhibiting herbicides has allowed for the use of several post-emergence applied (POST) ALS-inhibitors to control weeds in the crop. Field experiments were conducted at four sites in Kansas in 2008 to evaluate the efficacy of nicosulfuron and nicosulfuron+rimsulfuron applied alone or in combination with dicamba, metsulfuron methyl, and atrazine. All POST treatments slightly injured sorghum 2 weeks after treatment (WAT) at Garden City and Hesston, whereas at Hays and Manhattan, only treatments that included dicamba caused injury. Nicosulfuron+rimsulfuron applied alone provided 41, 83, 74, and 93% control of grasses 4 WAT at Garden City, Hays, Hesston, and Manhattan, respectively. However, to obtain the highest level broadleaf weed control, nicosulfuron or nicosulfuron+rimsulfuron need to be applied with other broadleaf herbicides. POST treatment of nicosulfuron+metsulfuron methyl+dicamba+atrazine provided 90% or greater control of all broadleaf weeds at sorghum flowering. Sorghum grain yield was greater following all herbicide treatments compared with the weedy check. The POST treatment that provided the highest yield at Garden City was nicosulfuron+rimsulfuron+atrazine, whereas in Hesston and Manhattan, nicosulfuron+metsulfuron methyl+dicamba+atrazine provided the highest yields. This research showed that many grasses can be effectively controlled with POST applications of nicosulfuron or nicosulfuron+rimsulfuron in ALS-resistant sorghum. The research also indicated that broadleaf weed control is greater when nicosulfuron or nicosulfuron+rimsulfuron are applied with other broadleaf-control herbicides such as dicamba, metsulfuron methyl, and atrazine.

Hennigh DShane; Al-Khatib Kassim; Currie RandallS; Tuinstra MitchellR; Geier PatrickW; Stahlman PhillipW; Claassen MarkM

2010-08-01

 
 
 
 
21

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker.  

UK PubMed Central (United Kingdom)

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.

Ogawa T; Kawahigashi H; Toki S; Handa H

2008-08-01

22

Differential germination pattern of rice cultivars resistant to imidazolinone herbicides carrying different acetolactate synthase gene mutations  

UK PubMed Central (United Kingdom)

Goulart ICGR, Matzenbacher FO & Merotto A JR (2012). Differential germination pattern of rice cultivars resistant to imidazolinone herbicides carrying different acetolactate synthase gene mutations. Weed Research52, 224–232. SUMMARY: Different mutations in the acetolactate synthase (ALS) gene can affect the germination pattern and facilitate the occurrence of ALS?inhibiting herbicide resistance in weedy red rice. The aim of this study was to evaluate the germination rates in imidazolinone?resistant rice cultivars carrying three different ALS gene mutations. Plant material consisted of the imidazolinone?resistant rice cultivars IRGA 422 CL, PUITÁ INTA CL and SATOR CL that carry the ALS mutations Gly654Glu, Ala122Thr and Ser653Asn, respectively, and the susceptible cultivar IRGA 417. Initially, the germination pattern of these cultivars was evaluated at temperatures of 15, 20, 25 and 30°C. Afterwards, five lots of each cultivar were evaluated at temperatures of 20 and 25°C to separate the environmental and genotypic effects. The environmental effect related to the different seed origin did not affect the germination rates of the rice cultivars. However, the germination pattern of the evaluated cultivars was different, mainly at 20°C. The time to reach 50% germination at 20°C for the imidazolinone?resistant rice cultivars IRGA 422 CL, SATOR CL and PUITÁ INTA CL was 6, 16 and 24?h earlier, respectively, than the susceptible cultivar IRGA 417. The PUITÁ INTA CL cultivar, which carries the mutation Ala122Thr, showed faster germination than the other herbicide–resistant and the susceptible cultivars at 20°C. The faster germination that resulted from the resistance to ALS?inhibiting herbicides could have different consequences for the establishment and competition of weedy red rice.

GOULART ICGR; MATZENBACHER FO; MEROTTO AJ

2012-06-01

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Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.  

Science.gov (United States)

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8). PMID:10329466

Namgoong, S K; Lee, H J; Kim, Y S; Shin, J H; Che, J K; Jang, D Y; Kim, G S; Yoo, J W; Kang, M K; Kil, M W; Choi, J D; Chang, S I

1999-05-19

24

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

Science.gov (United States)

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

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

2012-10-18

25

Roles of lysine 219 and 255 residues in tobacco acetolactate synthase.  

Science.gov (United States)

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. The ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The roles of three well-conserved lysine residues (K219, K255, K299) in tobacco ALS were determined using site-directed mutagenesis. The mutation of K219Q inactivated the enzyme and abolished the binding affinity for cofactor FAD. However, the secondary structure of the enzyme was not changed significantly by the mutation. Both mutants, K255F and K255Q, showed strong resistance to three classes of herbicides Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In addition, there was no difference in the secondary structures of wALS and K255F. On the other hand, the mutation of K299Q did not show any significant effect on the kinetic properties or any sensitivity to the herbicides. These results suggest that Lys219 is located at the active site and is likely involved in the binding of FAD, and that Lys255 is located at a binding site common for the three herbicides in tobacco ALS. PMID:12054619

Yoon, Tae-Yeol; Chung, Sun-Mee; Chang, Soo-Ik; Yoon, Moon-Young; Hahn, Tae-Ryong; Choi, Jung-Do

2002-04-26

26

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker.  

Science.gov (United States)

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin. PMID:18449542

Ogawa, Taiichi; Kawahigashi, Hiroyuki; Toki, Seiichi; Handa, Hirokazu

2008-05-01

27

A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice.  

UK PubMed Central (United Kingdom)

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.

Okuzaki A; Shimizu T; Kaku K; Kawai K; Toriyama K

2007-05-01

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

Directory of Open Access Journals (Sweden)

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

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

2002-01-01

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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  

Directory of Open Access Journals (Sweden)

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; R. Victória Filho; M.C.S.S. Novo; M.L. Adoryan

2002-01-01

30

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

UK PubMed Central (United Kingdom)

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.

van der Vyver C; Conradie T; Kossmann J; Lloyd J

2013-04-01

31

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

Science.gov (United States)

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

Mourad, G; Williams, D; King, J

1995-01-01

32

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

Science.gov (United States)

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

Délye, Christophe; Pernin, Fanny; Scarabel, Laura

2010-10-16

33

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

UK PubMed Central (United Kingdom)

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

Délye C; Pernin F; Scarabel L

2011-02-01

34

Isolation and expression of genes for acetolactate synthase and acetyl-CoA carboxylase in Echinochloa phyllopogon, a polyploid weed species.  

UK PubMed Central (United Kingdom)

BACKGROUND: Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS: Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION: E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Iwakami S; Uchino A; Watanabe H; Yamasue Y; Inamura T

2012-07-01

35

The mutant form acetolactate synthase genomic DNA from rice is an efficient selectable marker for genetic transformation.  

UK PubMed Central (United Kingdom)

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

Osakabe K; Endo M; Kawai K; Nishizawa Y; Ono K; Abe K; Ishikawa Y; Nakamura H; Ichikawa H; Nishimura S

2005-11-01

36

Interactions between the root pathogen Rhizoctonia solani AG-8 and acetolactate-synthase-inhibiting herbicides in barley.  

UK PubMed Central (United Kingdom)

BACKGROUND: The widespread acceptance of reduced-tillage farming in cereal cropping systems in the Pacific Northwest of the United States has resulted in increased use of herbicides for weed control. However, soil residual concentrations of widely used imidazalone herbicides limit the cultivation of barley, which is more sensitive than wheat. In addition, increased severity of the root rot disease caused by Rhizoctonia solani is associated with reduction in tillage. Many crops exhibit altered disease responses after application of registered herbicides. In this study, the injury symptoms in barley caused by sublethal rates of two acetolactate synthase (ALS)-inhibiting herbicides, imazamox and propoxycarbazone-sodium, were assessed in factorial combinations with a range of inoculum concentrations of the root rot pathogen Rhizoctonia solani AG-8. RESULTS: Both herbicides and pathogen had negative impacts on plant growth parameters such as root and shoot dry weight, shoot height and first leaf length, and interactions between pathogen and herbicide were detected. CONCLUSIONS: The results suggested that sublethal rates of herbicides and R. solani could alter severity of injury symptoms, possibly owing to the herbicide predisposing the plant to the pathogen.

Lee H; Ullrich SE; Burke IC; Yenish J; Paulitz TC

2012-06-01

37

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

UK PubMed Central (United Kingdom)

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 ilv2Delta 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 drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Kingsbury JM; McCusker JH

2010-03-01

38

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 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. (more) 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. Euphorbia heterophylla is common specie in soybean fields of Brazil. The study reports about a population of Euphorbia heterophylla resistant to imazethapyr. The objectives of the pr (more) esent work were to quantify the level of sensitivity to this herbicide in imazethapyr-resistant and -susceptible E. heterophylla populations evaluate the role of differential penetration into leaves as determining plant resistance to imazethapyr, and compare the waxy cells of R and S populations. The R population had a lower penetration rate compared with that of S population during the six first hours of incubation with the herbicide. Further studies indicated that R population was not different from S population in terms of translocation, metabolism, or target site (ALS enzyme) of imazethapyr action. Analysis of the leaf cuticle surface by scanning electron microscopy revealed higher wax density in the leaf cuticles of population R than that in S population. Thus, it is suggested that R population is resistant to imazethapyr because increased wax content of its cuticle permits less penetration of herbicide into the plant.

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

2006-07-01

39

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides  

UK PubMed Central (United Kingdom)

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I??), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro??? in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro???[rightward arrow]Ser???), Shiwa (Pro???[rightward arrow]His???), and Matsuyama (Pro???[rightward arrow]Leu???). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp???[rightward arrow]Leu??? substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro??? and Trp??? are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.

UCHINO AKIRA; OGATA SHIGERU; KOHARA HIROSHI; YOSHIDA SHUICHI; YOSHIOKA TOSHIHITO; WATANABE HIROAKI

2007-06-01

40

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

Science.gov (United States)

Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala(122) to Val and Ala(122) to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of (14)C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. (14)C-Penoxsulam translocation out of the treated leaf (?2%) was similar among all biotypes. (14)C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. (14)C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1. PMID:23237199

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

2013-01-04

 
 
 
 
41

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

UK PubMed Central (United Kingdom)

Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala(122) to Val and Ala(122) to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of (14)C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. (14)C-Penoxsulam translocation out of the treated leaf (?2%) was similar among all biotypes. (14)C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. (14)C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1.

Riar DS; Norsworthy JK; Srivastava V; Nandula V; Bond JA; Scott RC

2013-01-01

42

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

UK PubMed Central (United Kingdom)

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

Osuna MD; Fischer AJ; De Prado R

2003-11-01

43

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

Science.gov (United States)

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

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

2003-11-01

44

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans.  

UK PubMed Central (United Kingdom)

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Tougou M; Yamagishi N; Furutani N; Kaku K; Shimizu T; Takahata Y; Sakai J; Kanematsu S; Hidaka S

2009-05-01

45

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.  

UK PubMed Central (United Kingdom)

KEY MESSAGE: Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 ?g L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 ?g L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 ?g L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Yao JL; Tomes S; Gleave AP

2013-05-01

46

Resistance evaluation for herbicide resistance–endowing acetolactate synthase (ALS) gene mutations using Raphanus raphanistrum populations homozygous for specific ALS mutations  

UK PubMed Central (United Kingdom)

Yu Q, Han H, Li M, Purba E, Walsh MJ & Powles SB (2012). Resistance evaluation for herbicide resistance–endowing acetolactate synthase (ALS) gene mutations using Raphanus raphanistrum populations homozygous for specific ALS mutations. Weed Research 52, 178–186. SUMMARY: Acetolactate synthase (ALS)?inhibiting herbicide resistance is common in Raphanus raphanistrum (wild radish) populations across the Western Australian (WA) grain belt. This study investigates the molecular and biochemical basis of ALS herbicide resistance in five R. raphanistrum populations. Five known ALS herbicide resistance–endowing mutations (Pro?197?Ala, Pro?197?Thr, Pro?197?Ser, Asp?376?Glu and Trp?574?Leu) were identified, and their resistance spectrum to ALS?inhibiting herbicides was determined using purified populations individually homozygous for each mutation (except for Pro?197?Ala). Plants homozygous for ALS mutations at Pro?197 were found to be cross?resistant to ALS?inhibiting sulfonylurea (SU) and triazolopyrimidine (TP) herbicides, while plants homozygous for Trp?574?Leu were resistant to SU, TP and imidazolinone (IMI) ALS herbicide classes. The Asp?376?Glu mutation is reported here for the first time in R. raphanistrum populations and characterised at both the whole?plant and enzyme level. Plants homozygous for Asp?376?Glu were highly resistant to SU and TP herbicides, based on LD50 R/S ratios (>130 and 128 respectively) and I50 R/S ratios (170 and >110 respectively). In contrast, these plants were moderately resistant to the IMI imazamox (LD50 R/S ratio of 8, I50 R/S ratio of 3) and imazethapyr (I50 R/S ratio of 8) and susceptible to imazapyr (I50 R/S ratio of 0.76). A novel observation in this study is that resistance of homozygous Glu?376 plants is associated with a remarkable growth reduction in the presence of the ALS herbicides tested, making early resistance diagnosis and management difficult.

YU Q; HAN H; LI M; PURBA E; WALSH MJ; POWLES SB

2012-04-01

47

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 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 (more) 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 S (more) tate, 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.

Brighenti, Alexandre Magno

2012-06-01

48

Expression of Flavonoid 3',5'-Hydroxylase and Acetolactate Synthase Genes in Transgenic Carnation: Assessing the Safety of a Nonfood Plant.  

Science.gov (United States)

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

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

2013-05-15

49

Expression of flavonoid 3',5'-hydroxylase and acetolactate synthase genes in transgenic carnation: Assessing the safety of non-food plant.  

UK PubMed Central (United Kingdom)

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

Chandler SF; Senior M; Nakamura N; Tsuda S; Tanaka Y

2013-05-01

50

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

Directory of Open Access Journals (Sweden)

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

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

2013-01-01

51

Inhibitors of specific ceramide synthases.  

UK PubMed Central (United Kingdom)

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

Schiffmann S; Hartmann D; Fuchs S; Birod K; Ferreiròs N; Schreiber Y; Zivkovic A; Geisslinger G; Grösch S; Stark H

2012-02-01

52

Determination of acetolactate synthase activity and protein content of oilseed rape (Brassica napus L.) leaves using visible/near-infrared spectroscopy.  

UK PubMed Central (United Kingdom)

A new acetolactate synthase (ALS)-inhibiting herbicide, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273), was applied to oilseed rape (Brassica napus L.) leaves in different leaf positions. Visible/near-infrared (Vis/NIR) spectroscopy was investigated for fast and non-destructive determination of ALS activity and protein content in rapeseed leaves. Partial least squares (PLS) analysis was the calibration method with comparison of different spectral preprocessing by Savitzky-Golay (SG) smoothing, standard normal variate (SNV), first and second derivative. The best PLS models were obtained by first-derivative spectra for ALS, whereas original spectra for soluble, non-soluble and total protein contents. Simultaneously, certain latent variables (LVs) were used as the inputs of back-propagation neural network (BPNN) and least squares-support vector machine (LS-SVM) models. All LS-SVM models outperformed PLS models and BPNN models. The correlation coefficient (r), root mean square error of prediction (RMSEP) and bias in validation set by LS-SVM were 0.998, 0.715 and 0.079 for ALS, 0.999, 33.084 and 1.178 for soluble protein, 0.997, 42.773 and 6.244 for non-soluble protein, 0.999, 59.562 and 7.437 for total protein, respectively. The results indicated that Vis/NIR spectroscopy combined with LS-SVM could be successfully applied for the determination of ALS activity and protein content of rapeseed leaves. The results would be helpful for further on field analysis of using Vis/NIR spectroscopy to monitor the growing status and physiological properties of oilseed rape.

Liu F; Zhang F; Jin Z; He Y; Fang H; Ye Q; Zhou W

2008-11-01

53

Progress towards clinically useful aldosterone synthase inhibitors.  

UK PubMed Central (United Kingdom)

Owing to the high degree of similarity between aldosterone synthase (CYP11B2) and cortisol synthase (CYP11B1), the design of selective inhibitors of one or the other of these two enzymes was, at one time, thought to be impossible. Through development of novel enzyme screening assays and significant medicinal chemistry efforts, highly potent inhibitors of CYP11B2 have been identified with selectivities approaching 1000-fold between the two enzymes. Many of these molecules also possess selectivity against other steroidogenic cytochromes P450 (e.g. CYP17A1 and CYP19A1) as well as hepatic drug metabolizing P450s. Though not as well developed or explored, inhibitors of CYP11B1, with selectivities approaching 50-fold, have also been identified. The therapeutic benefits of affecting the renin-angiotensin-aldosterone system have been well established with the therapeutically useful angiotensin-converting enzymes inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists. Data regarding the additional benefits of an aldosterone synthase inhibitor (ASi) are beginning to emerge from animal models and human clinical trials. Despite great promise and much progress, additional challenges still exist in the path towards development of a therapeutically useful ASi.

Cerny MA

2013-01-01

54

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

UK PubMed Central (United Kingdom)

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.

Sondhia S; Waseem U; Varma RK

2013-08-01

55

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; A.M. Brighenti; E. Voll

2006-01-01

56

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 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 losna-branca (Parthenium hysterophorus) aos herbicidas inibidores da enzima acetolactato sintase (ALS), proveniente de uma propriedade rural no município de Mandaguari, (more) 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 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 (more) , north of Parana State, Brazil. Plants with suspicious resistance were treated with several herbicides and rates and compared with those of a susceptible population. The herbicide treatments were established considering the recommended rates, double and four times higher than the recommended rate as follows: cloransulam-methyl 0.0, 33.6, 67.2 and 134.4 g a.i. ha-1 plus adjuvant 0.2% v/v, chlorimuron-ethyl 0.0, 20.0, 40.0 and 80.0 g a.i., imazethapyr 0.0, 100.0, 200.0 and 400.0 g a.i. ha-1, iodosulfuron-methyl-sodium plus foramsulfuron 0.0, 3.0 + 45.0 ga.i. ha-1 (150.0 g c.p. ha-1), 6.0 + 90.0 g a.i. ha-1 (300.0 g c.p. ha-1) and 12.0 + 180.0 g a.i. ha¹ (600.0 g c.p. ha-1). In addition, a treatment with 2,4-D (536.0 g a.e. ha¹) was applied. Resistant plant dose-response curves presented lower values when compared to the susceptible population, in all rates and herbicides studied. The ragweed biotype was confirmed as resistant to the ALS inhibiting herbicides. Cross-resistance was observed with herbicides belonging to the chemical groups of imidazolinones (imazethapyr), triazolopyrimidines (cloransulam-methyl), sulfonylureas (chlorimuron-ethyl and iodosulfuron-methyl-sodium plus foramsulfuron). 2,4-D has a different mechanism of action, presenting high values of control, and thus being a management alternative in areas with ragweed resistant population.

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

2006-01-01

57

Catalepsy induced by nitric oxide synthase inhibitors.  

UK PubMed Central (United Kingdom)

1. Previous study showed that N(G)-nitro-L-arginine (L-NOARG), an inhibitor of nitric bxide synthase, induces catalepsy in a dose-dependent manner in male albino-Swiss mice. 2. The objective of the present work was to further investigate this effect, extending it to other NOS inhibitors. 3. Results showed that L-NOARG (40-80 mg/kg i.p.), N(G)-nitro-L-arginine methylester (L-NAME, 40-160 mg/kg i.p.) or N(G)-monomethyl-L-arginine (L-NMMA, 80 mg/kg i.p.) were able to induce catalepsy in mice. The effect of L-NOARG (40 mg/kg) was antagonized by pretreatment with L-arginine (300 mg/kg), but not by D-arginine (300 mg/kg). The catalepsy-inducing effect of L-NOARG suffered rapid tolerance, showing a significant decrease after two days of chronic treatment (40 mg/kg i.p., twice a day). 4. The results suggest that interference with the formation of nitric oxide induces significant motor effects in mice.

Del Bel EA; da Silva CA; Guimarães FS

1998-02-01

58

Farnesyl diphosphate synthase inhibitors from in silico screening.  

UK PubMed Central (United Kingdom)

The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of farnesyl diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads.

Lindert S; Zhu W; Liu YL; Pang R; Oldfield E; McCammon JA

2013-06-01

59

Farnesyl Diphosphate Synthase Inhibitors from In Silico Screening  

Science.gov (United States)

The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of farnesyl diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads.

Lindert, Steffen; Zhu, Wei; Liu, Yi-Liang; Pang, Ran; Oldfield, Eric; McCammon, J Andrew

2013-01-01

60

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

Directory of Open Access Journals (Sweden)

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

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

2003-01-01

 
 
 
 
61

Inhibitors of the catalytic domain of mitochondrial ATP synthase.  

UK PubMed Central (United Kingdom)

An understanding of the mechanism of ATP synthase requires an explanation of how inhibitors act. The catalytic F1-ATPase domain of the enzyme has been studied extensively by X-ray crystallography in a variety of inhibited states. Four independent inhibitory sites have been identified by high-resolution structural studies. They are the catalytic site, and the binding sites for the antibiotics aurovertin and efrapeptin and for the natural inhibitor protein, IF1.

Gledhill JR; Walker JE

2006-11-01

62

Aminotetrazole derivatives useful as nitric oxide synthase inhibitors  

UK PubMed Central (United Kingdom)

PCT No. PCT/US95/14001 Sec. 371 Date Apr. 30, 1997 Sec. 102(e) Date Apr. 30, 1997 PCT Filed Nov. 8, 1995 PCT Pub. No. WO96/15120 PCT Pub. Date May 23, 1996Aminotetrazole derivatives of the formula (I) wherein the variables are as defined in the disclosure are useful as nitric oxide synthase inhibitors. (I)

HALLINAN ANN E; HANSEN JR DONALD W; TSYMBALOV SOFYA

63

2-ACYLAMINOPROPOANOL-TYPE GLUCOSYLCERAMIDE SYNTHASE INHIBITORS  

UK PubMed Central (United Kingdom)

A compound is represented by Structural Formula (I): or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof. A method of treating a subject in need thereof comprises administering to the subject a therapeutically effective amount of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof. The subject has type 2 diabetes renal hypertrophy or hyperplasia associated with diabetic nephropathy Tay-Sachs Gaucher's or Fabry's disease. Methods of decreasing plasma TNF-[alpha], lowering blood glucose levels, decreasing glycated hemoglobin levels, inhibiting glucosylceramide synthase, and lowering glycosphingolipid concentrations in a subject in need thereof respectively comprise administering to the subject a therapeutically effective amount of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof.

SIEGEL CRAIG; BASTOS CECILIA M; HARRIS DAVID J; DIOS ANGELES; LEE EDWARD; SILVA RICHARD; CUFF LISA M; LEVINE MIKAELA; CELATKA CASSANDRA A; VINICK FREDERIC; JOZEFIAK THOMAS H; XIANG YIBIN; KANE JOHN; LIAO JUNKAI

64

Inhibitors of glycogen synthase 3 kinase  

Energy Technology Data Exchange (ETDEWEB)

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

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

2000-01-01

65

Enhancement of vascular targeting by inhibitors of nitric oxide synthase  

International Nuclear Information System (INIS)

[en] 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

2002-12-01

66

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

Scientific Electronic Library Online (English)

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

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

2013-09-01

67

Biochemistry: Acetohydroxyacid Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS) is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA) isoleucine, leucine and valine. The enzyme is inhibited by several comme...

Pham Ngoc Chien

68

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

69

Fatty acid synthase inhibitors isolated from Punica granatum L  

International Nuclear Information System (INIS)

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

2012-01-01

70

Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase  

Energy Technology Data Exchange (ETDEWEB)

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

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

2010-01-12

71

Structural studies of pterin-based inhibitors of dihydropteroate synthase.  

UK PubMed Central (United Kingdom)

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

Hevener KE; Yun MK; Qi J; Kerr ID; Babaoglu K; Hurdle JG; Balakrishna K; White SW; Lee RE

2010-01-01

72

Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments.  

UK PubMed Central (United Kingdom)

Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions.

King MK; Pardo M; Cheng Y; Downey K; Jope RS; Beurel E

2013-07-01

73

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

2010-01-01

74

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; Luciane Vilela Resende; Paulo Roberto Da-Silva; Ribas Antonio Vidal; Anderson Luis Nunes

2006-01-01

75

Screening assay for the identification of deoxyhypusine synthase inhibitors.  

Science.gov (United States)

The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV. PMID:15296643

Sommer, Marc-Nicola; Bevec, Dorian; Klebl, Bert; Flicke, Birgit; Hölscher, Kerstin; Freudenreich, Tatjana; Hauber, Ilona; Hauber, Joachim; Mett, Helmut

2004-08-01

76

Identification of cystathionine ?-synthase inhibitors using a hydrogen sulfide selective probe.  

UK PubMed Central (United Kingdom)

Buzzing with activity: A hydrogen sulfide selective fluorogenic probe, 7-azido-4-methylcoumarin (AzMC), serves as a highly sensitive assay for cystathionine ?-synthase activity, and is suitable for the high-throughput discovery of novel enzyme inhibitors.

Thorson MK; Majtan T; Kraus JP; Barrios AM

2013-04-01

77

Fatty acid synthase inhibitors isolated from Punica granatum L.  

Scientific Electronic Library Online (English)

Full Text Available 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 compostos incluindo principalmente triternóides e compostos fenólicos, dos quais um novo triterpeno do tipo oleanano (punicaone) juntamente com quatorze compostos conhecidos fora (more) m 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 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 evalu (more) ated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC50 value of 10.3 µmol L-1.

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

2012-05-01

78

Acetohydroxyacid Synthase: A Target for Antimicrobial Drug Discovery.  

UK PubMed Central (United Kingdom)

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 potentand 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) andhave low mammalian toxicity (LD50values 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 onAHAS inhibitors that have potential to be developed into antimicrobial therapeutics.

Pue N; Guddat LW

2013-05-01

79

Characteristics of the squalene synthase inhibitors produced by a Streptomyces species isolated from soils.  

UK PubMed Central (United Kingdom)

Microorganisms producing squalene synthase inhibitors were screened from soils. A high producer was selected and identified as a Streptomyces species. Two active inhibitors were obtained from culture broths via a series of purification processes involving solvent extraction, WK-10 cation-exchange column chromatography, HP-20 adsorption column chromatography, silica-gel column chromatography, preparative HPLC, and crystallization. The inhibitors were confirmed as macrolactins A and F with molecular weights of 402 by UV-absorption spectrometry, fast atom bombardment mass spectometry, and 13C- and 1H-NMR analyses. Kinetic results for macrolactins A and F showed that they appear to be noncompetitive inhibitors of rat liver squalene synthase with IC50 values of 1.66 and 1.53 micromol/L, respectively. Since mammalian squalene synthase was used, these inhibitors have significant potential as therapeutic agents for hyperlipemia and suppression of cholesterol biosynthesis.

Choi SW; Bai DH; Yu JH; Shin CS

2003-11-01

80

Characteristics of the squalene synthase inhibitors produced by a Streptomyces species isolated from soils.  

Science.gov (United States)

Microorganisms producing squalene synthase inhibitors were screened from soils. A high producer was selected and identified as a Streptomyces species. Two active inhibitors were obtained from culture broths via a series of purification processes involving solvent extraction, WK-10 cation-exchange column chromatography, HP-20 adsorption column chromatography, silica-gel column chromatography, preparative HPLC, and crystallization. The inhibitors were confirmed as macrolactins A and F with molecular weights of 402 by UV-absorption spectrometry, fast atom bombardment mass spectometry, and 13C- and 1H-NMR analyses. Kinetic results for macrolactins A and F showed that they appear to be noncompetitive inhibitors of rat liver squalene synthase with IC50 values of 1.66 and 1.53 micromol/L, respectively. Since mammalian squalene synthase was used, these inhibitors have significant potential as therapeutic agents for hyperlipemia and suppression of cholesterol biosynthesis. PMID:14735215

Choi, Sung-Won; Bai, Dong-Hoon; Yu, Ju-Hyun; Shin, Chul Soo

2003-11-01

 
 
 
 
81

Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.  

UK PubMed Central (United Kingdom)

We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

Morii H; Okauchi T; Nomiya H; Ogawa M; Fukuda K; Taniguchi H

2013-03-01

82

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

Directory of Open Access Journals (Sweden)

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

Chia-Hsien Lee; Hsuan-Cheng Huang; Hsueh-Fen Juan

2011-01-01

83

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

International Nuclear Information System (INIS)

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

1986-01-01

84

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

Energy Technology Data Exchange (ETDEWEB)

Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

1986-05-01

85

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

Science.gov (United States)

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

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

2013-07-02

86

Spirohexalines, new inhibitors of bacterial undecaprenyl pyrophosphate synthase, produced by Penicillium brasilianum FKI-3368.  

UK PubMed Central (United Kingdom)

An enzyme assay for bacterial undecaprenyl pyrophosphate (UPP) synthase was performed to screen microbial culture broths for inhibitors of UPP synthase. During the course of this screening program, an EtOH extract of a rice culture of Penicillium brasilianum FKI-3368 was found to inhibit UPP synthase activity. From activity-guided purification, a new compound-designated spirohexaline was isolated together with the structurally related and known viridicatumtoxin by ethyl acetate extraction silica gel and octadecylsilane column chromatographies and high-performance liquid chromatography. The structure of spirohexaline was elucidated by spectroscopic analysis, including NMR. Spirohexaline and viridicatumtoxin have a common hexacycline structure produced by fusion of a tetracycline-type ring with a spiro-type ring. They inhibited UPP synthase activity with IC?? values of 9.0 and 4.0??M, respectively.

Inokoshi J; Nakamura Y; Hongbin Z; Uchida R; Nonaka K; Masuma R; Tomoda H

2013-01-01

87

The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase.  

UK PubMed Central (United Kingdom)

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.

García JJ; Morales-Ríos E; Cortés-Hernandez P; Rodríguez-Zavala JS

2006-10-01

88

The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase.  

Science.gov (United States)

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane. PMID:17042487

García, José J; Morales-Ríos, Edgar; Cortés-Hernandez, Paulina; Rodríguez-Zavala, José S

2006-10-24

89

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

UK PubMed Central (United Kingdom)

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

Sathler PC; Santana M; Lourenço AL; Rodrigues CR; Abreu P; Cabral LM; Castro HC

2013-08-01

90

Synthesis of Bi-substrate State Mimics of Dihydropteroate Synthase as Potential Inhibitors and Molecular Probes  

Science.gov (United States)

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.

Qi, Jianjun; Virga, Kristopher G.; Das, Sourav; Zhao, Ying; Yun, Mi-Kyung; White, Stephen W.; Lee, Richard E.

2010-01-01

91

[Research and development of ozagrel, a highly selective inhibitor of TXA2 synthase  

UK PubMed Central (United Kingdom)

Highly selective inhibitors of thromboxane (TX) A2 synthase were noted as a therapeutic agent for ischemic heart diseases, thromboembolic disorders, cerebral circulatory disorders, and asthma. The 1-substituted imidazoles and beta-substituted pyridines showed high inhibitory potency on TXA2 synthase. The structure-activity relationships of the imidazole and pyridine derivatives as inhibitors of TXA2 synthase were investigated. Introduction of various substituents into the carboxy-bearing side chain of 1-(7-carboxyheptyl) imidazole and beta-(7-carboxyheptyl) pyridine was found to increase the inhibitory potency. The length of the side chains with the phenylene group was optimum in the region of 8.5 to 10 A for the inhibitory potency on TXA2 synthase. Among the tested imidazole and pyridine derivatives, (E)-4-(1-imidazolylmethyl)cinnamic acid (44) and (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methylacrylic acid (56) showed the highest potency (IC50 = 1.1 x 10(-8) and 3 x 10(-9) M). The inhibition by these derivatives was highly selective for TXA2 synthase, since other enzymes which are involved in the arachidonic acid cascade, such as fatty acid cyclooxygenase, 5-lipoxygenase, prostacyclin (PGI2) synthase, and PGE2 isomerase were not affected. On the basis of the results obtained from the pharmacological, physicochemical and toxicological studies on the two compounds (44 and 56), (E)-4-(1-imidazolylmethyl) cinnamic acid (44; OKY-046, ozagrel) was selected as the best compound of highly selective inhibitors of TXA2 synthase. The pharmacological properties of ozagrel are as follows. The inhibition of TXA2 synthase by ozagrel was more effective on human and rabbit enzymes than those of other species. Ozagrel increased 6-keto-PGF1 alpha, one of stable metabolites of PGI2, in various isolated cells and tissues perhaps via accumulated PG endoperoxides resulted by the inhibition of TXA2 synthase. Such an increase in PGI2 production by ozagrel was also observed in various experimental animals. We obtained the suggestion that, by the reduction of TXA2 production and increment of PGI2 production, ozagrel inhibits the spasms of basilar artery and the decreases in regional cerebral blood flow in dogs which received autologous blood into cisterna magna, and inhibits the decreases in motor function and regional cerebral blood flow, and the formation of infarcted area in the animals of cerebral ischemic treatment. It was also suggested that ozagrel inhibits leukotriene-, platelet-activating factor-, and antigen-induced bronchoconstriction in guinea-pigs and inhibits the induction of airway hyperresponsiveness by various stimuli in several species of animals by both mechanisms. The summarized results of ADME, toxicological, and clinical studies were also described.

Nakazawa M; Iizuka K; Ujiie A; Hiraku S; Ohki S

1994-12-01

92

Cyclopropyl- and methyl-containing inhibitors of neuronal nitric oxide synthase.  

UK PubMed Central (United Kingdom)

Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-cyclopropyl- and methyl-containing nNOS inhibitors have been synthesized. The insertion of a rigid electron-withdrawing cyclopropyl ring decreases the basicity of the adjacent amino group, which resulted in decreased inhibitory activity of these inhibitors compared to the parent compound. Nonetheless, three of them exhibited double-digit nanomolar inhibition with high nNOS selectivity on the basis of in vitro enzyme assays. Crystal structures of nNOS and eNOS with these inhibitors bound provide a basis for detailed structure-activity relationship (SAR) studies. The conclusions from these studies will be used as a guide in the future development of selective NOS inhibitors.

Li H; Xue F; Kraus JM 2nd; Ji H; Labby KJ; Mataka J; Delker SL; Martásek P; Roman LJ; Poulos TL; Silverman RB

2013-03-01

93

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

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Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mi...

Tomishima Yoshiro; Ishitsuka Yoichi; Matsunaga Naoya; Nagatome Minako; Furusho Hirokazu; Irikura Mitsuru; Ohdo Shigehiro

94

Evaluation of the susceptibility of Coccidioides immitis to lufenuron, a chitin synthase inhibitor.  

UK PubMed Central (United Kingdom)

The activity of the chitin synthase inhibitor lufenuron was evaluated in vitro using the spherule-endospore (SE) phase of Coccidioides immitis. The lufenuron was also used to treat mice infected with C. immitis by the respiratory route. In vitro, lufenuron had no effect upon fungal cell growth. Two formulations of lufenuron were evaluated in vivo. Neither the oral nor the injectable lufenuron extended the survival of mice infected with C. immitis when compared with placebo-treated mice.

Johnson SM; Zimmermann CR; Kerekes KM; Davidson A; Pappagianis D

1999-12-01

95

Intracoronary administration of a thromboxane A2 synthase inhibitor relieves acetylcholine-induced coronary spasm.  

Science.gov (United States)

This study sought to clarify the effectiveness of intracoronary administration of a thromboxane (TX) A2 synthase inhibitor, Ozagrel Na, to relieve coronary spasms induced by intracoronary injection of acetylcholine (ACh). An ACh spasm provocation test was performed in 92 consecutive patients with coronary spastic angina using incremental doses of 20, 50, and 80 microg into the right coronary artery, and 20, 50, and 100 microg into the left coronary artery within 20s. A coronary spasm was defined as TIMI 0 or 1 flow and an intracoronary injection of 20 mg Ozagrel Na was administered when it was provoked. Within 2 min of the administration of the TXA2 synthase inhibitor, ACh-induced coronary spasms were relieved (TIMI 3 flow) in 88.1% of procedures without complications. In only 4 cases (4.3%), it took more than 3 min to relieve the coronary spasms. Intracoronary administration of 20mg Ozagrel Na when ACh-induced spasms occurred, shortened the spasm relief time in all 7 patients (200 +/- 59s vs 111 +/- 23s, p Ozagrel Na into the left coronary artery (463 +/- 562 vs 96 +/- 45, p < 0.01). In conclusion, intracoronary administration of a TXA2 synthase inhibitor can relieve ACh-induced coronary spasms by inhibiting TXA2 synthesis in the local coronary circulation. PMID:12224820

Sueda, Shozo; Kohno, Hiroaki; Inoue, Katsuji; Fukuda, Hiroshi; Suzuki, Jun; Watanabe, Kouki; Ochi, Naoto; Kawada, Hiroyuki; Uraoka, Tadao

2002-09-01

96

2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase.  

UK PubMed Central (United Kingdom)

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.

Hagen TJ; Bergmanis AA; Kramer SW; Fok KF; Schmelzer AE; Pitzele BS; Swenton L; Jerome GM; Kornmeier CM; Moore WM; Branson LF; Connor JR; Manning PT; Currie MG; Hallinan EA

1998-09-01

97

An improved method for screening alpha-acetolactate producing mutants.  

UK PubMed Central (United Kingdom)

Alpha-Acetolactate-deficient Lactococcus lactis ssp. lactis biovar. diacelylactis are utilised in several industrial processes for producing diacetyl and alpha-acetolactate. They can be selected by screening after random mutagenesis. We improved a previously described screening method [Monnet, C., Schmitt, P., Diviès, C., 1997. Appl. Environ. Microbiol. 63, 793-795], which makes it possible to screen up to 1000 colonies per agar plate, whereas the previous method allowed to screen only 60 colonies per agar plate. The new screening method facilitates selection of alpha-acetolactate-deficient mutants.

Monnet C; Haddad S; Corrieu G

1999-08-01

98

The thymidylate synthase inhibitor ZD1694 potently inhibits murine and human cytomegalovirus replication in quiescent fibroblasts.  

UK PubMed Central (United Kingdom)

Tomudex (ZD1694) is a quinazoline-based folate analog and a powerful inhibitor of cellular thymidylate synthase and is approved in Europe for use in oncology. Here the first evidence of its activity against murine and human cytomegalovirus (MCMV and HCMV) is reported. ZD1694 irreversibly inhibited the replication and DNA synthesis of both viruses in quiescent fibroblasts. The corresponding 50% effective concentrations were 0.006 and 0.002 microM respectively, whereas the 50% cytotoxic concentration was >10 microM for both murine and human quiescent fibroblasts. A similar antiviral effect was observed against two ganciclovir-resistant HCMV strains isolated from AIDS patients. Taken as a whole these results demonstrate that cellular thymidylate synthase plays an essential role in viral replication and that ZD1694 merits further investigation as anticytomegaloviral agent.

Lembo D; Gribaudo G; Riera L; Mondo A; Cavallo R; Angeretti A; Landolfo S

2000-08-01

99

The thymidylate synthase inhibitor ZD1694 potently inhibits murine and human cytomegalovirus replication in quiescent fibroblasts.  

Science.gov (United States)

Tomudex (ZD1694) is a quinazoline-based folate analog and a powerful inhibitor of cellular thymidylate synthase and is approved in Europe for use in oncology. Here the first evidence of its activity against murine and human cytomegalovirus (MCMV and HCMV) is reported. ZD1694 irreversibly inhibited the replication and DNA synthesis of both viruses in quiescent fibroblasts. The corresponding 50% effective concentrations were 0.006 and 0.002 microM respectively, whereas the 50% cytotoxic concentration was >10 microM for both murine and human quiescent fibroblasts. A similar antiviral effect was observed against two ganciclovir-resistant HCMV strains isolated from AIDS patients. Taken as a whole these results demonstrate that cellular thymidylate synthase plays an essential role in viral replication and that ZD1694 merits further investigation as anticytomegaloviral agent. PMID:10996399

Lembo, D; Gribaudo, G; Riera, L; Mondo, A; Cavallo, R; Angeretti, A; Landolfo, S

2000-08-01

100

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.(1,2) 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 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±0.67nM 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

2013-07-24

 
 
 
 
101

Intracoronary administration of a thromboxane A2 synthase inhibitor relieves acetylcholine-induced coronary spasm.  

UK PubMed Central (United Kingdom)

This study sought to clarify the effectiveness of intracoronary administration of a thromboxane (TX) A2 synthase inhibitor, Ozagrel Na, to relieve coronary spasms induced by intracoronary injection of acetylcholine (ACh). An ACh spasm provocation test was performed in 92 consecutive patients with coronary spastic angina using incremental doses of 20, 50, and 80 microg into the right coronary artery, and 20, 50, and 100 microg into the left coronary artery within 20s. A coronary spasm was defined as TIMI 0 or 1 flow and an intracoronary injection of 20 mg Ozagrel Na was administered when it was provoked. Within 2 min of the administration of the TXA2 synthase inhibitor, ACh-induced coronary spasms were relieved (TIMI 3 flow) in 88.1% of procedures without complications. In only 4 cases (4.3%), it took more than 3 min to relieve the coronary spasms. Intracoronary administration of 20mg Ozagrel Na when ACh-induced spasms occurred, shortened the spasm relief time in all 7 patients (200 +/- 59s vs 111 +/- 23s, p < 0.01), improved the maximal ST segment elevation in 5 of them (3.9 +/- 3.7 mm vs 0.7 +/- 1.5 mm, p < 0.05), and stopped chest pain in 4 patients. In 4 patients who had ACh-induced coronary spasm of the left anterior descending artery, the TXB2 concentration in the coronary sinus decreased after intracoronary administration of Ozagrel Na into the left coronary artery (463 +/- 562 vs 96 +/- 45, p < 0.01). In conclusion, intracoronary administration of a TXA2 synthase inhibitor can relieve ACh-induced coronary spasms by inhibiting TXA2 synthesis in the local coronary circulation.

Sueda S; Kohno H; Inoue K; Fukuda H; Suzuki J; Watanabe K; Ochi N; Kawada H; Uraoka T

2002-09-01

102

Structure-based inhibitors exhibit differential activities against Helicobacter pylori and Escherichia coli undecaprenyl pyrophosphate synthases.  

UK PubMed Central (United Kingdom)

Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

Kuo CJ; Guo RT; Lu IL; Liu HG; Wu SY; Ko TP; Wang AH; Liang PH

2008-01-01

103

Structure-Based Inhibitors Exhibit Differential Activities against Helicobacter pylori and Escherichia coli Undecaprenyl Pyrophosphate Synthases  

Directory of Open Access Journals (Sweden)

Full Text Available Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

Chih-Jung Kuo; Rey-Ting Guo; I-Lin Lu; Hun-Ge Liu; Su-Ying Wu; Tzu-Ping Ko; Andrew H.-J. Wang; Po-Huang Liang

2008-01-01

104

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.  

Science.gov (United States)

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-08-21

105

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.  

UK PubMed Central (United Kingdom)

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Wu YH; Hu CW; Chien CW; Chen YJ; Huang HC; Juan HF

2013-01-01

106

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

UK PubMed Central (United Kingdom)

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

Huang H; Silverman RB

2013-01-01

107

Attenuation of acetic acid-induced gastric ulcer formation in rats by glucosylceramide synthase inhibitors.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Ceramide has been suggested to play a role in apoptosis during gastric ulcerogenesis. The present study is designed to investigate whether accumulated ceramide could serve as the effector molecules of ulcer formation in a rat model of acetic acid-induced gastric ulcer. METHODS: The effect of fumonisin B1, an inhibitor of ceramide synthase, and of d,l,-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) and N-butyldeoxynojirimycin (NB-DNJ), both inhibitors of glucosylceramide synthase, on the accumulation of ceramide and formation of gastric ulcer were examined in the rat model of acetic acid-induced gastric ulcer. RESULTS: Fumonisin B1 attenuated acetic acid-induced gastric ulcer formation, associated with a decrease in the number of apoptotic cells. Our results showed that it is neither the C18- nor the C24-ceramide itself, but the respective metabolites that were ulcerogenic, because PPMP and NB-DNJ attenuated gastric mucosal apoptosis and the consequent mucosal damage in spite of their reducing the degradation of ceramide. CONCLUSION: The ceramide pathway, in particular, the metabolites of ceramide, significantly contributes to acetic acid-induced gastric damage, possibly via enhancing apoptosis. On the other hand, PPMP and NB-DNJ treatment attenuated gastric mucosal apoptosis and ulcer formation despite increasing the ceramide accumulation, suggesting that it was not the ceramides themselves, but their metabolites that contributed to the ulcer formation in the acetic acid-induced gastric ulcer model.

Nakashita M; Suzuki H; Miura S; Taki T; Uehara K; Mizushima T; Nagata H; Hibi T

2013-02-01

108

Systemic injection of a nitric oxide synthase inhibitor suppresses sleep responses to sleep deprivation in rats.  

UK PubMed Central (United Kingdom)

We hypothesized that nitric oxide (NO) may play a role in homeostatic sleep regulation. To test this hypothesis, we studied the sleep deprivation (SD)-induced homeostatic sleep responses after intraperitoneal administration of an NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, a cumulative dose of 100 mg/kg). Amounts and intensity of sleep were increased in response to 8 h of SD in control rats (n = 8). Sleep amounts remained above baseline for 16 h after SD followed by a negative rebound. Rapid eye movement sleep (REMS) and non-REMS (NREMS) intensities were elevated for 16 and 4 h, respectively. L-NAME treatment (n = 8) suppressed the rebound increases in NREMS amount and intensity. REMS rebound was attenuated by L-NAME in the first dark period after SD; however, a second rebound appeared in the subsequent dark period. REMS intensity did not increase after SD in L-NAME-injected rats. The finding that the NO synthase inhibitor suppressed rebound increases in NREMS suggests that NO may play a role as a signaling molecule in homeostatic regulation of NREMS.

Ribeiro AC; Gilligan JG; Kapás L

2000-04-01

109

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

Directory of Open Access Journals (Sweden)

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

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

2011-01-01

110

Effects of NO synthase inhibitors on the synovial microcirculation in the mouse knee joint.  

UK PubMed Central (United Kingdom)

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors [selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo.

Veihelmann A; Krombach F; Refior HJ; Messmer K

1999-09-01

111

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

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Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of severa...

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

112

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

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Introduction: Increase in the nitric oxide in the spermatic veins of men by varicocele has been reported. Although Several studies have considered the relationship between varicocele and semen NO concentrations, no study on the effects of nitric oxide synthase inhibitor (L-NAME) on epididymal sperm ...

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

113

Glycogen synthase kinase-3 inhibitors as potent therapeutic agents for the treatment of Parkinson disease.  

UK PubMed Central (United Kingdom)

Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of the nigrostriatal dopaminergic pathway. Because the current therapies only lead to temporary, limited improvement and have severe side effects, new approaches to treat PD need to be developed. To discover new targets for potential therapeutic intervention, a chemical genetic approach involving the use of small molecules as pharmacological tools has been implemented. First, a screening of an in-house chemical library on a well-established cellular model of PD was done followed by a detailed pharmacological analysis of the hits. Here, we report the results found for the small heterocyclic derivative called SC001, which after different enzymatic assays was revealed to be a new glycogen synthase kinase-3 (GSK-3) inhibitor with IC(50) = 3.38 ± 0.08 ?M. To confirm that GSK-3 could be a good target for PD, the evaluation of a set of structurally diverse GSK-3 inhibitors as neuroprotective agents for PD was performed. Results show that inhibitors of GSK-3 have neuroprotective effects in vitro representing a new pharmacological option for the disease-modifying treatment of PD. Furthermore, we show that SC001 is able to cross the blood-brain barrier, protects dopaminergic neurons, and reduces microglia activation in in vivo models of Parkinson disease, being a good candidate for further drug development.

Morales-García JA; Susín C; Alonso-Gil S; Pérez DI; Palomo V; Pérez C; Conde S; Santos A; Gil C; Martínez A; Pérez-Castillo A

2013-02-01

114

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

Science.gov (United States)

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

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

2013-07-08

115

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

Science.gov (United States)

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

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

2007-08-01

116

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

UK PubMed Central (United Kingdom)

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

Behery FA; Naeem ZE; Maatooq GT; Amer MM; Wen ZH; Sheu JH; Ahmed AF

2007-08-01

117

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

Directory of Open Access Journals (Sweden)

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

Stevanovi? Ivana; Jovanovi? Marina; Ninkovi? Milica

2013-01-01

118

Day- and nighttime injection of a nitric oxide synthase inhibitor elicits opposite sleep responses in rats.  

UK PubMed Central (United Kingdom)

Previous studies suggest that nitric oxide (NO) may play a role in sleep regulation, particularly in the homeostatic process. The present studies were undertaken to compare the sleep effects of injecting a NO synthase (NOS) inhibitor when homeostatic sleep pressure is naturally highest (light onset) or when it is at its nadir (dark onset) in rats. Sleep, electroencephalogram delta-wave activity during nonrapid eye movement sleep (NREMS), also known as slow-wave activity (SWA), and brain temperature responses to three doses of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME ; 5, 50, and 100 mg/kg) injected intraperitoneally at light or dark onset were examined in rats (n = 6 to 8). The effects of 5 mg/kg L-NAME were determined in both normal and vagotomized (VX) rats. Light onset administration of 50 mg/kg L-NAME decreased NREMS amounts and suppressed SWA and increased rapid eye movement sleep (REMS) amounts. At dark onset, L-NAME injection also dose dependently suppressed SWA; however, unlike light onset injections, both NREMS and REMS amounts were increased after all three doses. Sleep responses to 5 mg/kg L-NAME were not different in control and VX rats, suggesting that the sleep effects of L-NAME are not mediated through the activation of sensory vagal mechanisms. The present findings suggest that timing of the injection is a major determinant of the sleep responses observed after systemic L-NAME injection in rats.

Ribeiro AC; Kapás L

2005-08-01

119

Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells.  

UK PubMed Central (United Kingdom)

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.

Kirby LA; Schott JT; Noble BL; Mendez DC; Caseley PS; Peterson SC; Routledge TJ; Patel NV

2012-01-01

120

Selectivity of commonly used pharmacological inhibitors for cystathionine ? synthase (CBS) and cystathionine ? lyase (CSE).  

UK PubMed Central (United Kingdom)

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2 S are cystathionine ? synthase (CBS) and cystathionine ? lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2 S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ?-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 ?M vs. 40 ± 8 ?M respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1??M); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 ?M. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2 S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Asimakopoulou A; Panopoulos P; Chasapis CT; Coletta C; Zhou Z; Cirino G; Giannis A; Szabo C; Spyroulias GA; Papapetropoulos A

2013-06-01

 
 
 
 
121

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

UK PubMed Central (United Kingdom)

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.

Chen CN; Chen Q; Liu YC; Zhu XL; Niu CW; Xi Z; Yang GF

2010-07-01

122

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

2010-06-11

123

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.

Rossana Domenis; Elena Bisetto; Davide Rossi; Marina Comelli; Irene Mavelli

2012-01-01

124

Pharmacological profile of JTE-522, a novel prostaglandin H synthase-2 inhibitor, in rats.  

UK PubMed Central (United Kingdom)

OBJECTIVE AND DESIGN: The antinociceptive, antipyretic, and anti-inflammatory effects of JTE-522, a novel selective prostaglandin H synthase (PGHS)-2 inhibitor, were examined in rats. MATERIALS: Sheep seminal vesicle PGHS-1 and placenta PGHS-2 were used for in vitro assay, while for in vivo experiments, male rats (4-8 weeks old) were used. TREATMENT: JTE-522 and reference compounds (0.01-100 microM) were subjected to enzyme assay. JTE-522 (0.3-30 mg/kg) and indomethacin (0.3-10 mg/kg) were administered orally. RESULTS: JTE-522 inhibited PGHS-2 (IC50: 0.64 microM) without affecting PGHS-1 activity at 100 microM. In rats with yeast-induced hyperalgesia, JTE-522 showed a dose-dependent antinociceptive effect (ED50: 4.4 mg/kg). In rats with yeast-induced pyrexia, JTE-522 significantly reversed the pyrexic response (ED50: 3.9 mg/kg). Orally administered JTE-522 dose-dependently inhibited carrageenin-induced rat paw edema (ED30: 4.7 mg/kg). In rats with adjuvant-induced arthritis, JTE-522 showed a significant inhibitory effect at daily doses of 0.3-3 mg/kg. JTE-522 did not cause severe gastric lesions at oral doses up to 300 mg/kg. CONCLUSIONS: Our results indicate that the selective PGHS-2 inhibitor JTE-522 may represent a novel type of anti-inflammatory drug without adverse effects on the gastrointestinal tract. JTE-522 may thus be a promising agent for treating both acute inflammatory disease and chronic inflammatory diseases such as rheumatoid arthritis.

Matsushita M; Masaki M; Yagi Y; Tanaka T; Wakitani K

1997-11-01

125

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

126

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

Science.gov (United States)

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

Conrad, John A; Moran, Graham R

2008-03-01

127

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

Scientific Electronic Library Online (English)

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

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

1999-11-01

128

Selective aldosterone synthase inhibitors reduce aldosterone formation in vitro and in vivo.  

UK PubMed Central (United Kingdom)

Aldosterone plays a crucial role in salt and water homeostasis but in case of pathologically increased plasma aldosterone levels it is also involved in the development and the progression of severe cardiovascular diseases like heart failure and myocardial fibrosis. For the treatment of these diseases we propose inhibition of the aldosterone forming enzyme CYP11B2 as a new pharmacological strategy. We recently developed in vitro highly potent and selective inhibitors of human CYP11B2, but the evidence of their in vivo activity is still missing. For this purpose, rat aldosterone synthase gene was cloned and expressed in V79MZ cells to establish a new screening assay for the identification of "rat-active" substances. Compound 7 from the class of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones showed a moderate inhibitory effect (65% at 2 microM) on rat CYP11B2 in vitro. Furthermore, it diminished the conversion of deoxycorticosterone to aldosterone in rat adrenals and significantly reduced plasma aldosterone levels in vivo.

Ries C; Lucas S; Heim R; Birk B; Hartmann RW

2009-09-01

129

Glycogen synthase kinase 3 inhibitor attenuates endotoxin-induced liver injury.  

UK PubMed Central (United Kingdom)

BACKGROUND/AIMS: Endotoxin (lipopolysaccharide, LPS)-induced acute liver injury was attenuated by endotoxin tolerance (ET), which is characterized by phosphatidylinositol 3-kinase pathway/Akt signaling. Glycogen synthase kinase 3 (GSK-3) acts downstream of phosphatidylinositol 3-kinase pathway/Akt and GSK-3 inhibitor protects against organic injury. This study evaluates the hypothesis that ET attenuated LPS-induced liver injury through inhibiting GSK-3 functional activity and downstream signaling. METHODS: Sprague-Dawley rats with or without low-dose LPS pretreatment were challenged with or without large dose of LPS and subsequently received studies. Serum tumor necrosis factor-alpha, interleukin-10, alanine aminotransferase, lactate dehydrogenase, and total bilirubin levels were analyzed, morphology of liver tissue was performed, glycogen content, myeloperoxidase content, phagocytosis activity of Kupffer cells, and the expression and inhibitory phosphorylation as well as kinase activity of GSK-3 were examined. Survival after LPS administration was also determined. RESULTS: LPS induced significant increases of serum TNF-?, alanine aminotransferase, lactate dehydrogenase, and total bilirubin (P < 0.05), which were companied by obvious alterations in liver: the injury of liver tissue, the decrease of glycogen, the infiltration of neutrophils, and the enhancement of phagocytosis of Kupffer cells (P < 0.05). LPS pretreatment significantly attenuated these alterations, promoted the inhibitory phosphorylation of GSK-3 and inhibited its kinase activity, and improved the survival rate (P < 0.05). CONCLUSIONS: ET attenuated LPS-induced acute liver injury through inhibiting GSK-3 functional activity and its downstream signaling.

Gong JH; Gong JP; Li JZ; He K; Li PZ; Jiang XW

2013-10-01

130

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

UK PubMed Central (United Kingdom)

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

Manos-Turvey A; Cergol KM; Salam NK; Bulloch EM; Chi G; Pang A; Britton WJ; West NP; Baker EN; Lott JS; Payne RJ

2012-12-01

131

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

Science.gov (United States)

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

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

2012-10-29

132

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

133

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

Science.gov (United States)

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

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

2012-06-07

134

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

UK PubMed Central (United Kingdom)

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

Chi G; Manos-Turvey A; O'Connor PD; Johnston JM; Evans GL; Baker EN; Payne RJ; Lott JS; Bulloch EM

2012-06-01

135

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

Directory of Open Access Journals (Sweden)

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

2008-01-01

136

Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.  

UK PubMed Central (United Kingdom)

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Renodon A; Boucher JL; Sari MA; Delaforge M; Ouazzani J; Mansuy D

1997-04-01

137

[Effects of glycogen synthase kinase 3? overexpression in rat and glycogen synthase kinase 3? inhibitor SB-216763 on proliferation of hepatic oval cells].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To research the effects of glycogen synthase kinase (GSK3?) overexpression and GSK3? inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway. METHODS: The hepatic oval cells WBF-344 were divided into the blank control group, GSK3? over-expression group, DMSO control group and GSK3? inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3? over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3?, ?-catenin and cyclin D1 were detected by Western blot. RESULTS: The cells of GSK3? over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3? over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3? over-expression group. Western blot showed that the expression of GSK3? was increased, while the expression of ?-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3? had no significant difference among the control group and inhibitor groups. However, the expression of ?-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing. CONCLUSIONS: The overexpression of GSK3? can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Zhong JQ; Xie YK; Ji XK; Fu JH; Wang Y; Zhang QY; Shi HQ; Shan YF

2012-11-01

138

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice.  

UK PubMed Central (United Kingdom)

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of reactive oxygen species generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-knockout mouse strain grew and bred without defect. The general behaviors, blood test results and responses to starvation of the IF1-knockout mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-knockout mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Nakamura J; Fujikawa M; Yoshida M

2013-07-01

139

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice  

Science.gov (United States)

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Nakamura, Junji; Fujikawa, Makoto; Yoshida, Masasuke

2013-01-01

140

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice  

Directory of Open Access Journals (Sweden)

Full Text Available IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Junji Nakamura; Makoto Fujikawa; Masasuke Yoshida

2013-01-01

 
 
 
 
141

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

UK PubMed Central (United Kingdom)

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

Capasso C; Supuran CT

2013-04-01

142

Discovery of novel acetohydroxyacid synthase inhibitors as active agents against Mycobacterium tuberculosis by virtual screening and bioassay.  

UK PubMed Central (United Kingdom)

Acetohydroxyacid synthase (AHAS) has been regarded as a promising drug target against Mycobacterium tuberculosis (MTB) as it catalyzes the biosynthesis of branched-chain amino acids. In this study, 23 novel AHAS inhibitors were identified through molecular docking followed by similarity search. The determined IC(50) values range from 0.385 ± 0.026 ?M to >200 ?M against bacterium AHAS. Five of the identified compounds show significant in vitro activity against H37Rv strains (MICs in the range of 2.5-80 mg/L) and clinical MTB strains, including MDR and XDR isolates. More impressively, compounds 5 and 7 can enhance the killing ability against macrophages infected pathogen remarkably. This study suggests our discovered inhibitors can be further developed as novel anti-MTB therapeutics targeting AHAS.

Wang D; Zhu X; Cui C; Dong M; Jiang H; Li Z; Liu Z; Zhu W; Wang JG

2013-02-01

143

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1986-05-01

144

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.

1986-01-01

145

Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3.  

UK PubMed Central (United Kingdom)

Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3?. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and ?-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of ?-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment.

Lee S; Yang WK; Song JH; Ra YM; Jeong JH; Choe W; Kang I; Kim SS; Ha J

2013-04-01

146

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

International Nuclear Information System (INIS)

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

2007-08-15

147

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

UK PubMed Central (United Kingdom)

Arsenic toxicity has been reported to damage all the major organs including 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 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), 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 & brain GSH levels along with increase in serum & 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.

Sharma B; Sharma PM

2013-08-01

148

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

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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 e...

Caplin, B; Leiper, J

149

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

UK PubMed Central (United Kingdom)

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.

Caberlotto L; Carboni L; Zanderigo F; Andreetta F; Andreoli M; Gentile G; Razzoli M

2013-10-01

150

4- [HETEROCYCLYL-METHYL] -8-FLUORO-QUINOLIN-2-ONES USEFUL AS NITRIC OXIDE SYNTHASE INHIBITORS  

UK PubMed Central (United Kingdom)

Novel compounds of formulae (II, III) and pharmaceutical compositions have been found to inhibit inducible NOS synthase wherein: R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen, lower alkyl, and halogen; and, R8 has the structure whrein X1, X2, X3, X4, X5, X6, R9, R13, R14 and n are as described herein.

SMITH NICHOLAS D; BONNEFOUS CELINE; DURON SERGIO

151

Effects of S-ethylisothiourea, a potent inhibitor of nitric oxide synthase, alone or in combination with a nitric oxide donor in splanchnic artery occlusion shock.  

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1. The aim of this study was to compare the effects of an intravenous infusion of a potent and non selective nitric oxide synthase inhibitor S-ethylisothiourea (Ethyl-TU) with that of a nitric oxide (NO) donor on the pathological sequelae associated with splanchnic artery occlusion (SAO) shock. In a...

Squadrito, F.; Altavilla, D.; Squadrito, G.; Campo, G. M.; Ioculano, M.; Canale, P.; Rossi, F.; Saitta, A.; Caputi, A. P.

152

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

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The exact functional role of nitric oxide (NO) in liver injury is currently a source of controversy. NO is enzymatically synthesized by nitric oxide synthase (NOS). In this study, we assessed the role of inducible NOS (iNOS) in carbon tetrachloride (CCl 4)-induced acute liver injury using inhibitors...

Tipoe, GL; Leung, TM; Liong, E; So, H; Leung, KM; Lau, TYH; Tom, WM; Fung, ML; Fan, ST; Nanji, AA

153

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

UK PubMed Central (United Kingdom)

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.

Ashe KM; Bangari D; Li L; Cabrera-Salazar MA; Bercury SD; Nietupski JB; Cooper CG; Aerts JM; Lee ER; Copeland DP; Cheng SH; Scheule RK; Marshall J

2011-01-01

154

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

UK PubMed Central (United Kingdom)

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

Kapilashrami K; Bommineni GR; Machutta CA; Kim P; Lai CT; Simmerling C; Picart F; Tonge PJ

2013-03-01

155

The nitric oxide synthase inhibitor L-NAME suppresses androgen-induced male-like pseudocopulatory behavior in whiptail lizards.  

Science.gov (United States)

The synthesis of nitric oxide by the enzyme nitric oxide synthase (NOS) is involved in the androgen-dependent gating of male-typical copulatory behavior, both centrally, particularly in the preoptic area, and peripherally, notably through its role in penile erection. In the all-female whiptail lizard species Cnemidophorus uniparens, individuals display copulatory behaviors indistinguishable from males of similar species if gonadectomized and treated with testosterone. In this experiment, androgenized individuals were treated with a NOS inhibitor, which eliminated male-like behavior in half the individuals, suggesting that the central role of nitric oxide synthesis is conserved in this species. The deficit was principally in mounting, suggesting that sexual motivational systems were affected, rather than consummatory mechanisms. PMID:16023092

Sanderson, Nicholas S R; Weissler, Erik; Crews, David

2005-08-01

156

The nitric oxide synthase inhibitor L-NAME suppresses androgen-induced male-like pseudocopulatory behavior in whiptail lizards.  

UK PubMed Central (United Kingdom)

The synthesis of nitric oxide by the enzyme nitric oxide synthase (NOS) is involved in the androgen-dependent gating of male-typical copulatory behavior, both centrally, particularly in the preoptic area, and peripherally, notably through its role in penile erection. In the all-female whiptail lizard species Cnemidophorus uniparens, individuals display copulatory behaviors indistinguishable from males of similar species if gonadectomized and treated with testosterone. In this experiment, androgenized individuals were treated with a NOS inhibitor, which eliminated male-like behavior in half the individuals, suggesting that the central role of nitric oxide synthesis is conserved in this species. The deficit was principally in mounting, suggesting that sexual motivational systems were affected, rather than consummatory mechanisms.

Sanderson NS; Weissler E; Crews D

2005-08-01

157

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

UK PubMed Central (United Kingdom)

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

Vasan M; Neres J; Williams J; Wilson DJ; Teitelbaum AM; Remmel RP; Aldrich CC

2010-12-01

158

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

Science.gov (United States)

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

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

2010-12-01

159

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

2001-01-01

160

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; Haitao Ji; Pavel Martásek; Linda J. Roman; Richard B. Silverman

2009-01-01

 
 
 
 
161

Beneficial effect of CV-4151 (Isbogrel), a thromboxane A2 synthase inhibitor, in a rat middle cerebral artery thrombosis model.  

UK PubMed Central (United Kingdom)

Effects of thromboxane A2 (TXA2) synthase inhibitors (CV-4151 and ozagrel) on cerebral thrombosis and cerebral damage were examined in a rat middle cerebral artery (MCA) thrombosis model and their potencies were compared with the conventional antithrombotic agents, aspirin and ticlopidine. CV-4151 significantly inhibited photochemically induced MCA thrombosis by oral (1 and 10 mg/kg) and intravenous (1 mg/kg) administration. Ozagrel (10 mg/kg, p.o.) also inhibited it. The potency of CV-4151 was about 10 times stronger than that of ozagrel, being comparable with the inhibition of blood TXA2 generation. Aspirin (100 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) showed an inhibitory tendency on MCA thrombosis. Twenty-four h after photochemical stimulation, cerebral edema and cerebral infarction were observed, and the lactate content in the brain increased. CV-4151 and ozagrel prevented this edema, and the antiedema effects of the drugs were correlated with the antithrombotic effect on thrombotic MCA occlusion. CV-4151 (10 mg/kg, p.o.), furthermore, significantly reduced the infarct size and inhibited the increase in lactate content. These results indicate that TXA2 synthase inhibitors inhibit cerebral damage by inhibition of MCA occlusion with thrombosis, probably resulting from the inhibition of TXA2 generation, and their effects are superior to those of aspirin and ticlopidine. TXA2 might play an important role in cerebral damage in the MCA thrombosis model. CV-4151 might be a useful drug for the treatment of cerebral thrombosis and for the prevention of cerebral infarction.

Imura Y; Kiyota Y; Nagai Y; Nishikawa K; Terashita Z

1995-07-01

162

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

Scientific Electronic Library Online (English)

Full Text Available 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-nitrotyrosine (NT) in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NO (more) S, 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.

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

2005-03-01

163

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

Directory of Open Access Journals (Sweden)

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

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

2005-01-01

164

Amide hydrolysis of a novel chemical series of microsomal prostaglandin E synthase-1 inhibitors induces kidney toxicity in the rat.  

UK PubMed Central (United Kingdom)

A novel microsomal prostaglandin E synthase 1 (mPGES-1) inhibitor induced kidney injury at exposures representing less than 4 times the anticipated efficacious exposure in man during a 7-day toxicity study in rats. The findings consisted mainly of tubular lesions and the presence of crystalline material and increases in plasma urea and creatinine. In vitro and in vivo metabolic profiling generated a working hypothesis that a bis-sulfonamide metabolite (determined M1) formed by amide hydrolysis caused this toxicity. To test this hypothesis, rats were subjected to a 7-day study and were administered the suspected metabolite and two low-potency mPGES-1 inhibitor analogs, where amide hydrolysis was undetectable in rat hepatocyte experiments. The results suggested that compounds with a reduced propensity to undergo amide hydrolysis, thus having less ability to form M1, reduced the risk of inducing kidney toxicity. Rats treated with M1 alone showed no histopathologic change in the kidney, which was likely related to underexposure to M1. To circumvent rat kidney toxicity, we identified a potent mPGES-1 inhibitor with a low propensity for amide hydrolysis and superior rat pharmacokinetic properties. A subsequent 14-day rat toxicity study showed that this compound was associated with kidney toxicity at 42, but not 21, times the anticipated efficacious exposure in humans. In conclusion, by including metabolic profiling and exploratory rat toxicity studies, a new and active mPGES-1 inhibitor with improved margins to chemically induced kidney toxicity in rats has been identified.

Bylund J; Annas A; Hellgren D; Bjurström S; Andersson H; Svanhagen A

2013-03-01

165

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

Science.gov (United States)

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

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

166

Effects of ozagrel (OKY-046), a thromboxane synthase inhibitor, on oxidative drug-metabolizing enzymes in mouse hepatic microsomes.  

UK PubMed Central (United Kingdom)

The inhibitory effects of ozagrel (OZA), an imidazole derivative and a specific thromboxane synthase inhibitor, on a monooxygenase system in mouse hepatic microsomes were studied. Pentobarbital sleeping time was significantly prolonged by i.p. administration of a single dose of 100 mg/kg of OZA, and the potency of OZA for the prolongation of sleeping time was similar to that of cimetidine. In vitro, OZA inhibited aminopyrine N-demethylase, aniline hydroxylase and testosterone 6 beta- and 7 alpha-hydroxylase activities in hepatic microsomes with inhibition constants (Ki) of 0.19-3.72 mM. The potency and the mode of inhibition of OZA for these enzyme activities were similar to those of cimetidine, while no inhibitory effect of OZA on testosterone 16 alpha-hydroxylase activity was found. A spectrophotometric study revealed that the imidazole moiety of OZA binds to cytochrome P-450 and has little effect on reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity. These results indicated that OZA is an inhibitor of some cytochrome P-450-mediated drug metabolism in hepatic microsomes.

Morita K; Ono T; Shimakawa H

1988-07-01

167

Effect of JGK-263 as a new glycogen synthase kinase-3? inhibitor on extrinsic apoptosis pathway in motor neuronal cells.  

Science.gov (United States)

Glycogen synthase kinase-3? (GSK-3?) has been identified as one of the important pathogenic mechanisms in motor neuronal death. GSK-3? inhibitor has been investigated as a modulator of apoptosis and has been shown to confer significant protective effects on cell death in neurodegenerative diseases. However, GSK-3? is known to have paradoxical effects on apoptosis subtypes, i.e., pro-apoptotic in mitochondrial-associated intrinsic apoptosis, but anti-apoptotic in death receptor-related extrinsic apoptosis. In this study, we evaluated the effect of a new GSK-3? inhibitor (JGK-263) on motor neuron cell survival and apoptosis, by using low to high doses of JGK-263 after 48h of serum withdrawal, and monitoring changes in extrinsic apoptosis pathway components, including Fas, FasL, cleaved caspase-8, p38?, and the Fas-Daxx interaction. Cell survival peaked after treatment of serum-deprived cells with 50?M JGK-263. The present study showed that treatment with JGK-263 reduced serum-deprivation-induced motor neuronal apoptosis by inactivating not only the intrinsic, but also the extrinsic apoptosis pathway. These results suggest that JGK-263 has a neuroprotective effect through effective modulation of the extrinsic apoptosis pathway in motor neuron degeneration. PMID:23899525

Jeon, Gye Sun; Kim, Jee-Eun; Ahn, Suk-Won; Park, Kyung-Seok; Hong, Yoon-Ho; Ye, In-Hae; Park, Ji-Seon; Kim, Seung Hyun; Lee, Kwang-Woo; Kim, Sung-Min; Sung, Jung-Joon

2013-07-27

168

Effect of JGK-263 as a new glycogen synthase kinase-3? inhibitor on extrinsic apoptosis pathway in motor neuronal cells.  

UK PubMed Central (United Kingdom)

Glycogen synthase kinase-3? (GSK-3?) has been identified as one of the important pathogenic mechanisms in motor neuronal death. GSK-3? inhibitor has been investigated as a modulator of apoptosis and has been shown to confer significant protective effects on cell death in neurodegenerative diseases. However, GSK-3? is known to have paradoxical effects on apoptosis subtypes, i.e., pro-apoptotic in mitochondrial-associated intrinsic apoptosis, but anti-apoptotic in death receptor-related extrinsic apoptosis. In this study, we evaluated the effect of a new GSK-3? inhibitor (JGK-263) on motor neuron cell survival and apoptosis, by using low to high doses of JGK-263 after 48h of serum withdrawal, and monitoring changes in extrinsic apoptosis pathway components, including Fas, FasL, cleaved caspase-8, p38?, and the Fas-Daxx interaction. Cell survival peaked after treatment of serum-deprived cells with 50?M JGK-263. The present study showed that treatment with JGK-263 reduced serum-deprivation-induced motor neuronal apoptosis by inactivating not only the intrinsic, but also the extrinsic apoptosis pathway. These results suggest that JGK-263 has a neuroprotective effect through effective modulation of the extrinsic apoptosis pathway in motor neuron degeneration.

Jeon GS; Kim JE; Ahn SW; Park KS; Hong YH; Ye IH; Park JS; Kim SH; Lee KW; Kim SM; Sung JJ

2013-09-01

169

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

Science.gov (United States)

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

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

2009-12-28

170

Epi-trichosetin, a new undecaprenyl pyrophosphate synthase inhibitor, produced by Fusarium oxysporum FKI-4553.  

UK PubMed Central (United Kingdom)

A new compound, designated epi-trichosetin (1), was isolated along with the known compound trichosetin (2) from the culture broth of Fusarium oxysporum FKI-4553 by solvent extraction, silica gel column chromatography and reversed-phase HPLC. The structure of 1 was elucidated by comparing various spectral data with those of 2, revealing that 1 was a stereoisomer of 2. Compounds 1 and 2 inhibited the undecaprenyl pyrophosphate synthase activity of Staphylococcus aureus with IC50 values of 83 and 30??M, respectively, and showed antimicrobial activity, particularly against Gram-positive bacteria, including methicillin-sensitive and -resistant S. aureus.The Journal of Antibiotics advance online publication, 29 May 2013; doi:10.1038/ja.2013.44.

Inokoshi J; Shigeta N; Fukuda T; Uchida R; Nonaka K; Masuma R; Tomoda H

2013-05-01

171

Inducible nitric oxide synthase inhibitors from Saposhnikovia divaricata and Panax quinquefolium.  

Science.gov (United States)

A series of polyacetylenes, falcarinone, panaxynol, falcarindiol, panaxydol, and panaxytriol, were isolated from Saposhnikovia divaricata (Turcz.) Schischk and Panax quinquefolium L. These polyacetylenes were identified as active principles on the inhibition of nitrite production by inducible nitric oxide synthase (iNOS). Treatment with 10 microM of panaxynol, falcarindiol, panaxydol and panaxytriol decreased the LPS/IFN-gamma-stimulated accumulation of nitrite by 71.92 +/- 3.07, 69.95 +/- 3.68, 45.48 +/- 6.11 and 36.85 +/- 8.80%, respectively. The IC50 value of falcarinone, panaxynol, falcarindiol, panaxydol and panaxytriol was > 20, 2.23, 1.98, 6.58 and 9.85 microM, respectively. PMID:11105571

Wang, C N; Shiao, Y J; Kuo, Y H; Chen, C C; Lin, Y L

2000-10-01

172

Inducible nitric oxide synthase inhibitors from Saposhnikovia divaricata and Panax quinquefolium.  

UK PubMed Central (United Kingdom)

A series of polyacetylenes, falcarinone, panaxynol, falcarindiol, panaxydol, and panaxytriol, were isolated from Saposhnikovia divaricata (Turcz.) Schischk and Panax quinquefolium L. These polyacetylenes were identified as active principles on the inhibition of nitrite production by inducible nitric oxide synthase (iNOS). Treatment with 10 microM of panaxynol, falcarindiol, panaxydol and panaxytriol decreased the LPS/IFN-gamma-stimulated accumulation of nitrite by 71.92 +/- 3.07, 69.95 +/- 3.68, 45.48 +/- 6.11 and 36.85 +/- 8.80%, respectively. The IC50 value of falcarinone, panaxynol, falcarindiol, panaxydol and panaxytriol was > 20, 2.23, 1.98, 6.58 and 9.85 microM, respectively.

Wang CN; Shiao YJ; Kuo YH; Chen CC; Lin YL

2000-10-01

173

Endothelium-dependent hyperpolarization in isolated arteries taken from animals treated with NO-synthase inhibitors.  

Science.gov (United States)

To study the effects of chronic in vivo inhibition of NO synthase on endothelium-dependent hyperpolarization, cell-membrane potential (in individual vascular smooth-muscle cells) and changes in tension (in isolated rings) were recorded from isolated canine coronary arteries and guinea-pig carotid arteries and aortas. In coronary arteries taken from control dogs and contracted with U46619, acetylcholine- and bradykinin-induced endothelium-dependent relaxations, which were unaffected by short-term in vitro exposure to indomethacin but were inhibited partially by L-nitro-arginine (LNA). In coronary arteries taken from dogs treated over the long term in vivo with LNA (30 mg/kg on the first day and 20 mg/kg the 7 following days, i.v.), the response to acetylcholine and bradykinin was inhibited when compared with arteries from control dogs. Short-term in vitro exposure to LNA or indomethacin or both did not influence the effects of either agonist. In these arteries, the hyperpolarizing response to acetylcholine, observed in the presence of LNA and indomethacin, was enhanced, whereas that to bradykinin was partially inhibited. In the guinea pig isolated aorta, the relaxation to bradykinin was abolished by long-term in vivo treatment with L-nitro-arginine-methyl-ester (L-NAME; 1.5 mg/ml, in the drinking water for > or =4 days). In the isolated guinea pig carotid artery studied in the presence of LNA and indomethacin, acetylcholine induced a hyperpolarization that was not significantly affected by long-term in vivo treatment with L-NAME. These findings indicate that endothelium-dependent hyperpolarizations are maintained during long-term inhibition of NO synthase and probably act as a back-up mechanism to elicit endothelium-dependent relaxations. PMID:9869500

Corriu, C; Félétou, M; Puybasset, L; Bea, M L; Berdeaux, A; Vanhoutte, P M

1998-12-01

174

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

UK PubMed Central (United Kingdom)

BACKGROUND: The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide-dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. METHODS AND RESULTS: Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography-mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg(-1) · min(-1)) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 ?mol · kg(-1) · d(-1)) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO(-/-) mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. CONCLUSIONS: ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.

von Leitner EC; Klinke A; Atzler D; Slocum JL; Lund N; Kielstein JT; Maas R; Schmidt-Haupt R; Pekarova M; Hellwinkel O; Tsikas D; D'Alecy LG; Lau D; Willems S; Kubala L; Ehmke H; Meinertz T; Blankenberg S; Schwedhelm E; Gadegbeku CA; Böger RH; Baldus S; Sydow K

2011-12-01

175

Decreased spontaneous motor activity and startle response in nitric oxide synthase inhibitor-treated rats.  

Science.gov (United States)

In the central nervous system, nitric oxide has been proposed to be a retrograde messenger mediating learning and synaptic plasticity. Since only pretraining injections of nitric oxide synthesis inhibitors were shown to impair learning, we examined the possibility that systemic administration of these inhibitors might influence some non-specific aspects related to the organism's general psychophysiological status. Intraperitoneal administration of NG-nitro-L-arginine methyl ester (30 or 100 mg/kg) 60 min pre-test to adult rats resulted in: (i) altered exploratory pattern and reduced locomotion in a novel environment; (ii) reduced startle response to either acoustic or electric stimuli; and (iii) cardiovascular alterations. In addition, intracerebroventricular administration of N-nitro-L-arginine (10 microliters of a 10 mM solution) diminished the acoustic startle response. Specificity of these effects through nitric oxide was supported by the ability of the nitric oxide precursor, L-arginine, to prevent the inhibitors actions. These findings indicate that nitric oxide inhibitors interfere with the general psychophysiological status of the organism. PMID:7543413

Sandi, C; Venero, C; Guaza, C

1995-04-13

176

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

International Nuclear Information System (INIS)

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

2004-10-01

177

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

UK PubMed Central (United Kingdom)

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

Tomishima Y; Ishitsuka Y; Matsunaga N; Nagatome M; Furusho H; Irikura M; Ohdo S; Irie T

2013-01-01

178

Thromboxane A(2) synthase inhibitor enhanced antithrombotic efficacy of GPIIb-IIIa receptor antagonist without increasing bleeding.  

UK PubMed Central (United Kingdom)

The advantage of platelet integrin GPIIb-IIIa receptor antagonists in the prevention of thrombotic occlusion was clearly proven in patients who underwent interventional treatment of the coronary artery, but its value in cerebral ischemia is still under investigation. The expectation of intracranial hemorrhage on strong inhibition of platelet function restricts its application in cerebral ischemia. To minimize bleeding while keeping antithrombotic activity, we have tried to find an appropriate approach using a combination of platelet integrin GPIIb-IIIa receptor antagonist and some other antithrombotic agents. The time to thrombotic occlusion was measured using a photothrombotic occlusion model of guinea pig middle cerebral artery. A platelet integrin GPIIb-IIIa receptor antagonist, ME3277 (sodium hydrogen [4-[(4,5,6,7-tetrahydrothieno [3,2-c] pyridin-2-yl) carbonylamino] acetyl-o-phenylene] dioxydiacetate), delayed occlusion time from 7.3 min in vehicle to 15.0, 20.6 and 25.9 min (P<0.05) at 0.1, 0.3 and 1 mg/kg, respectively. ME3277 profoundly inhibited ex vivo platelet aggregation and the highest dose of ME3277 prolonged (3.5 folds, P<0.01) the bleeding time measured in the hind paw. A thromboxane A(2) synthase inhibitor, sodium ozagrel, significantly delayed occlusion time to 19.5 min at 30 mg/kg (P<0.05) while it did not affect bleeding time or platelet aggregation. ME3277 (0.1 mg/kg) in combination with 10 mg/kg sodium ozagrel synergistically delayed occlusion time (sodium ozagrel alone; 7.9 min, combination; 26.1 min, P<0.05 vs. ME3277 alone). Sodium ozagrel did not affect ex vivo platelet aggregation or bleeding time when combined with 0.1 mg/kg of ME3277. This synergy was cancelled by combination with 30 mg/kg aspirin (14.7 min). A thromboxane A(2) receptor antagonist, vapiprost (0.1 mg/kg), did not enhance the antithrombotic efficacy of ME3277. These results imply that local prostacyclin production enhances the in vivo antithrombotic effect of the platelet integrin GPIIb-IIIa receptor antagonist. Therefore, the thromboxane A(2) synthase inhibitor allowed a reduction in the dose level of the platelet integrin GPIIb-IIIa receptor antagonist for cerebral thrombosis, which resulted in a reduced risk of bleeding.

Kawano KI; Hokamura K; Kondo K; Ikeda Y; Suzuki Y; Umemura K

2001-04-01

179

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

Directory of Open Access Journals (Sweden)

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

Tomishima Yoshiro; Ishitsuka Yoichi; Matsunaga Naoya; Nagatome Minako; Furusho Hirokazu; Irikura Mitsuru; Ohdo Shigehiro; Irie Tetsumi

2013-01-01

180

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

Science.gov (United States)

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

2013-01-01

 
 
 
 
181

Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor.  

UK PubMed Central (United Kingdom)

The inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are overexpressed in colonic tumors of humans, as well as in colon tumors that develop in rats after the administration of the colon-specific carcinogen, azoxymethane (AOM). iNOS may regulate COX-2 production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to assess the potential chemopreventive properties of highly selective iNOS inhibitors, administered individually and in combination with a selective COX-2 inhibitor, on the development of AOM-induced colonic aberrant crypt foci (ACF). F344 rats were fed experimental diets containing one of the following: 0, 10, 30, or 100 parts/million (ppm) of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); 1800 ppm of the less potent, selective iNOS inhibitor aminoguanidine (AG); 500 ppm of the COX-2 inhibitor celecoxib; 320 ppm of the nonsteroidal anti-inflammatory sulindac (positive control); or 30 ppm of SC-51 with 500 ppm of celecoxib, and 100 ppm of SC-51 with 500 ppm of celecoxib. One and 2 weeks later, rats received s.c. injections of AOM at a dose of 15 mg/kg of body weight. At 17 weeks of age, all rats were sacrificed. Colons were evaluated for ACF, and colonic mucosae were assayed for COX and NOS isoform enzyme activities. Samples of venous blood, collected at various time points, were analyzed for these agents. SC-51, administered alone, demonstrated dose-dependent inhibition of the incidence of colonic ACF. The highest doses of SC-51 (100 ppm) and AG (1800 ppm) significantly suppressed the incidence of colonic ACF (P < 0.01 and < 0.001, respectively) and crypt multiplicity in terms of numbers of aberrant crypts/focus (P < 0.0001). Importantly, the combination of either low or high effective doses of SC-51 (30 or 100 ppm) and celecoxib (500 ppm) suppressed AOM-induced colonic ACF formation (P < 0.05 and < 0.001, respectively) and reduced multiplicity of four or more aberrant crypts/focus (P < 0.0001) to a greater extent than did these agents administered individually. As expected, sulindac inhibited colonic ACF formation (P < 0.001) and reduced the multiplicity of four or more aberrant crypts (P < 0.0001) to approximately 45%. The enzymatic activities of COX-2 and iNOS were significantly induced in the AOM-treated animals, and administration of the iNOS inhibitors, SC-51 and AG, significantly inhibited the activities of both iNOS and COX-2 in the colonic mucosa. The combined administration of SC-51 and celecoxib inhibited the COX-2 activity to a greater extent than did either of these agents administered alone. These findings support the hypothesis that selective iNOS inhibitors may have chemopreventive properties and that coadministration with a selective COX-2 inhibitor may have additional chemopreventive potential.

Rao CV; Indranie C; Simi B; Manning PT; Connor JR; Reddy BS

2002-01-01

182

Counteraction by Nitric Oxide Synthase Inhibitor of Neurochemical Alterations of Dopaminergic System in 6-OHDA-Lesioned Rats Under L-DOPA Treatment.  

UK PubMed Central (United Kingdom)

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

Del-Bel E; Padovan-Neto FE; Szawka RE; da-Silva CA; Raisman-Vozari R; Anselmo-Franci J; Romano-Dutra AC; Guimaraes FS

2013-06-01

183

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

UK PubMed Central (United Kingdom)

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.

Szlachcic A; Krzysiek-Maczka G; Pajdo R; Targosz A; Magierowski M; Jasnos K; Drozdowicz D; Kwiecien S; Brzozowski T

2013-01-01

184

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

UK PubMed Central (United Kingdom)

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

Leung CY; Langille AM; Mancuso J; Tsantrizos YS

2013-04-01

185

Plasma levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, are elevated in sickle cell disease.  

Science.gov (United States)

In recent years an important role has been ascribed to a reduced nitric oxide (NO) availability in the pathophysiology of sickle cell disease (SCD). Endogenously produced inhibitors of NO synthase, in particular asymmetric dimethylarginine (ADMA), are currently considered of importance in various vascular disease states characterized by reduced NO availability. We determined ADMA levels in plasma of 12 adult sickle cell patients (eight HbSS and four HbSC), and compared these to plasma levels in race- and age-matched controls. Sickle cell patients were characterized by strongly elevated levels of ADMA [HbSS: median 0.63 micromol/l (interquartile range 0.54-0.85), HbSC: 0.43 micromol/l (0.40-0.46), HbAA: 0.33 micromol/l (0.32-0.35) p<0.001]. ADMA levels were highest in HbSS patients with lowest hemoglobin levels and highest leukocyte counts, and in HbSS patients ADMA levels were positively associated with serum levels of soluble vascular cell adhesion molecule-1. These results suggest an important role of ADMA in limiting NO availability in SCD, and its role in the pathophysiology of SCD should be further investigated. PMID:15599544

Schnog, J B; Teerlink, T; van der Dijs, F P L; Duits, A J; Muskiet, F A J

2004-12-14

186

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

Science.gov (United States)

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

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

2006-11-29

187

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1).  

UK PubMed Central (United Kingdom)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.

Bernert B; Porsch H; Heldin P

2011-12-01

188

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1).  

Science.gov (United States)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion. PMID:22016393

Bernert, Berit; Porsch, Helena; Heldin, Paraskevi

2011-10-20

189

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.

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

190

An evaluation of thymidine phosphorylase as a means of preventing thymidine rescue from the thymidylate synthase inhibitor raltitrexed.  

UK PubMed Central (United Kingdom)

The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.

Graham-Cole CL; Thomas HD; Taylor GA; Newell DR; Melton RG; Hesp R; Boddy AV

2007-02-01

191

Immediate force loss after eccentric contractions is increased with L-NAME administration, a nitric oxide synthase inhibitor.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Nitric oxide (NO) signaling regulates many biological processes in skeletal muscle, wherein aberrant signaling contributes to myopathic conditions (e.g., Duchenne muscular dystrophy). NO has been shown to play a role in muscle regeneration after injury. However, less is known about its role during injury. In this study we aimed to determine whether NO synthase (NOS) inhibition exacerbates functional deficits immediately after the performance of eccentric contractions. METHODS: Wild-type mouse extensor digitorum longus (EDL) muscles underwent in vitro functional testing in the presence or absence of a non-specific NOS inhibitor (L-NAME, 10 mM) before and after performance of 10 eccentric contractions. RESULTS: After eccentric contractions, P(o) was reduced by ?25% for muscle in regular physiological solution but by ?50% with the addition of L-NAME (P = 0.009). CONCLUSIONS: Non-specific blockade of NOS exacerbates functional deficits immediately after eccentric contractions, suggesting that NO signaling protects skeletal muscle from excessive injury in healthy muscle.

Corona BT; Ingalls CP

2013-02-01

192

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 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 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.

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

2012-01-01

193

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

UK PubMed Central (United Kingdom)

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

Salykina MA; Sorokina EG; Krasilnikova IA; Reutov VP; Pinelis VG

2013-05-01

194

Enhanced antitumor activity for the thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid.  

Science.gov (United States)

The purpose of this investigation was to determine whether antitumor selectivity of the third generation thymidylate synthase inhibitor 1843U89 could be enhanced by a combination of the drug with folic acid. The effects of folic acid on toxicity of 1843U89 to the dog and mouse and on antitumor efficacy of 1843U89 in the mouse were studied. These data were compared to the effect of folic acid on the in vitro cell culture antitumor activity of 1843U89. The sensitivity of eight cancer cell lines (three ovarian, one colon, one ileocecal, one epidermoid, one osteosarcoma, and one breast line) to 1843U89 was tested in vitro in the presence and absence of folic acid. Folic acid concentrations greater than 100 microM were required to decrease 1843U89 activity in seven of the cell lines. Only the activity in HCT-8, the ileocecal line, was reserved at folic acid concentrations below 100 microM. Oral folic acid given 30 min prior to an i.v. dose of 1843U89 increased the maximally tolerated dose and the lethal dose of 1843U89, both in dogs and in thymidine-depleted mice. In mice, oral folic acid produced little or no effect upon the antitumor efficacy of 1843U89 in two of three tumor cell lines in vivo. HCT-8, the line that was sensitive to folate reversal in vitro, was also sensitive in vivo. The results show that an oral dose of folic acid 30 min prior to i.v. 1843U89 can block mouse and dog intestinal toxicity without decreasing efficacy of 1843U89 in two of three human tumor lines in the nude mouse. Thus, the data reported here indicate that the antitumor selectivity of 1843U89 may be enhanced through a combination of 1843U89 with oral folic acid. PMID:8521402

Smith, G K; Amyx, H; Boytos, C M; Duch, D S; Ferone, R; Wilson, H R

1995-12-15

195

Enhanced antitumor activity for the thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid.  

UK PubMed Central (United Kingdom)

The purpose of this investigation was to determine whether antitumor selectivity of the third generation thymidylate synthase inhibitor 1843U89 could be enhanced by a combination of the drug with folic acid. The effects of folic acid on toxicity of 1843U89 to the dog and mouse and on antitumor efficacy of 1843U89 in the mouse were studied. These data were compared to the effect of folic acid on the in vitro cell culture antitumor activity of 1843U89. The sensitivity of eight cancer cell lines (three ovarian, one colon, one ileocecal, one epidermoid, one osteosarcoma, and one breast line) to 1843U89 was tested in vitro in the presence and absence of folic acid. Folic acid concentrations greater than 100 microM were required to decrease 1843U89 activity in seven of the cell lines. Only the activity in HCT-8, the ileocecal line, was reserved at folic acid concentrations below 100 microM. Oral folic acid given 30 min prior to an i.v. dose of 1843U89 increased the maximally tolerated dose and the lethal dose of 1843U89, both in dogs and in thymidine-depleted mice. In mice, oral folic acid produced little or no effect upon the antitumor efficacy of 1843U89 in two of three tumor cell lines in vivo. HCT-8, the line that was sensitive to folate reversal in vitro, was also sensitive in vivo. The results show that an oral dose of folic acid 30 min prior to i.v. 1843U89 can block mouse and dog intestinal toxicity without decreasing efficacy of 1843U89 in two of three human tumor lines in the nude mouse. Thus, the data reported here indicate that the antitumor selectivity of 1843U89 may be enhanced through a combination of 1843U89 with oral folic acid.

Smith GK; Amyx H; Boytos CM; Duch DS; Ferone R; Wilson HR

1995-12-01

196

Induction of hypoxia in experimental murine tumors by the nitric oxide synthase inhibitor, NG-nitro-L-arginine.  

Science.gov (United States)

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis. PMID:7987843

Wood, P J; Sansom, J M; Butler, S A; Stratford, I J; Cole, S M; Szabo, C; Thiemermann, C; Adams, G E

1994-12-15

197

Induction of hypoxia in experimental murine tumors by the nitric oxide synthase inhibitor, NG-nitro-L-arginine.  

UK PubMed Central (United Kingdom)

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis.

Wood PJ; Sansom JM; Butler SA; Stratford IJ; Cole SM; Szabo C; Thiemermann C; Adams GE

1994-12-01

198

Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.  

Science.gov (United States)

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target. PMID:20669893

Pagnozzi, Daniela; Birolo, Leila; Leo, Gabriella; Contessi, Stefania; Lippe, Giovanna; Pucci, Pietro; Mavelli, Irene

2010-09-01

199

Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.  

UK PubMed Central (United Kingdom)

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.

Pagnozzi D; Birolo L; Leo G; Contessi S; Lippe G; Pucci P; Mavelli I

2010-09-01

200

INTEGRATION OF A POLYNUCLEOTIDE ENCODING A POLYPEPTIDE THAT CATALYZES PYRUVATE TO ACETOLACTATE CONVERSION  

UK PubMed Central (United Kingdom)

The invention relates to recombinant host cells having at least one integrated polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactate conversion. The invention also relates to methods of increasing the biosynthetic production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone using such host cells.

ANTHONY LARRY CAMERON; MAGGIO-HALL LORI ANN; PAUL BRIAN JAMES

 
 
 
 
201

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

UK PubMed Central (United Kingdom)

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

Zabalza A; Zulet A; Gil-Monreal M; Igal M; Royuela M

2013-06-01

202

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.

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

203

Inducible nitric oxide synthase inhibitor aminoguanidine, differently affects Morris water maze tasks of ovariectomized and naive female rats.  

UK PubMed Central (United Kingdom)

The role of ovarian hormones, nitric oxide, and their interaction on learning and memory has been widely investigated. The objective of present study was to investigate different effects of chronic administration of inducible nitric oxide synthase inhibitor, aminoguanidine (AM) on learning and memory of ovariectomized (OVX) and naïve (Sham) female rats. Thirty-two rats were divided into four groups: 1) Sham, 2) OVX, 3) Sham-AM and 4) OVX-AM. The animals of Sham-AM and OVX-AM chronically received 100 mg/kg/day of aminoguanidine during 8 weeks before 5 test days. The animals in Sham and OVX groups received 1 ml/kg saline instead of aminoguanidine. The animals were tested in Morris water maze and the escape latency and traveled path to reach the platform were compared between groups. On the fifth day, the platform was removed, and the animals were allowed to swim for 60 s ( prob trial). The time spent in the target quadrant (Q1) was compared between groups.Results showed that the escape latency and traveled path in OVX group were significantly higher than in the Sham group (p<0.01). Both escape latency and traveled path in the Sham-AM group was significantly higher than in the Sham group (p<0.01) however, there was no significant difference between OVX-AM and OVX groups.The time spent by the animals of OVX group in the target quadrant (Q1) during the probe trial was significantly lower than that in the Sham group (p<0.01). The animals of the Sham-AM group spent shorter times in the target quadrant in comparison with the Sham group (p<0.01). There was no significant difference between the OVX and OVX-AM groups in the time spent in tarthe get quadrant. It is concluded that the effect of aminoguanidine on learning and memory is different in the presence or absence of ovarian hormones but it needs further investigation.

Hosseini M; Nemati Karimooy HA; Hadjzadeh MA; Safari V

2011-12-01

204

Multivariate SAR/QSAR of 3-aryl-4-hydroxyquinolin-2(1H)-one derivatives as type I fatty acid synthase (FAS) inhibitors.  

UK PubMed Central (United Kingdom)

Two multivariate studies, a PCA-SAR and a PLS-QSAR, of 3-aryl-4-hydroxyquinolin-2(1H)-one derivatives described as type I fatty acid synthase (FAS) inhibitors, are presented in this work. The variable selection was performed with the Fisher's weight and Ordered Predictors Selection (OPS) algorithm, respectively. In the PCA, a separation between active and inactive compounds was obtained by six descriptors (topological and geometrical). The PLS model presented five descriptors and two Latent Variables. Leave-N-out cross validation and y-randomization test showed that the model presented robustness and no chance correlation, respectively, and the descriptors indicated that the FAS inhibition depends on electronic distribution of the investigated compounds. The model obtained in this study may provide a guidance for proposition of new FAS inhibitors.

de Melo EB

2010-12-01

205

Design, synthesis and biological evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3? (GSK-3?).  

UK PubMed Central (United Kingdom)

Glycogen synthase kinase-3? (GSK-3?) plays a key role in type II diabetes and Alzheimer's diseases, to which non-ATP competitive inhibitors represent an effectively therapeutical approach due to their good specificity. Herein, a series of small molecules benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of GSK-3? have been designed and synthesized. The in vitro evaluation performed by luminescent assay showed most BTZ derivatives have inhibitory effects in micromolar scale. Among them compounds 6l, 6t and 6v have the IC50 values of 25.0 ?M, 27.8 ?M and 23.0 ?M, respectively. Moreover 6v is devoid of any inhibitory activity in the assays to other thirteen protein kinases. Besides, SAR is analyzed and a hypothetical enzymatic binding mode is proposed by molecular docking study, which would be useful for new candidates design.

Zhang P; Hu HR; Bian SH; Huang ZH; Chu Y; Ye DY

2013-03-01

206

A novel lumazine synthase inhibitor derived from oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine to a tetraazaperylenehexaone derivative.  

Science.gov (United States)

Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66+/-13 microM in Tris buffer and 22+/-4 microM in phosphate buffer. This is significantly more potent than the reactant (Ki 350+/-76 microM, competitive inhibition), which had previously been identified as a lumazine synthase inhibitor by high-throughput screening. Ab initio calculations indicate that the meso form is slightly less stable than the enantiomeric form, and that the two forms interconvert rapidly at room temperature. PMID:17348709

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

2007-03-10

207

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

208

Chemical marker for ALS-inhibitor herbicides: 2-aminobutyric acid proportional in sub-lethal applications.  

Science.gov (United States)

A chemical profiling technique for sub-lethal acetolactate synthase (ALS)-inhibitor herbicides (e.g., sulfonylureas, imidazolines, triazolopyrimidine sulfonanilides, and pyrimidyloxy salicylic) was developed using 2-aminobutyric acid, and was found to be directly proportional to application rates in field studies on two varieties of potato plants. An uncomplicated, benign-by-design analytical method for the determination of 2-aminobutyric acid in plant tissue was developed. The method is simple, fast, and automated, entailing a water-trichloroacetic acid extraction followed by precolumn on-line derivatization using o-phthalaldehyde (OPA) solution and liquid chromatographic analyses. Use of reagents and chlorinated organic solvents, and generation of waste, are minimized as compared to other ALS-inhibitor herbicide analytical techniques. Recoveries for a series of fortified plant tissues ranged from 82 to 103%. Two 20-day field trials on two potato varieties, Russet Burbank and Shepody, were conducted during the 2000 and 2001 growing seasons. The study demonstrated that the 2-aminobutyric acid method is an excellent, selective chemical marker technique for ALS-inhibitor herbicides for real world plant matrixes. PMID:11958629

Loper, Bob R; Cobb, William T; Anderson, Kim A

2002-04-24

209

3-Aryl-4-hydroxyquinolin-2(1H)-one derivatives as type I fatty acid synthase inhibitors.  

UK PubMed Central (United Kingdom)

A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).

Rivkin A; Kim YR; Goulet MT; Bays N; Hill AD; Kariv I; Krauss S; Ginanni N; Strack PR; Kohl NE; Chung CC; Varnerin JP; Goudreau PN; Chang A; Tota MR; Munoz B

2006-09-01

210

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

UK PubMed Central (United Kingdom)

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.

Arooj M; Sakkiah S; Cao Gp; Lee KW

2013-01-01

211

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

UK PubMed Central (United Kingdom)

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.

Zhao G; Haskins N; Jin Z; M Allewell N; Tuchman M; Shi D

2013-08-01

212

Molecular dynamic simulation and inhibitor prediction of cysteine synthase structured model as a potential drug target for trichomoniasis.  

UK PubMed Central (United Kingdom)

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10?ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was -0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (-13.0?Kcal/Mol) and a log?P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection.

Singh S; Sablok G; Farmer R; Singh AK; Gautam B; Kumar S

2013-01-01

213

A novel role of andrographolide, an NF-kappa B inhibitor, on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP.  

Science.gov (United States)

Andrographolide is a novel NF-?B inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25-75 ??) exhibited a more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca(2+) mobilization; thromboxane A(2) formation; and phospholipase C (PLC)?2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless, a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH([Symbol: see text])) formation. In vivo studies revealed that andrographolide (22 and 55 ?g/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism and significantly prolonged platelet plug formation in mice. This study demonstrates for the first time that andrographolide possesses a novel role of antiplatelet activity, which may involve the activation of the eNOS-NO/cyclic GMP pathway, resulting in the inhibition of the PI3 kinase/Akt-p38 MAPK and PLC?2-PKC cascades, thereby leading to inhibition of platelet aggregation. PMID:21822619

Lu, Wan-Jung; Lee, Jie-Jen; Chou, Duen-Suey; Jayakumar, Thanasekaran; Fong, Tsorng-Han; Hsiao, George; Sheu, Joen-Rong

2011-08-06

214

6-PYRIDIN-3-YL-3,4-DIHYDRO-1H-QUINOLIN-2-ONE DERIVATIVES AND RELATED COMPOUNDS AS INHIBITORS OF THE HUMAN ALDOSTERONE SYNTHASE CYP11B2  

UK PubMed Central (United Kingdom)

The invention provides compounds of the general formula (I) which are inhibitors of the human aldosterone synthase, and also pharmaceutical compositions containing these compounds, and the use of these compounds and other heteroaryl substituted quinolinone derivatives for the treatment of hyperaldosteronism and/or disorders or diseases that are mediated by 11 ss-hydroxylase (CYP11 B1 ).

HARTMANN ROLF W; HEIM RALF; LUCAS SIMON

215

6-Pyridin-3-YL-3,4-Dihydro-1H-Quinolin-2-One Derivatives and Related Compounds as Inhibitors of the Human Aldosterone Synthase CYP11B2  

UK PubMed Central (United Kingdom)

The invention provides compounds of the general formula (I) which are inhibitors of the human aldosterone synthase, and also pharmaceutical compositions containing these compounds, and the use of these compounds and other heteroaryl substituted quinolinone derivatives for the treatment of hyperaldosteronism and/or disorders or diseases that are mediated by 11 [beta]-hydroxylase (CYP11 B1).

HARTMANN ROLF W; HEIM RALF; LUCAS SIMON

216

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

UK PubMed Central (United Kingdom)

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

Chen CN; Lv LL; Ji FQ; Chen Q; Xu H; Niu CW; Xi Z; Yang GF

2009-04-01

217

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

Science.gov (United States)

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

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

2009-03-14

218

Feeding the nitric oxide synthase inhibitor L-N(omega)nitroarginine elevates serum very low density lipoprotein and hepatic triglyceride synthesis in rats.  

UK PubMed Central (United Kingdom)

This study was conducted to study the influence of dietary L-N(omega)nitroarginine (L-NNA), a nitric oxide (NO) synthase inhibitor, on serum lipids and lipoproteins and on the activities of enzymes related to lipid metabolism in rats. Feeding rats a diet containing 0.2 g/kg L-NNA for 5 weeks elevated serum concentrations of triglyceride, cholesterol, phospholipid, and free fatty acid and reduced serum nitrate (an oxidation product of NO). The elevation in serum triglyceride was mainly due to the elevation in very low density lipoprotein (VLDL) triglyceride. Contents of cholesterol and phospholipid in the VLDL fraction also were elevated by L-NNA. L-NNA treatment caused significantly higher activity of hepatic microsomal phosphatidate phosphohydrolase (the rate-limiting enzyme in triglyceride synthesis) and lower activity of hepatic carnitine palmitoyltransferase (the rate-limiting enzyme in fatty acid oxidation). Activities of hepatic enzymes responsible for fatty acid synthesis such as glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase were unaffected by L-NNA. The activity of hepatic microsomal phosphocholine cytidyltransferase (the rate-limiting enzyme in phosphatidylcholine synthesis) was reduced significantly by L-NNA. Our results suggest that lower NO production caused the elevations in hepatic triglyceride synthesis by higher esterification of fatty acid and lower fatty acid oxidation, leading to an enrichment of VLDL triglyceride.

Goto T; Ohnomi S; Khedara A; Kato N; Ogawa H; Yanagita T

1999-05-01

219

Feeding the nitric oxide synthase inhibitor L-N(omega)nitroarginine elevates serum very low density lipoprotein and hepatic triglyceride synthesis in rats.  

Science.gov (United States)

This study was conducted to study the influence of dietary L-N(omega)nitroarginine (L-NNA), a nitric oxide (NO) synthase inhibitor, on serum lipids and lipoproteins and on the activities of enzymes related to lipid metabolism in rats. Feeding rats a diet containing 0.2 g/kg L-NNA for 5 weeks elevated serum concentrations of triglyceride, cholesterol, phospholipid, and free fatty acid and reduced serum nitrate (an oxidation product of NO). The elevation in serum triglyceride was mainly due to the elevation in very low density lipoprotein (VLDL) triglyceride. Contents of cholesterol and phospholipid in the VLDL fraction also were elevated by L-NNA. L-NNA treatment caused significantly higher activity of hepatic microsomal phosphatidate phosphohydrolase (the rate-limiting enzyme in triglyceride synthesis) and lower activity of hepatic carnitine palmitoyltransferase (the rate-limiting enzyme in fatty acid oxidation). Activities of hepatic enzymes responsible for fatty acid synthesis such as glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase were unaffected by L-NNA. The activity of hepatic microsomal phosphocholine cytidyltransferase (the rate-limiting enzyme in phosphatidylcholine synthesis) was reduced significantly by L-NNA. Our results suggest that lower NO production caused the elevations in hepatic triglyceride synthesis by higher esterification of fatty acid and lower fatty acid oxidation, leading to an enrichment of VLDL triglyceride. PMID:15539300

Goto, T; Ohnomi, S; Khedara, A; Kato, N; Ogawa, H; Yanagita, T

1999-05-01

220

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma, epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase.  

UK PubMed Central (United Kingdom)

The location of the endogenous inhibitor protein (IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of alpha-IF1, IF1-IF1, gamma-IF1, and epsilon-IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance < or = 12 angstroms to gamma and epsilon subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic beta subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

Minauro-Sanmiguel F; Bravo C; García JJ

2002-12-01

 
 
 
 
221

Effect of N1-guanyl-1,7-diaminoheptane, an inhibitor of deoxyhypusine synthase, on endothelial cell growth, differentiation and apoptosis.  

UK PubMed Central (United Kingdom)

An unusual amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A (eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although eIF5A and its hypusine modification are essential for eukaryotic cell viability, the true physiological function of eIF5A is yet unknown. We have examined the effects of N1-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of deoxyhypusine synthase, on endothelial cell proliferation, differentiation and apoptosis. Upon treatment of human umbilical vein endothelial cells (HUVEC) with GC7, dose-dependent inhibition of hypusine formation and cellular proliferation was observed. GC7 at 10 microM caused almost complete inhibition of cellular hypusine synthesis and led to cytostasis of HUVEC. Pretreatment of HUVEC with GC7 up to 50 microM for 4 days had little effect on the attachment and differentiation of these cells on Matri-gel and did not cause induction of apoptosis. Instead, the GC7 pretreatment (96 h at 5-50 microM) elicited protective effects against apoptotic death of HUVEC induced by serum starvation. These results suggest that eIF-5A may be involved in expression of proteins essential for apoptosis of endothelial cells as well as those for cellular proliferation.

Lee Y; Kim HK; Park HE; Park MH; Joe YA

2002-08-01

222

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

Science.gov (United States)

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

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

2008-07-01

223

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

UK PubMed Central (United Kingdom)

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

Landburg PP; Teerlink T; Muskiet FA; Duits AJ; Schnog JJ

2008-07-01

224

Efficacy of FK463, a (1,3)-?-d-Glucan Synthase Inhibitor, in Disseminated Azole-Resistant Candida albicans Infection in Mice  

Science.gov (United States)

The efficacy of FK463, a new (1,3)-?-d-glucan synthase inhibitor, against azole-resistant Candida albicans strains has been studied. The MIC of FK463 was lower than those of azoles and amphotericin B against CDR1-expressing C26 and CaMDR-expressing C40 strains. All mice treated with FK463 (1 mg/kg) survived disseminated murine candidiasis. The fungal burden in the kidney after 6 days was markedly reduced after therapy with FK463 and amphotericin B sodium deoxycholate, and plasma (1,3)-?-d-glucan concentration was found to be lower in FK463-treated mice. In our study, FK463 was found to be a potent antifungal agent against disseminated infection with azole-resistant C. albicans.

Maesaki, Shigefumi; Hossain, Mohammad Ashraf; Miyazaki, Yoshitsugu; Tomono, Kazunori; Tashiro, Takayoshi; Kohno, Shigeru

2000-01-01

225

In vitro activity of a new oral glucan synthase inhibitor (MK-3118) tested against Aspergillus spp. by CLSI and EUCAST broth microdilution methods.  

UK PubMed Central (United Kingdom)

MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ? 4 ?g/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC(90))/MIC(90) values (?g/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fumigatus SC; 0.12, 2, and 0.06 for Aspergillus terreus SC; and 0.06, 1, and 0.03 for Aspergillus niger SC. Essential agreement between the values found by CLSI and EUCAST (± 2 log(2) dilution steps) was 94.3%. MK-3118 was determined to be a potent agent regardless of the in vitro method applied, with excellent activity against contemporary wild-type and itraconazole-resistant strains of Aspergillus spp.

Pfaller MA; Messer SA; Motyl MR; Jones RN; Castanheira M

2013-02-01

226

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

International Nuclear Information System (INIS)

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

2008-01-01

227

Ni(2+), a double-acting inhibitor of neuronal nitric oxide synthase interfering with L-arginine binding and Ca(2+)/calmodulin-dependent enzyme activation.  

Science.gov (United States)

Ni(2+), a toxic and carcinogenic pollutant and one of the leading causes of contact dermatitis, is shown to inhibit neuronal nitric oxide synthase (nNOS) in a competitive, reversible manner with respect to the substrate l-arginine (K(i) = 30 +/- 4 microM). The IC(50) values were dependent on calmodulin (CaM) concentration, but proved independent of Ca(2+), tetrahydrobiopterin (BH(4)) and other essential cofactors. Ni(2+) also inhibited CaM-dependent cytochrome c reduction, NADPH oxidation, and H(2)O(2) production by nNOS. Overall, the action profile of Ni(2+) was suggestive of an unusual, double-acting inhibitor of nNOS affecting l-arginine-binding and Ca(2+)/CaM-dependent enzyme activation. PMID:11437384

Palumbo, A; Astarita, G; Picardo, M; d'Ischia, M

2001-07-01

228

Ni(2+), a double-acting inhibitor of neuronal nitric oxide synthase interfering with L-arginine binding and Ca(2+)/calmodulin-dependent enzyme activation.  

UK PubMed Central (United Kingdom)

Ni(2+), a toxic and carcinogenic pollutant and one of the leading causes of contact dermatitis, is shown to inhibit neuronal nitric oxide synthase (nNOS) in a competitive, reversible manner with respect to the substrate l-arginine (K(i) = 30 +/- 4 microM). The IC(50) values were dependent on calmodulin (CaM) concentration, but proved independent of Ca(2+), tetrahydrobiopterin (BH(4)) and other essential cofactors. Ni(2+) also inhibited CaM-dependent cytochrome c reduction, NADPH oxidation, and H(2)O(2) production by nNOS. Overall, the action profile of Ni(2+) was suggestive of an unusual, double-acting inhibitor of nNOS affecting l-arginine-binding and Ca(2+)/CaM-dependent enzyme activation.

Palumbo A; Astarita G; Picardo M; d'Ischia M

2001-07-01

229

Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats.  

UK PubMed Central (United Kingdom)

Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N(G)-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5-2.5 ?g) and endomorphin-1 (2.5-20 ?g) in acute pain and morphine (10-40 ?g) and endomorphin-1 (5-20 ?g) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED50 for endomorphin-1 (7.1 ?g) was higher than that of morphine (1.3 ?g) in the tail-flick test. For neuropathic pain, the ED50 value for morphine was much higher (43.2 ?g) than that of endomorphin-1 (9.2 ?g) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by l-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the effects of endomorphin-1, an opioid ligand with a peptidergic structure.

Makuch W; Mika J; Rojewska E; Zychowska M; Przewlocka B

2013-09-01

230

Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.  

UK PubMed Central (United Kingdom)

Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.

Moujalled D; James JL; Parker SJ; Lidgerwood GE; Duncan C; Meyerowitz J; Nonaka T; Hasegawa M; Kanninen KM; Grubman A; Liddell JR; Crouch PJ; White AR

2013-01-01

231

Effects of aminoguanidine and cyclooxygenase inhibitors on nitric oxide and prostaglandin production, and nitric oxide synthase and cyclooxygenase expression induced by lipopolysaccharide in the estrogenized rat uterus.  

UK PubMed Central (United Kingdom)

BACKGROUND/OBJECTIVE: The aim of our study was first to investigate if there exists an interaction between nitric oxide (NO) and prostaglandin (PG) generation in the estrogenized rat uterus challenged by lipopolysaccharide (LPS), and, secondly, which isoforms of nitric oxide synthase (NOS) and cyclooxygenase (COX) participate in this process. METHODS: To study the effect of LPS and to characterize the isoenzymes involved in the process, specific inhibitors of iNOS (aminoguanidine) and COX-II (meloxicam, nimesulide) and non-specific of COX (indomethacin) were injected intraperitoneally to determine their effect on NO and PG production, and on NOS and COX expression induced by LPS in estrogenized rat uterus. NO production was measured by arginine-citrulline conversion assay and PGE(2)/PGF(2alpha,)by radioconversion. Enzyme expression was evaluated by Western blot analysis. RESULTS: The present work shows that iNOS inhibitor, aminoguanidine, reduced NO and PGE(2)/PGF(2alpha) production induced by LPS injection. Aminoguanidine exerts its effect over the PG metabolism by inhibiting COX-II activity and expression. On the other hand, both indomethacin, a non-selective PG inhibitor, and meloxicam, a COX-II inhibitor, stimulated NO production and reduced PGE(2)/PGF(2alpha) generation. Indomethacin also reduced COX-II and iNOS expression. CONCLUSION: These results indicate that in the estrogenized rat uterus challenged with LPS, PG and NO interact affecting each other's metabolic pathways. The above findings indicate that the interaction between NOS and COX might be important in the regulation of physiopathologic events during pregnancy.

Ribeiro M; Cella M; Farina M; Franchi A

2004-01-01

232

Purification, kinetics, inhibitors and CD for recombinant ?-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases.  

Science.gov (United States)

?-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant ?-amyrin synthase. The ?-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 ?m and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The ?-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations. PMID:23294602

Ito, Ryousuke; Masukawa, Yukari; Hoshino, Tsutomu

2013-02-13

233

Purification, kinetics, inhibitors and CD for recombinant ?-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases.  

UK PubMed Central (United Kingdom)

?-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant ?-amyrin synthase. The ?-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 ?m and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The ?-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.

Ito R; Masukawa Y; Hoshino T

2013-03-01

234

Complete amino acid sequence of alpha-acetolactate decarboxylase from Bacillus brevis.  

UK PubMed Central (United Kingdom)

The complete amino acid sequence of acetolactate decarboxylase (EC 4.1.1.5) from Bacillus brevis has been determined by sequencing of the intact enzyme and of peptides obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin, respectively. Determination of the C-terminal part was made by treatment with carboxypeptidases Y and M II. The enzyme has a molecular weight of 29,093 and consists of 260 amino acid residues arranged in a single peptide chain without disulphide bonds.

Svendsen I; Jensen BR; Ottesen M

1989-01-01

235

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2012-05-29

236

In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.  

UK PubMed Central (United Kingdom)

Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

Lucas S; Heim R; Ries C; Schewe KE; Birk B; Hartmann RW

2008-12-01

237

In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.  

Science.gov (United States)

Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats. PMID:19049427

Lucas, Simon; Heim, Ralf; Ries, Christina; Schewe, Katarzyna E; Birk, Barbara; Hartmann, Rolf W

2008-12-25

238

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

239

Long-lasting antidepressant action of ketamine, but not glycogen synthase kinase-3 inhibitor SB216763, in the chronic mild stress model of mice.  

UK PubMed Central (United Kingdom)

BACKGROUND: Clinical studies demonstrate that the N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine, induces rapid antidepressant effects in patients with refractive major depressive disorder and bipolar depression. This rapid onset of action makes ketamine a highly attractive drug for patients, particularly those who do not typically respond to therapy. A recent study suggested that glycogen synthase kinase (GSK)-3 may underlie the rapid antidepressant action of ketamine, although the precise mechanisms are unclear. In this study, we examined the effects of ketamine and GSK-3 inhibitor SB216763 in the unpredictable, chronic mild stress (CMS) mouse model of mice. METHODOLOGY/PRINCIPAL FINDINGS: Adult C57/B6 male mice were divided into 2 groups, a non-stressed control group and the unpredictable CMS (35 days) group. Then, either vehicle, ketamine (10 mg/kg), or the established GSK-3 inhibitor, SB216763 (10 mg/kg), were administered into mice in the CMS group, while vehicle was administered to controls. In the open field test, there was no difference between the four groups (control+vehicle, CMS+vehicle, CMS+ketamine, CMS+SB216763). In the sucrose intake test, a 1% sucrose intake drop, seen in CMS mice, was significantly attenuated after a single dose of ketamine, but not SB216763. In the tail suspension test (TST) and forced swimming test (FST), the increased immobility time seen in CMS mice was significantly attenuated by a single dose of ketamine, but not SB216763. Interestingly, the ketamine-induced increase in the sucrose intake test persisted for 8 days after a single dose of ketamine. Furthermore, a single administration of ketamine, but not SB216763, significantly attenuated the immobility time of the TST and FST in the control (non-stressed) mice. CONCLUSIONS/SIGNIFICANCE: These findings suggest that a single administration of ketamine, but not GSK-3 inhibitor SB216763, produces a long-lasting antidepressant action in CMS model mice.

Ma XC; Dang YH; Jia M; Ma R; Wang F; Wu J; Gao CG; Hashimoto K

2013-01-01

240

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

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

Henry JL; Dableh LJ

2011-01-01

 
 
 
 
241

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

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1. Formyl-methionyl-leucyl-phenylalanine (FMLP) or arachidonic acid (AA) induced luminol dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was found to be inhibited by nitric oxide synthease inhibitors and their D-enantiomers. 2. Rat PMNLs LCL response was inhibi...

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

242

ACETOLACTATE METABOLISM AND THE PRESENCE OF A DIHYDROXY ACID DEHYDRATASE IN MICRO-ORGANISMS.  

UK PubMed Central (United Kingdom)

1. The growth characteristics of nine micro-organisms on complex broth and defined media, usually with a single nitrogen source (other than vitamins), were examined as a necessary step before growth of cells for enzyme assays. Six of these bacteria gave a positive colour test with a creatine-potassium hydroxide reagent, indicating the presence of acetoin, which other investigators have shown is formed via the intermediate, alpha-acetolactate. 2. Cell-free extracts of exponential-phase cells of Bacillus subtilis, Staphylococcus aureus, Proteus morganii, Acetobacter rancens (two strains), A. kuetzingianus, A. acetosus, Acetomonas (Acetobacter) melanogenus and Acetomonas (Acetobacter) suboxydans (A.T.C.C. no. 621) were found to contain the enzyme, dihydroxy acid dehydratase (2,3-dihydroxy acid hydro-lyase). 3. The specific activity of the dehydratase from organisms grown on valine- and isoleucine-deficient media was greater than those grown on a complex broth or media containing complete amino acid mixtures. The omission of valine plus isoleucine from a medium containing 19 amino acids caused an increase in the dehydratase specific activity of Staphylococcus aureus and Proteus morganii. 4. The rate of keto acid formation from alphabeta-dihydroxyisovalerate by extracts of six of the above-named organisms was faster than, but somewhat proportional to, the similar rate from alphabeta-dihydroxy-beta-methyl-n-valerate as substrate. 5. These findings may be related to acetolactate synthesis, acetoin formation and valine-isoleucine biosynthesis in the above-mentioned micro-organisms.

WIXOM RL

1965-02-01

243

VALENCENE SYNTHASE  

UK PubMed Central (United Kingdom)

The present invention relates to a novel valencene synthase, to a nucleic acid encoding such valencene synthase, to a host cell comprising said encoding nucleic acid sequence and to a method for preparing valencene, comprising converting farnesyl diphosphate to valencene in the presence of a valencene synthase according to the invention.

ACHKAR JIHANE; SONKE THEODORUS

244

Valencene synthase  

UK PubMed Central (United Kingdom)

The present invention relates to a novel valencene synthase, to a nucleic acid encoding such valencene synthase, to a host cell comprising said encoding nucleic acid sequence and to a method for preparing valencene, comprising converting farnesyl diphosphate to valencene in the presence of a valencene synthase according to the invention.

ACHKAR JIHANE; SONKE THEODORUS; BEEKWILDER MARTINUS JULIUS; BOUWMEESTER HENDRIK JAN; BOSCH HENDRIK JAN

245

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

UK PubMed Central (United Kingdom)

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

Park J; Lin YS; De Schutter JW; Tsantrizos YS; Berghuis AM

2012-01-01

246

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli.  

UK PubMed Central (United Kingdom)

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Gollop N; Damri B; Barak Z; Chipman DM

1989-07-01

247

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli.  

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Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2675968

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

1989-07-25

248

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

UK PubMed Central (United Kingdom)

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 28days exposure in rats. After 7days, 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.

Gilmour PS; O'Shea PJ; Fagura M; Pilling JE; Sanganee H; Wada H; Courtney PF; Kavanagh S; Hall PA; Escott KJ

2013-10-01

249

Anti-dyskinetic effect of the neuronal nitric oxide synthase inhibitor is linked to decrease of FosB/deltaFosB expression.  

UK PubMed Central (United Kingdom)

Rodents with lesion of dopaminergic pathway when receiving repeated l-3,4-dihydroxiphenylalanine (l-DOPA) treatment develop abnormal involuntary movements called dyskinesia. We demonstrated that nitric oxide synthase (NOS) inhibitors mitigate l-DOPA-induced dyskinesia in rodents. The aim of the present study was to verify if the in vivo preferential neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI) affect the expression of the transcription factor FosB/?FosB in the lesioned striatum, an indicator of neuronal activity associated with dyskinesia. Male Wistar rats with unilateral microinjection (medial forebrain bundle) of either the neurotoxin 6-hydroxidopamine (6-OHDA; n=4-6/group) or saline (sham; n=6/group) were provided with l-DOPA (30mg/kg plus benserazide 7.5mg/kg/day, oral gavage), once a day during 22 days. 6-OHDA-lesioned animals developed abnormal involuntary movements (AIMs) classified as axial, limb, orofacial and locomotive dyskinesia and presented FosB/?FosB increase in the dopamine-depleted striatum. Administration of 7-NI (30mg/kg, i.p.), 30min prior to l-DOPA reduced the severity of AIMs (?65% for axial, limb and orofacial and 74% for locomotive AIMs scores), without interfering with the rotarod performance. Simultaneously, 7-NI attenuated the expression of FosB/?FosB in dopamine-depleted striatum (?65% in medial and ?54% in lateral striatum, bregma 0.48mm). FosB/?FosB expression in lateral striatum was correlated with l-DOPA-induced dyskinesia. The findings described here corroborate a new approach to the management of l-DOPA-therapy in Parkinson's disease (PD) treatment.

Padovan-Neto FE; Ferreira NR; de Oliveira-Tavares D; de Aguiar D; da Silva CA; Raisman-Vozari R; Del Bel E

2013-04-01

250

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 28days exposure in rats. After 7days, 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

2013-07-18

251

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

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Full Text Available 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 interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA ( (more) 30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P

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

2010-11-01

252

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 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 (more) ), (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

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

2011-01-01

253

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

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

Cho Yu; Kim Ji; Yoon Jiyeon; Cho Sung; Ko Young; Park Jong-Wan; Lee Hye; Lee Hee; Kim Woo; Lee Byung

2010-01-01

254

Inhibition of antigen-induced airway hyperresponsiveness in rats: effects of ozagrel (a thromboxane A2 synthase inhibitor) and of CV-3988 (a platelet activating factor antagonist).  

UK PubMed Central (United Kingdom)

The effects of ozagrel, a thromboxane A2 (TXA2) synthase inhibitor, and CV-3988, a platelet activating factor (PAF) antagonist, was investigated on the repeatedly antigenic challenge-induced airway hyperresponsiveness (AHR) in rats. Rats were actively sensitized with DNP-Ascaris antigen and received 3 inhalations of antigen (challenges) or saline (sensitized control) every 48 hr. These animals were also pretreated with ozagrel (100 mg/kg, p.o., 30 min before), CV-3988 (3 mg/kg, i.v., 5 min before) or respective vehicle (water and saline, respectively) before each inhalation of antigen or saline. The in vivo airway responsiveness to cumulatively inhaled acetylcholine (ACh; 0.001-0.03%, each for 3 min) was measured 24 hr after the last inhalation of antigen or saline under anesthesia. A marked AHR was observed after repeated antigenic challenge when compared with the sensitized control group (5.5-9.5 times in order). This AHR was significantly, but partly, attenuated by pretreatment with ozagrel although this treatment alone had no effect on the airway responsiveness to inhaled ACh in sensitized control animals. On the other hand, CV-3988 had no inhibitory effect on this AHR. These findings suggest that TXA2, but not PAF, is one of the most important mediators participating in the pathogenesis of the antigen-induced AHR in rats.

Misawa M; Chiba Y

1994-06-01

255

The possibility of clinical application of the thromboxane A2 synthase inhibitor, ozagrel, for the treatment and prevention of preeclampsia: a preliminary report.  

UK PubMed Central (United Kingdom)

OBJECTIVES: This study was performed to investigate whether the TXA2 synthase inhibitor, ozagrel, was effective in the treatment and prevention of pre-eclampsia. STUDY DESIGN: Ozagrel was administrated therapeutically to 4 severely pre-eclamptic women, and prophylactically to 5 pregnant women with histories of severe preeclampsia and complications. RESULTS: The therapeutic administration (TA) of ozagrel improved hypertension and proteinuria. Two patients delivered appropriate-for-date (AFD) infants, whereas the other 2 patients delivered light-for-date (LFD) infants. Mean plasma concentrations of TXB2 (plasma TXB2) decreased, whereas plasma 6-keto PGF1 alpha were almost unchanged. The prophylactic administration (PA) of ozagrel prevented the occurrence of preeclampsia in 3 of the 5 patients. All delivered AFD infants. The duration of pregnancy was prolonged more than that of previous pregnancies in all patients. Plasma TXB2 decreased, whereas plasma 6-keto PGF1 alpha increased. CONCLUSIONS: PA prevented preeclampsia and intrauterine growth retardation, whereas TA improved only maternal symptoms. These results might justify a large prospective study to determine whether ozagrel is an effective prophylactic.

Seki H; Kuromaki K; Takeda S; Kinoshita K; Satoh K

1995-08-01

256

In vitro activity of a new oral glucan synthase inhibitor (MK-3118) tested against Aspergillus spp. by CLSI and EUCAST broth microdilution methods.  

Science.gov (United States)

MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ? 4 ?g/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC(90))/MIC(90) values (?g/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fumigatus SC; 0.12, 2, and 0.06 for Aspergillus terreus SC; and 0.06, 1, and 0.03 for Aspergillus niger SC. Essential agreement between the values found by CLSI and EUCAST (± 2 log(2) dilution steps) was 94.3%. MK-3118 was determined to be a potent agent regardless of the in vitro method applied, with excellent activity against contemporary wild-type and itraconazole-resistant strains of Aspergillus spp. PMID:23229479

Pfaller, Michael A; Messer, Shawn A; Motyl, Mary R; Jones, Ronald N; Castanheira, Mariana

2012-12-10

257

Inhibitory effects of JTE-522, a novel prostaglandin H synthase-2 inhibitor, on adjuvant-induced arthritis and bone changes in rats.  

UK PubMed Central (United Kingdom)

OBJECTIVE AND DESIGN: To investigate the effect of JTE-522, a novel selective prostaglandin H synthase (PGHS)-2 inhibitor, on adjuvant-induced arthritis and bone changes. SUBJECTS: Male Lewis rats at 8 weeks old were immunized with heat-killed mycobacteria. TREATMENT: JTE-522 (0.1-30 mg/kg) and indomethacin (0.1-3 mg/kg) were administered orally once-daily after immunization. METHODS: Paw swelling, bone changes in arthritic paws and vertebrae, urinary levels of deoxypyridinoline and pyridinium crosslinks, and the incidence of gastric lesions were determined in arthritic rats. RESULTS: JTE-522 (from 0.3 mg/kg) suppressed the development of paw swelling, and also reduced bone damage (score and bone mineral density) in arthritic paws and the urinary excretion of deoxypyridinoline and pyridinium crosslinks. However, JTE-522 did not cause gastric lesions even at 30 mg/kg in arthritic rats. CONCLUSIONS: These results suggest that JTE-522 possesses potent anti-arthritic activities and suppressive activity on inflammatory bone resorption without gastric side effects.

Masaki M; Matsushita M; Wakitani K

1998-04-01

258

A QSAR and molecular modeling study on a series of 3, 4-dihydro-1-isoquinolinamines and thienopyridines acting as nitric oxide synthase inhibitors.  

UK PubMed Central (United Kingdom)

A quantitative structure-activity relationship (QSAR) and molecular modeling study were performed on a series of 3,4-dihyro-1-isoquinolinamines and thienopyridines reported by Beaton et al. [Beaton et al. (2001) Bioorg Med Chem Lett 11, 1023-1026, 1027-1030] as potent, highly selective inhibitors of two isoforms of nitric oxide synthase (NOS)--neuronal NOS (nNOS) and endothelial NOS (eNOS), in order to find the physicochemical properties that governed their activity and the mode of interaction with the receptors, so that still more potent compounds in the series could be suggested. A multiple regression analysis revealed that nNOS and eNOS inhibition potency of these compounds could be controlled by their hydrophobic property and molar refractivity, respectively. Thus, nNOS and eNOS inhibition was indicated to involve the hydrophobic interaction and steric effects, respectively, suggesting some structural differences of the two isoforms of NOS. Based on the correlations obtained, some new, more potent compounds belonging to the series were predicted. These compounds were then docked into the receptors to see their interactions and find out the docking scores. The docked structures of two representative compounds, whose interaction energies with nNOS and eNOS, respectively were found to be the lowest, were given as an example to exhibit the possible orientation of the compounds to interact with the receptors.

Kumar V; Gupta SP

2013-02-01

259

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

UK PubMed Central (United Kingdom)

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

Di Gennaro E; Bruzzese F; Pepe S; Leone A; Delrio P; Subbarayan PR; Avallone A; Budillon A

2009-05-01

260

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

Science.gov (United States)

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

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

2009-05-09

 
 
 
 
261

Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester.  

UK PubMed Central (United Kingdom)

The role of nitric oxide (NO) in libido and fertility of male rats was investigated by administration of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (25 or 50 mg/kg/day). L-NAME caused marked reduction of precoital sexual behaviour, and a failure of most rats to mount or ejaculate during the test interval. In most matings that were successful, recovered spermatozoa were present in normal numbers. In mating experiments, L-NAME profoundly reduced the fertility of male rats. In those animals that did succeed in mating, the quantal pregnancy and the number of implants were reduced. After cessation of treatment with L-NAME, the fertility parameters returned close to normal. The inactive stereoisomer, D-NAME, caused none of the above effects when administered to rats. The results suggest that NO is essential for the expression of normal libido and fertility in male rats. It is likely that NO is required both in the male reproductive tract and in the brain.

Ratnasooriya WD; Dharmasiri MG; Wadsworth RM

2000-06-01

262

Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester.  

Science.gov (United States)

The role of nitric oxide (NO) in libido and fertility of male rats was investigated by administration of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (25 or 50 mg/kg/day). L-NAME caused marked reduction of precoital sexual behaviour, and a failure of most rats to mount or ejaculate during the test interval. In most matings that were successful, recovered spermatozoa were present in normal numbers. In mating experiments, L-NAME profoundly reduced the fertility of male rats. In those animals that did succeed in mating, the quantal pregnancy and the number of implants were reduced. After cessation of treatment with L-NAME, the fertility parameters returned close to normal. The inactive stereoisomer, D-NAME, caused none of the above effects when administered to rats. The results suggest that NO is essential for the expression of normal libido and fertility in male rats. It is likely that NO is required both in the male reproductive tract and in the brain. PMID:10844545

Ratnasooriya, W D; Dharmasiri, M G; Wadsworth, R M

2000-06-01

263

Opioids are non-competitive inhibitors of nitric oxide synthase in T47D human breast cancer cells.  

Science.gov (United States)

Opioids and nitric oxide (NO) interact functionally in different systems. NO-generating agents decrease the activity of opioid agonists, prevent opioid tolerance, and are used in opioid withdrawal syndromes. There exist, however, few reports indicating a direct interaction of the two systems. T47D human breast cancer cells in culture express opioid receptors, and opioid agonists inhibit their growth, while they release high amounts of the NO-related molecules NO(2-)/NO(3-)to the culture medium. We have used this system to assay a possible direct interaction of opiergic and nitric oxide systems. Our results show that delta- or mu-acting opioid agonists do not modify the release of NO(2-)/NO(3-). In contrast, kappa-acting opioid agonists (ethylketocyclazocine, and alpha(S1)-casomorphine) decrease the release of NO(2-)/NO(3-), in a time- and dose-dependent manner. The general opioid antagonist diprenorphine (10(-6) M) produce a similar NO(2-)/NO(3-)release inhibition, indicating a possible non-opioid-receptor mediated phenomenon. In addition, ethylketocyclazocine, alpha(S1)-casomorphin and diprenorphine directly inhibit NOS activity: agonists, interact with both calcium-dependent and independent NOS-isoforms, while the antagonist diprenorphine modifies only the activity of the calcium-dependent fraction of the enzyme. Analysis of this interaction revealed that opioids modify the dimeric active form of NOS, through binding to the reductase part of the molecule, acting as non-competitive inhibitors of the enzyme. This interaction opens interesting new possibilities for tumor biology and breast cancer therapy. PMID:11526449

Kampa, M; Hatzoglou, A; Notas, G; Niniraki, M; Kouroumalis, E; Castanas, E

2001-09-01

264

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; Mahmoud Hosseini; Fatima Saffarzadeh; Reza Karami; Fatimeh Khodabandehloo

2011-01-01

265

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

Directory of Open Access Journals (Sweden)

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; Maryam-Sadat Shahin; Mohamade Nasehi; Mohamd reza Zarrindast

2011-01-01

266

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

Science.gov (United States)

Glycogen synthase kinase-3? (GSK3?) role in human immunodeficiency virus (HIV)-associated neurodegeneration has been evidenced by previous investigations. In this study, we investigated the specificity of two GSK3?-specific inhibitors, AR-A014418 (A) and B6B30 (B) to prevent direct neurotoxicity in primary human neurons exposed to HIV (BaL). Neurons were exposed to HIV (500 pg/ml) for 12-h and 6-day periods in the presence and absence of A (1 µM, 100 nM, 10 nM) and B (50 nM, 5 nM, 500 pM) to investigate acute and ongoing mechanisms of HIV neurotoxicity. Using an lactate dehydrogenase (LDH) assay to assess cytotoxicity, we observed a significant neurotoxic effect of HIV from control values (P < .01) that was not restored via coexposures of all concentrations of A and B. Additionally, no change in LDH levels were observed after 6 days. However, activity of the acute proapoptotic markers caspases 3 and 7 using a luminescence assay were measured and found to be increased by exposure to HIV (BaL) compared to controls (P = .022). This effect was ameliorated via coexposure to all concentrations of A and 50 nM B after 12 h (P < .01) and to all concentrations of A and B after 6 days (P < .01). Overall, the results from this study provide further evidence for the ability of GSK3? inhibition to be neuroprotective against HIV-associated neurotoxicity by reducing HIV associated procaspase induction. These data support a role for GSK3? as a potential therapeutic target and may have important clinical implications for treatment of HIV-associated neurocognitive disorder.

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

2011-01-01

267

Valencene synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The present invention relates to a novel valencene synthase, to a nucleic acid encoding such valencene synthase, to a host cell comprising said encoding nucleic acid sequence and to a method for preparing valencene, comprising converting farnesyl diphosphate to valencene in the presence of a valence...

Achkar, A.; Sonke, Th.; Bouwmeester, H.J.; Bosch, H.J.

268

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

Czech Academy of Sciences Publication Activity Database

. Prague, 1999 - (Jen?, P.). s. 64[Czech-Swiss Symposium on Advanced Biotechnology /1./. 04.09.1999-07.09.1999, Prague]Výzkumný zám?r: CEZ:A53/98:Z5-020-9iiKód oboru RIV: EI - Biotechnologie a bionika

Kopecký, JanG; Janata, Ji?í

269

Prevention of early islet graft failure by selective inducible nitric oxide synthase inhibitors after pig to nude rat intraportal islet transplantation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Clinical and experimental data indicate that early failure of intraportally grafted islets is caused by inflammation including secretion of cytokines and nitric oxide. Direct inducible nitric oxide synthase suppression may avoid detrimental effects associated with steroid administration. We compared...

Brandhorst, D; Brandhorst, H; Zwolinski, A; Nahidi, F; Bretzel, RG

270

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.  

UK PubMed Central (United Kingdom)

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.

Ishitsuka Y; Moriuchi H; Isohama Y; Tokunaga H; Hatamoto K; Kurita S; Irikura M; Iyama K; Irie T

2009-10-01

271

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

2009-09-26

272

Effects of thromboxane A2 synthase inhibitors (CV-4151 and ozagrel), aspirin, and ticlopidine on the thrombosis caused by endothelial cell injury.  

UK PubMed Central (United Kingdom)

The antiplatelet and antithrombotic effects of CV-4151 (isbogrel), a potent selective thromboxane A2 (TXA2) synthase inhibitor, were compared with those of ozagrel (OKY-046), aspirin, and ticlopidine in rats. Two hours after oral administration, CV-4151, ozagrel and aspirin inhibited blood TXA2 generation with ID50 values of 0.04, 0.3 and 6.4 mg/kg, respectively. These values were similar to the oral ID50 values of CV-4151 (0.06 mg/kg), ozagrel (0.92 mg/kg) and aspirin (7.0 mg/kg) for arachidonic acid (AA)-induced platelet aggregation ex vivo. Two hours after p.o. administration, CV-4151 and ozagrel inhibited femoral vein platelet-rich thrombosis caused by endothelial injury with ID50 values of 2.46 and 13.7 mg/kg, respectively. However, aspirin (100 mg/kg, p.o.) only slightly inhibited the thrombosis. Ticlopidine (300 mg/kg, p.o.) slightly but significantly inhibited AA-induced and ADP-induced platelet aggregation, however, it potently inhibited the thrombosis. CV-4151 and ozagrel given by i.v. injection showed therapeutic effects on the thrombosis with ED50 values of 0.026 and 0.066 mg/kg, respectively. These values were similar to the i.v. ED50 values of CV-4151 (0.0056 mg/kg) and ozagrel (0.042 mg/kg) for blood TXA2 generation. However, aspirin (30 mg/kg, i.v.) only moderately reduced the thrombosis. CV-4151 (> 0.3 mg/kg, p.o.), ozagrel (> 3 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) all significantly prolonged tail bleeding time. Aspirin (100 mg/kg, p.o.) tended to prolong the bleeding time. The antiplatelet and antithrombotic effects of CV-4151 are more potent than those of ozagrel, aspirin and ticlopidine in rats. CV-4151 may therefore be a useful drug for the treatment of thrombotic diseases.

Terashita Z; Imura Y; Kawamura M; Kato K; Nishikawa K

1995-03-01

273

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

International Nuclear Information System (INIS)

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

2006-10-15

274

Spermine synthase.  

UK PubMed Central (United Kingdom)

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

Pegg AE; Michael AJ

2010-01-01

275

Involvement of thromboxane A2 (TXA2) in the early stages of oleic acid-induced lung injury and the preventive effect of ozagrel, a TXA2 synthase inhibitor, in guinea-pigs.  

Science.gov (United States)

An intravenous injection of oleic acid into animals can produce a lung injury with hypoxaemia and pulmonary vascular hyper-permeability. Although oleic acid lung injury is used as a model of acute respiratory distress syndrome (ARDS), the precise mechanisms of the lung injury are still unclear. We have investigated whether thromboxane A(2) (TXA(2)) participated in the lung injury and have evaluated the efficacy of ozagrel, a TXA(2) synthase inhibitor, on the lung injury in guinea-pigs. Oleic acid injection increased the plasma level of TXB(2), a stable metabolite of TXA(2), and the time-course of plasma TXB(2) was similar to that of the decreased partial oxygen pressure of arterial blood (Pao(2)) induced with oleic acid. Ozagrel administered intravenously 30 min before oleic acid injection prevented the decrease in Pao(2) and pulmonary vascular hyper-permeability. It also prevented increases in lactate dehydrogenase activity, a measure of lung cell injury, TXB(2 )and its weight ratio to 6-keto prostaglandin F(1alpha) in bronchoalveolar lavage fluid. Although ozagrel administered simultaneously with oleic acid ameliorated the decrease in Pao(2), post treatment showed little effect. We suggest that TXA(2) participated in the oleic acid lung injury, as an "early phase" mediator, and rapidly-acting TXA(2) synthase inhibitors were effective in the prevention of acute lung injury. PMID:15099446

Ishitsuka, Yoichi; Moriuchi, Hiroshi; Hatamoto, Keita; Yang, Changqing; Takase, Junko; Golbidi, Saeid; Irikura, Mitsuru; Irie, Tetsumi

2004-04-01

276

Involvement of thromboxane A2 (TXA2) in the early stages of oleic acid-induced lung injury and the preventive effect of ozagrel, a TXA2 synthase inhibitor, in guinea-pigs.  

UK PubMed Central (United Kingdom)

An intravenous injection of oleic acid into animals can produce a lung injury with hypoxaemia and pulmonary vascular hyper-permeability. Although oleic acid lung injury is used as a model of acute respiratory distress syndrome (ARDS), the precise mechanisms of the lung injury are still unclear. We have investigated whether thromboxane A(2) (TXA(2)) participated in the lung injury and have evaluated the efficacy of ozagrel, a TXA(2) synthase inhibitor, on the lung injury in guinea-pigs. Oleic acid injection increased the plasma level of TXB(2), a stable metabolite of TXA(2), and the time-course of plasma TXB(2) was similar to that of the decreased partial oxygen pressure of arterial blood (Pao(2)) induced with oleic acid. Ozagrel administered intravenously 30 min before oleic acid injection prevented the decrease in Pao(2) and pulmonary vascular hyper-permeability. It also prevented increases in lactate dehydrogenase activity, a measure of lung cell injury, TXB(2 )and its weight ratio to 6-keto prostaglandin F(1alpha) in bronchoalveolar lavage fluid. Although ozagrel administered simultaneously with oleic acid ameliorated the decrease in Pao(2), post treatment showed little effect. We suggest that TXA(2) participated in the oleic acid lung injury, as an "early phase" mediator, and rapidly-acting TXA(2) synthase inhibitors were effective in the prevention of acute lung injury.

Ishitsuka Y; Moriuchi H; Hatamoto K; Yang C; Takase J; Golbidi S; Irikura M; Irie T

2004-04-01

277

Synthesis with Good Enantiomeric Excess of Both Enantiomers of ?-Ketols and Acetolactates by Two Thiamin Diphosphate Dependent Decarboxylases.  

Science.gov (United States)

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21473-21482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and ?-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.

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

2006-01-01

278

Fine-tuning the selectivity of aldosterone synthase inhibitors: structure-activity and structure-selectivity insights from studies of heteroaryl substituted 1,2,5,6-tetrahydropyrrolo[3,2,1-ij]quinolin-4-one derivatives.  

UK PubMed Central (United Kingdom)

Pyridine substituted 3,4-dihydro-1H-quinolin-2-ones (e.g., 1-3) constitute a class of highly potent and selective inhibitors of aldosterone synthase (CYP11B2), a promising target for the treatment of hyperaldosteronism, congestive heart failure, and myocardial fibrosis. Among these, ethyl-substituted 3 possesses high selectivity against CYP1A2. Rigidification of 3 by incorporation of the ethyl group into a 5- or 6-membered ring affords compounds with a pyrroloquinolinone or pyridoquinolinone molecular scaffold (e.g., 4 and 5). It was found that these molecules are even more potent and selective CYP11B2 inhibitors than their corresponding open-chain analogues. Moreover, pyrroloquinolinone 4 exhibits no inhibition of the six most important hepatic CYP enzymes as well as a bioavailability in the range of the marketed drug fadrozole. The SAR studies disclose that subtle changes in the heterocyclic moiety are responsible for either a strong or a weak inhibition of the highly homologous 11?-hydroxylase (CYP11B1). These results are not only important for fine-tuning the selectivity of CYP11B2 inhibitors but also for the development of selective CYP11B1 inhibitors that are of interest for the treatment of Cushing's syndrome and metabolic syndrome.

Lucas S; Negri M; Heim R; Zimmer C; Hartmann RW

2011-04-01

279

The effects of early and late administration of inhibitors of inducible nitric oxide synthase in a thioacetamide-induced model of acute hepatic failure in the rat  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Background/aims: Nitric oxide (NO) is a pivotal mediator of inflammation. Its role in acute hepatic failure (AHF) is controversial. We investigated the role of NO, and the hypothesis that inhibition of inducible NO synthase (iNOS) activity would improve outcome in liver failure in rats, using the iN...

Rahman, T.; Hodgson, H.J.F.

280

Polyphosphoester-Based Cationic Nanoparticles Serendipitously Release Integral Biologically-Active Components to Serve as Novel Degradable Inducible Nitric Oxide Synthase Inhibitors.  

UK PubMed Central (United Kingdom)

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

Shen Y; Zhang S; Zhang F; Loftis A; Pavía-Sanders A; Zou J; Fan J; Taylor JS; Wooley KL

2013-09-01

 
 
 
 
281

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma.  

UK PubMed Central (United Kingdom)

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 micrograms/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.

Hamilton CL; Kirkwood JA

1994-04-01

282

Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-carboxamide and sulfonamide derivatives as thymidylate synthase A inhibitors.  

UK PubMed Central (United Kingdom)

Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A deconstruction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was identified and two compound libraries based on this scaffold were designed. The carboxamide library (Library B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)-sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant against hTS with respect to the starting lead and represent potential compounds for further lead development.

Ferrari S; Ingrami M; Soragni F; Wade RC; Costi MP

2013-02-01

283

INHIBITORS OF BIOFILM FORMATION  

UK PubMed Central (United Kingdom)

Disclosed herein is a composition for inhibiting bacterial biofilm formation comprising carrier and an effective amount of an inhibitor of squalene/phytoene synthesis. Inhibitors may inhibit, for example, HMG-CoA Reductase, squalene synthase, 1-deoxy-D-xylulose 5-phosphate synthase. Examples of such inhibitors are a phosphonosulfonate (e.g., BPH-652, BPH-689, BPH-700), a statin (e.g., mevastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin), zaragozic acid, clomazone, and lapaquistat acetate or a functional derivative thereof. Also disclosed are substrates comprising the inhibitor of squalene/phytoene synthesis, and methods of inhibiting bacterial biofilm formation.

LOPEZ DANIEL; HATTON BENJAMIN; KOLTER ROBERTO

284

Intrathecally injected Ile-Pro-Ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase IV, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 2.  

Science.gov (United States)

We have found recently that membrane-bound dipeptidyl peptidase IV (DPP-IV) generated extracellularly immunoreactive endomorphin-2 from Tyr-Pro precursor in a depolarisation-sensitive manner in rat isolated L4,5 dorsal root ganglia when the enzyme was switched to synthase mode by the hydrolase inhibitor Ile-Pro-Ile. Presently, we induced hyperalgesia in rats by injecting carrageenan into the right hindpaw and measured the reduction in nociceptive threshold (hyperalgesia) to pressure (Randall-Selitto test). The hyperalgesia, peaking at 180 min after injection, was fully reversed by intrathecal administration of 30 nmol/rat Ile-Pro-Ile. The antihyperalgesic action was antagonized by s.c. naloxone (1 mg/kg) and intrathecally injected specific antiserum to endomorphin-2 indicating that the opioid receptor-mediated effect was produced by an endogenously generated endomorphin-2-like immunoreactive substance. Intrathecal Ile-Pro-Ile was ineffective as an analgesic in the acute nociceptive test such as the rat tail-flick, whereas endomorphin-2 (EC(50)=13.3 nmol/rat), endomorphin-1 (6.8 nmol/rat), morphine (0.11 nmol/rat) and DAMGO (0.0059 nmol/rat) exerted opioid receptor-mediated analgesia given by the same route. We concluded that carrageenan-induced C-fiber barrage (wind-up) may create ideal conditions for the de novo synthesis of endomorphin-2 in rat spinal cord dorsal horns if the DPP-IV enzyme is switched to the synthase functional mode by Ile-Pro-Ile. PMID:19695241

Király, Kornél; Szalay, Balázs; Szalai, Judit; Barna, István; Gyires, Klára; Verbeken, Mathieu; Rónai, András Z

2009-08-18

285

Modulation of the protein kinase Cdelta interaction with the "d" subunit of F1F0-ATP synthase in neonatal cardiac myocytes: development of cell-permeable, mitochondrially targeted inhibitor and facilitator peptides.  

UK PubMed Central (United Kingdom)

The F(1)F(0)-ATP synthase provides approximately 90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cdelta (PKCdelta) inhibits F(1)F(0) activity via an interaction with the "d" subunit of F(1)F(0)-ATP synthase (dF(1)F(0)) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831-29840). We have now identified a dF(1)F(0)-derived peptide (NH(2)-(2)AGRKLALKTIDWVSF(16)-COOH) that inhibits PKCdelta binding to dF(1)F(0) in overlay assays. We have also identified a second dF(1)F(0)-derived peptide (NH(2)-(111)RVREYEKQLEKIKNMI(126)-COOH) that facilitates PKCdelta binding to dF(1)F(0). Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCdelta-dF(1)F(0) interaction inhibitor, decreased 100 nm 4beta-phorbol 12-myristate 13-acetate (4beta-PMA)-induced co-immunoprecipitation of PKCdelta with dF(1)F(0) by 50 +/- 15% and abolished the 30 nm 4beta-PMA-induced inhibition of F(1)F(0)-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCdelta-dF(1)F(0) facilitator peptide by itself induced the PKCdelta-dF(1)F(0) co-immunoprecipitation and inhibited F(1)F(0)-ATPase activity. In in vitro PKC add-back experiments, the PKCdelta-F(1)F(0) inhibitor blocked PKCdelta-mediated inhibition of F(1)F(0)-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCdelta-dF(1)F(0) interaction in NCMs. These novel peptides will improve our understanding of cardiac F(1)F(0) regulation and may have potential as therapeutics to attenuate cardiac injury.

Nguyen TT; Ogbi M; Yu Q; Fishman JB; Thomas W; Harvey BJ; Fulton D; Johnson JA

2010-07-01

286

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

Science.gov (United States)

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

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

2012-12-18

287

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

UK PubMed Central (United Kingdom)

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

Lu W; Li L; Chen M; Zhou Z; Zhang W; Ping S; Yan Y; Wang J; Lin M

2013-03-01

288

Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation.  

UK PubMed Central (United Kingdom)

The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking ?-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these ?-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

J Landau M; Sharma H; Anderson KS

2013-09-01

289

UDP-N-acetylmuramic acid (UDP-MurNAc) is a potent inhibitor of MurA (enolpyruvyl-UDP-GlcNAc synthase).  

UK PubMed Central (United Kingdom)

Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.

Mizyed S; Oddone A; Byczynski B; Hughes DW; Berti PJ

2005-03-01

290

Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation.  

Science.gov (United States)

The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking ?-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these ?-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance. PMID:23813474

J Landau, Mark; Sharma, Hitesh; Anderson, Karen S

2013-08-01

291

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

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

Park Jaeok; Lin Yih-Shyan; De Schutter Joris W; Tsantrizos Youla S; Berghuis Albert M

2012-01-01

292

Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21(waf1/cip1) expression.  

UK PubMed Central (United Kingdom)

It is desirable to find more appropriate therapeutic opportunities in non-small-cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S-1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth-inhibitory effect of 5-fluorouracil (5-FU), S-1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5-FU. Combined treatment with low-dose SAHA enhanced 5-FU- and S-1-mediated cytotoxicity and resulted in synergistic effects, especially in 5-FU-resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5-FU sensitivity/response, were analyzed in the cells undergoing treatment. 5-Fluorouracil-resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down-regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5-FU treatment, in all examined cell types. We also examined the status of the Rb-E2F1 pathway, with SAHA up-regulating p21(waf1/cip1) expression via promoter histone acetylation; this, in turn, blocked the Rb-E2F1 pathway. We conclude that combination therapy with SAHA and S-1 in lung cancer may be promising due to its potential to overcome S-1 resistance via modulation of 5-FU/S-1 sensitivity-associated biomarker (TS) by HDAC inhibitor.

Noro R; Miyanaga A; Minegishi Y; Okano T; Seike M; Soeno C; Kataoka K; Matsuda K; Yoshimura A; Gemma A

2010-06-01

293

The effect of N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, on respiratory mechanics in rats.  

UK PubMed Central (United Kingdom)

BACKGROUND: Data describing the inhibitory effects of nitric oxide synthase (NOS) on respiratory mechanics are conflicting, and no data are available concerning possible effects on the viscoelasticity of the respiratory system, on the inspiratory work of breathing (WOB) or on respiratory system hysteresis. OBJECTIVES: The aim of this study was to measure the effects of NOS inhibition by N(G)-nitro-L-arginine methyl ester (L-NAME) on respiratory mechanics in normal anesthetized rats. METHODS: Using the end-inflation occlusion method, it was possible to quantify the ohmic and viscoelastic airway resistance and elastance of the respiratory system. Ohmic resistance is the normalized-to-flow pressure dissipation due to viscous forces opposing the airflow in the airways, as predicted by the Poiseuille law. Viscoelastic resistance is the normalized-to-flow pressure dissipation due to the resistance of respiratory system tissue to deformation during inflation, which is recovered after the arrest of the inspiratory flow (stress relaxation). The inspiratory WOB, its elastic and resistive components, and hysteresis were also calculated. RESULTS: L-NAME induced an increment in the ohmic airway resistance and in the resistive ohmic inspiratory WOB. The viscoelastic resistance due to stress relaxation and the elastic properties of the respiratory system were not modified, and no effect was detected on the related components of the inspiratory WOB and on hysteresis. CONCLUSIONS: NO acts in normal rats to reduce the ohmic component of airway resistance, decreasing the ohmic inspiratory WOB. The elastic and viscoelastic components are unaltered. Hysteresis is also unaltered, suggesting that NO has negligible effects on alveolar surfactant activity.

Rubini A

2011-01-01

294

The nitric oxide synthase inhibitor, N-monomethyl-L-arginine blocks induction of a long-term potentiation-like phenomenon in rat medial frontal cortical neurons in vitro.  

UK PubMed Central (United Kingdom)

1. Nitric oxide has been implicated in the production of long-term depression (LTD) in the cerebellum and in the production of long-term potentiation (LTP) and LTD in the hippocampus. We now provide evidence of its involvement in the induction of long-term synaptic potentiation in in vitro slices in the cerebral cortex of the rat. 2. Intracellular recordings were made from layer V neurons in the medial frontal cortex, and excitatory synaptic potentials (EPSPs) were evoked by electrical stimulation of layers II/III. Tetanic stimulation of this pathway may induce LTD or LTP or no change at these synapses. First we established experimental conditions under which a long lasting potentiation could be induced with a high incidence (> 60%), namely perfusion of slices with 1 microM bicuculline methiodide, second the use of increased shock duration in the tetanic conditioning stimuli, third and most important the addition of QX-314 to the microelectrode to reduce potassium conductances. Because the potentiation of the mean EPSP slope was significantly greater than the control at 40-min postconditioning, but was declining throughout this period, we refer to it for brevity as LTP, but strictly class it as an LTP-like phenomenon. 3. The nitric oxide (NO) synthase inhibitor interfered with the production of LTP. In the control group of neurons (n = 13) the mean depolarizing slope of the EPSP at 30-min post-conditioning was 142.7 +/- 2% (mean +/- SE) of the prestimulation control.(ABSTRACT TRUNCATED AT 250 WORDS)

Nowicky AV; Bindman LJ

1993-09-01

295

Abnormalities of endothelium-dependent responses in mesenteric arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats are improved by chronic treatment with thromboxane A2 synthase inhibitor.  

Science.gov (United States)

Thromboxane A(2) (TXA(2)) is thought to contribute to the development of diabetic complications. We tested the hypothesis that the impaired endothelial function seen in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a type 2 diabetic model) might be improved by chronic treatment with ozagrel, a TXA(2) synthase inhibitor. In mesenteric arteries from OLETF rats (40-46 weeks old) [vs. those from age-matched Long-Evans Tokushima Otsuka (LETO) rats]: (1) ACh-induced endothelium-dependent relaxation, NO-mediated relaxation, and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation were all reduced; (2) ACh-induced cyclooxygenase-dependent contraction was enhanced; (3) endothelium-derived contracting factor (EDCF)-mediated contraction was enhanced; (4) ACh-stimulated nitrite production was reduced but the nitrate/nitrite ratio was increased; and (5) ACh-stimulated production of TXA(2) was increased. Chronic treatment with ozagrel (100mg/kg/day for 4 weeks, starting when they were 36-42 weeks of age) partly corrected the above abnormalities. These results suggest that ozagrel has normalizing effects on endothelial functions in OLETF mesenteric arteries, at least partly by increasing endothelium-derived relaxing factors (i.e., NO and EDHF) signaling and reducing EDCF signaling. PMID:19111834

Matsumoto, Takayuki; Takaoka, Eri; Ishida, Keiko; Nakayama, Naoaki; Noguchi, Eri; Kobayashi, Tsuneo; Kamata, Katsuo

2008-11-25

296

Abnormalities of endothelium-dependent responses in mesenteric arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats are improved by chronic treatment with thromboxane A2 synthase inhibitor.  

UK PubMed Central (United Kingdom)

Thromboxane A(2) (TXA(2)) is thought to contribute to the development of diabetic complications. We tested the hypothesis that the impaired endothelial function seen in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a type 2 diabetic model) might be improved by chronic treatment with ozagrel, a TXA(2) synthase inhibitor. In mesenteric arteries from OLETF rats (40-46 weeks old) [vs. those from age-matched Long-Evans Tokushima Otsuka (LETO) rats]: (1) ACh-induced endothelium-dependent relaxation, NO-mediated relaxation, and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation were all reduced; (2) ACh-induced cyclooxygenase-dependent contraction was enhanced; (3) endothelium-derived contracting factor (EDCF)-mediated contraction was enhanced; (4) ACh-stimulated nitrite production was reduced but the nitrate/nitrite ratio was increased; and (5) ACh-stimulated production of TXA(2) was increased. Chronic treatment with ozagrel (100mg/kg/day for 4 weeks, starting when they were 36-42 weeks of age) partly corrected the above abnormalities. These results suggest that ozagrel has normalizing effects on endothelial functions in OLETF mesenteric arteries, at least partly by increasing endothelium-derived relaxing factors (i.e., NO and EDHF) signaling and reducing EDCF signaling.

Matsumoto T; Takaoka E; Ishida K; Nakayama N; Noguchi E; Kobayashi T; Kamata K

2009-07-01

297

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.  

UK PubMed Central (United Kingdom)

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.

Pérez DI; Pistolozzi M; Palomo V; Redondo M; Fortugno C; Gil C; Felix G; Martinez A; Bertucci C

2012-04-01

298

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

2012-01-28

299

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.  

UK PubMed Central (United Kingdom)

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.

Montori-Grau M; Tarrats N; Osorio-Conles O; Orozco A; Serrano-Marco L; Vázquez-Carrera M; Gómez-Foix AM

2013-05-01

300

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

2013-02-20

 
 
 
 
301

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

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

Alao John P; Stavropoulou Alexandra V; Lam Eric; Coombes R Charles

2006-01-01

302

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; Lisa Samuelson; Cai-Bin Cui; Yang-Zhong Sun; David A. Gerber

2013-01-01

303

Two squalene synthase inhibitors, E5700 and ER-119884, interfere with cellular proliferation and induce ultrastructural and lipid profile alterations in a Candida tropicalis strain resistant to fluconazole, itraconazole, and amphotericin B.  

UK PubMed Central (United Kingdom)

Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16 ?g/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492 nm, respectively, after 48 h of incubation) of 1 and 4 ?g/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species.

Ishida K; Visbal G; Rodrigues JC; Urbina JA; de Souza W; Rozental S

2011-08-01

304

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

Science.gov (United States)

Glycogen synthase kinase-3beta (GSK3beta) role in human immunodeficiency virus(HIV)-associated neurodegeneration has been evidenced by previous investigations. In this study, we investigated the specificity of two GSK3beta-specific inhibitors, AR-A014418 (A) and B6B30 (B) to prevent direct neurotoxicity in primary human neurons exposed to HIV (BaL). Neurons were exposed to HIV (500 pg/ml) for 12-h and 6-day periods in the presence and absence of A (1 microM, 100 nM, 10 nM) and B (50 nM, 5 nM, 500 pM) to investigate acute and ongoing mechanisms of HIV neurotoxicity. Using an lactate dehydrogenase (LDH) assay to assess cytotoxicity, we observed a significant neurotoxic effect of HIV from control values (P < .01) that was not restored via coexposures of all concentrations of A and B. Additionally, no change in LDH levels were observed after 6 days. However, activity of the acute proapoptotic markers caspases 3 and 7 using a luminescence assay were measured and found to be increased by exposure to HIV (BaL) compared to controls (P = .022). This effect was ameliorated via coexposure to all concentrations of A and 50 nM B after 12 h (P < .01) and to all concentrations of A and B after 6 days (P < .01). Overall, the results from this study provide further evidence for the ability of GSK3beta inhibition to be neuroprotective against HIV-associated neurotoxicity by reducing HIV associated procaspase induction. These data support a role for GSK3beta as a potential therapeutic target and may have important clinical implications for treatment of HIV-associated neurocognitive disorder. PMID:19688630

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

2009-09-01

305

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

Science.gov (United States)

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

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

2006-11-02

306

Mechanism of inhibition of mitochondrial ATP synthase by 17?-estradiol.  

UK PubMed Central (United Kingdom)

17?-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling ("slipping") of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and VFCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and Volig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the Fo component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F1-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.

Moreno AJ; Moreira PI; Custódio JB; Santos MS

2013-06-01

307

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

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

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

2006-01-01

308

The spread of resistance to acetolactate synthase inhibiting herbicides in a wind borne, self-pollinated weed species, Lactuca serriola L.  

UK PubMed Central (United Kingdom)

Resistance to ALS-inhibiting herbicides in Lactuca serriola first appeared in the northern Yorke Peninsula in South Australia in 1994, with resistance soon observed at a number of additional sites. The rapid appearance of resistance at many sites could be attributed to a number of independent selection events or to movement of resistant seed from the original field. ISSRs were used to genotype plants collected in 1999 and 2004 from roadsides or fields in an attempt to determine the importance of these two factors in the spread of herbicide resistance in L. serriola. In 1999 and 2004, chlorsulfuron-resistant L. serriola plants were found in both fields and roadsides with resistant plants being more frequent in fields than roadsides and more frequent in 2004 than in 1999. Genetic relationships generated using UPGMA analysis indicated the presence of more than one genotype within the herbicide resistant populations sampled for both years and suggested independent selection as well as movement of resistant seed had occurred. DNA extracted from samples collected in 1999 was used to sequence a highly conserved region of the ALS gene that coded for a single amino acid modification within the gene. Four different mutations were identified within the resistant samples and these mutations tended to cluster on a geographical basis. Together these data provide evidence for both multiple independent evolutionary events and for the potential movement of individual genotypes as far as 43 km in the region.

Lu YQ; Baker J; Preston C

2007-08-01

309

The spread of resistance to acetolactate synthase inhibiting herbicides in a wind borne, self-pollinated weed species, Lactuca serriola L.  

Science.gov (United States)

Resistance to ALS-inhibiting herbicides in Lactuca serriola first appeared in the northern Yorke Peninsula in South Australia in 1994, with resistance soon observed at a number of additional sites. The rapid appearance of resistance at many sites could be attributed to a number of independent selection events or to movement of resistant seed from the original field. ISSRs were used to genotype plants collected in 1999 and 2004 from roadsides or fields in an attempt to determine the importance of these two factors in the spread of herbicide resistance in L. serriola. In 1999 and 2004, chlorsulfuron-resistant L. serriola plants were found in both fields and roadsides with resistant plants being more frequent in fields than roadsides and more frequent in 2004 than in 1999. Genetic relationships generated using UPGMA analysis indicated the presence of more than one genotype within the herbicide resistant populations sampled for both years and suggested independent selection as well as movement of resistant seed had occurred. DNA extracted from samples collected in 1999 was used to sequence a highly conserved region of the ALS gene that coded for a single amino acid modification within the gene. Four different mutations were identified within the resistant samples and these mutations tended to cluster on a geographical basis. Together these data provide evidence for both multiple independent evolutionary events and for the potential movement of individual genotypes as far as 43 km in the region. PMID:17628783

Lu, Y-Q; Baker, J; Preston, C

2007-07-13

310

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

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The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source ...

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

311

Aldosterone synthase inhibition in humans.  

UK PubMed Central (United Kingdom)

Aldosterone synthase (CYP11B2) inhibition has emerged as a new option for the treatment of hypertension, heart failure and renal disorders, in addition to mineralocorticoid receptor (MR) blockade. The aim is to decrease aldosterone concentrations in both plasma and tissues, thereby decreasing MR-dependent and MR-independent effects in the cardiac, vascular and renal target organs. LCI699 was the first orally active aldosterone-synthase inhibitor to be developed for human use. Its structure is similar to that of FAD286, the dextroenantiomer of the aromatase inhibitor, fadrozole. It dose-dependently decreases plasma and urine aldosterone concentrations by up to 70 or 80% and increases plasma renin activity in healthy male subjects on a low-sodium diet. LCI699 does not decrease basal plasma cortisol concentrations at doses of 0.5-3 mg q.d., but it blocks the cortisol response to adrenocorticotropic hormone (ACTH) at doses ? 3 mg q.d. In a proof-of-concept study in patients with primary aldosteronism (PA), LCI699 (0.5-1 mg b.i.d.) induced a dose-dependent and reversible 70-80% decrease in plasma and urinary aldosterone concentration accompanied by a massive dose-dependent accumulation of deoxycorticosterone (>+700%), the aldosterone precursor, in the plasma, thereby confirming the inhibition of the CYP11B2 gene product. This effect was associated with a rapid correction of hypokalaemia, a modest decrease in blood pressure (BP) and a mild increase in plasma renin concentration in patients with PA. LCI699 administration induced biological signs of partial inhibition of the glucocorticoid axis, such as dose-dependent increases in both plasma ACTH and 11-deoxycortisol (the precursor of cortisol) concentrations, consistent with the inhibition of the CYP11B1 gene product. An 8-week placebo-controlled dose-response study on patients with Stage 1 and 2 essential hypertension reported an optimal decrease in BP with a dose of 1 mg LCI699 q.d., which had an antihypertensive effect similar to that of 50 mg b.i.d. eplerenone. A blunted cortisol response to ACTH was observed in 20% of patients, but the clinical and biological safety and tolerability of LCI699 were similar to those of placebo and eplerenone. The discovery of this first orally active aldosterone synthase inhibitor, LCI699, has provided new opportunities to assess the feasibility and the haemodynamic, biological and safety consequences as well as the limitations of this new approach to block the aldosterone pathway in hypertensive patients. However, as the effects of LCI699 on the glucocorticoid axis limit the use of higher doses range because of the loss of selectivity for CYP11B2, this aldosterone synthase inhibitor cannot replace the MR blockade in patients with hypertension, other cardiovascular or renal disorders. The development of second-generation aldosterone synthase inhibitors with a higher selectivity index for CYP11B2 than LCI699 should make it possible to test this approach at much higher doses in these patients, after the necessary toxicology and Phase I studies.

Azizi M; Amar L; Menard J

2013-01-01

312

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids.  

UK PubMed Central (United Kingdom)

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Grazzini R; Hesk D; Heininger E; Hildenbrandt G; Reddy CC; Cox-Foster D; Medford J; Craig R; Mumma RO

1991-04-01

313

Nitric Oxide Synthase Inhibitors as Antidepressants  

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

Gregers Wegener; Vallo Volke

2010-01-01

314

Inhibitors of Fatty Acid Synthase (Fas)  

UK PubMed Central (United Kingdom)

The instant invention provides for compounds which comprise substituted 3-aryl-4-hydroxyquinolin-2(1H)-ones that inhibit FAS activity. The invention also provides for compositions comprising such inhibitory compounds and methods of inhibiting FAS activity by administering the compound to a patient in need of treatment of cancer.

GOULET MARK T; MUNOZ BENITO; RIVKIN ALEXEY A

315

Nitric Oxide Synthase Inhibitors as Antidepressants  

DEFF Research Database (Denmark)

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

Wegener, Gregers; Volke, Vallo

2010-01-01

316

ATP synthase: a molecular therapeutic drug target for antimicrobial and antitumor peptides.  

UK PubMed Central (United Kingdom)

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimicrobial/ antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of ?/? subunits on the F(1) sector of the enzyme. Molecular interaction of peptides at the ? DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented.

Ahmad Z; Okafor F; Azim S; Laughlin TF

2013-01-01

317

Structural organization of mitochondrial ATP synthase.  

UK PubMed Central (United Kingdom)

Specific modules and subcomplexes like F(1) and F(0)-parts, F(1)-c subcomplexes, peripheral and central stalks, and the rotor part comprising a ring of c-subunits with attached subunits gamma, delta, and epsilon can be identified in yeast and mammalian ATP synthase. Four subunits, alpha(3)beta(3), OSCP, and h, seem to form a structural entity at the extramembranous rotor/stator interface (gamma/alpha(3)beta(3)) to hold and stabilize the rotor in the holo-enzyme. The intramembranous rotor/stator interface (c-ring/a-subunit) must be dynamic to guarantee unhindered rotation. Unexpectedly, a c(10)a-assembly could be isolated with almost quantitive yield suggesting that an intermediate step in the rotating mechanism was frozen under the conditions used. Isolation of dimeric a-subunit and (c(10))(2)a(2)-complex from dimeric ATP synthase suggested that the a-subunit stabilizes the same monomer-monomer interface that had been shown to involve also subunits e, g, b, i, and h. The natural inhibitor protein Inh1 does not favor oligomerization of yeast ATP synthase. Other candidates for the oligomerization of dimeric ATP synthase building blocks are discussed, e.g. the transporters for inorganic phosphate and ADP/ATP that had been identified as constituents of ATP synthasomes. Independent approaches are presented that support previous reports on the existence of ATP synthasomes in the mitochondrial membrane.

Wittig I; Schägger H

2008-07-01

318

Effects of ALS-inhibitor herbicides, crop sequence, and fertilization on natural soil suppressiveness to Striga hermonthica  

UK PubMed Central (United Kingdom)

Striga hermonthica remains one of the greatest biological threats to cereal production in the savannahs of sub-Saharan Africa. Control efforts at the International Institute of Tropical Agriculture (IITA), Nigeria, focus on developing integrated S. hermonthica management (ISM) options such as legume-cereal rotation, use of host-plant resistance, soil-based biological control exploiting enhancement of naturally occurring biotic soil suppressiveness, and use of acetolactate synthase (ALS)-inhibiting herbicides as host-crop seed treatments. We investigated, in pots, if soybean crops with or without fertilizer (N, P, or NPK) and preceding a maize crop used as a bioassay enhanced biotic soil suppressiveness to S. hermonthica, and if the ALS-inhibitor herbicides, imazaquin and nicosulfuron, used to control weeds in preceding crops constitute any risk to this biotic system. Factors tested included: (1) crop preceding bioassay maize (soybean [EMGOPA] versuss maize [8338-1]); (2) herbicide weed control in preceding crop (imazaquin in soybean and nicosulfuron in maize versus hand weeding); (3) fertilizer application to preceding crop (90 kg N ha-1, 40 kg P ha-1; 90 kg NPK; versus no fertilizer); (4) soil treatment before planting bioassay maize (pasteurized soil versus non-pasteurized soil). Effects of treatments on biotic suppressiveness were evaluated by comparing effects of treatments in non-pasteurized soil with those of the same treatments in pasteurized soil. Results indicated that biotic soil suppressiveness to S. hermonthica existed naturally in the soil used and was enhanced by a preceding soybean crop and application of N, P or NPK fertilizers. Weed control using ALS-inhibiting herbicides in the preceding crops, particularly imazaquin applied in soybean, had a negative effect on natural soil suppressiveness to S. hermonthica parasitism in maize. Results of this study further confirm the biotic nature of soil suppressiveness to S. hermonthica, and stress its important role in ISM. Land-based management strategies for S. hermonthica control, such as legume crops in rotation to enhance soil N and fertilizer application appear to directly enhance soil suppressiveness to S. hermonthica. Because ALS-inhibiting herbicides pose a risk to biotic soil suppressiveness, their use as a primary control measure for S. hermonthica control in Africa may not be a sustainable approach.

Ahonsi MO; Berner DK; Emechebe AM; Lagoke ST

2004-12-01

319

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  

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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

320

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

Directory of Open Access Journals (Sweden)

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

2008-01-01

 
 
 
 
321

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 (more) 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.

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

2008-01-01

322

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

2009-09-08

323

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

UK PubMed Central (United Kingdom)

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).

Gollop N; Damri B; Chipman DM; Barak Z

1990-06-01

324

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

Science.gov (United States)

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1). PMID:2345154

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

1990-06-01

325

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

326

Dimers of mitochondrial ATP synthase form the permeability transition pore.  

UK PubMed Central (United Kingdom)

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

Giorgio V; von Stockum S; Antoniel M; Fabbro A; Fogolari F; Forte M; Glick GD; Petronilli V; Zoratti M; Szabó I; Lippe G; Bernardi P

2013-04-01

327

Dimers of mitochondrial ATP synthase form the permeability transition pore.  

Science.gov (United States)

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

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

2013-03-25

328

Characterization of domain interfaces in monomeric and dimeric ATP synthase.  

UK PubMed Central (United Kingdom)

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.

Wittig I; Velours J; Stuart R; Schägger H

2008-05-01

329

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

2001-01-01

330

Folate binding site of flavin-dependent thymidylate synthase.  

UK PubMed Central (United Kingdom)

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

Koehn EM; Perissinotti LL; Moghram S; Prabhakar A; Lesley SA; Mathews II; Kohen A

2012-09-01

331

Folate binding site of flavin-dependent thymidylate synthase.  

Science.gov (United States)

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs. PMID:23019356

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

2012-09-10

332

Herbicide resistances in Amaranthus tuberculatus: a call for new options.  

UK PubMed Central (United Kingdom)

Amaranthus tuberculatus is a major weed of crop fields in the midwestern United States. Making this weed particularly problematic to manage is its demonstrated ability to evolve resistance to herbicides. Herbicides to which A. tuberculatus has evolved resistance are photosystem II inhibitors, acetolactate synthase inhibitors, protoporphyrinogen oxidase inhibitors, and glyphosate. Many populations of A. tuberculatus contain more than one of these resistances, severely limiting the options for effective herbicide control. A survey of multiple-herbicide resistance in A. tuberculatus revealed that all populations resistant to glyphosate contained resistance to acetolactate synthase inhibitors, and 40% contained resistance to protoporphyrinogen oxidase inhibitors. The occurrences of multiple-herbicide resistances in A. tuberculatus illustrate the need for continued herbicide discovery efforts and/or the development of new strategies for weed management.

Tranel PJ; Riggins CW; Bell MS; Hager AG

2011-06-01

333

Monoterpene synthases from common sage (Salvia officinalis)  

Energy Technology Data Exchange (ETDEWEB)

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

334

SPINOSYN-PRODUCING POLYKETIDE SYNTHASES  

UK PubMed Central (United Kingdom)

The invention provides, biologically active spinosyns, hybrid spinosyn polyketide synthases capable of functioning in Saccharopolyspora spinosa to produce the spinosyns, and methods of controlling insects using the spinosyns.

BURNS LESLEY S; GRAUPNER PAUL R; LEWER PAUL; MARTIN CHRISTINE J; VOUSDEN WILLIAM A; WALDRON CLIVE; WILKINSON BARRIE

335

Spinosyn-producing polyketide synthases  

UK PubMed Central (United Kingdom)

The invention provides, biologically active spinosyns, hybrid spinosyn polyketide synthases capable of functioning in Saccharopolyspora spinosa to produce the spinosyns, and methods of controlling insects using the spinosyns.

BURNS LESLEY S; GRAUPNER PAUL R; LEWER PAUL; MARTIN CHRISTINE J; VOUSDEN WILLIAM A; WALDRON CLIVE; WILKINSON BARRIE

336

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

Directory of Open Access Journals (Sweden)

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

Serracino-Inglott Ferdinand; Virlos Ioannis T; Habib Nagy A; Williamson Robin CN; Mathie Robert T

2002-01-01

337

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis.  

UK PubMed Central (United Kingdom)

The F1F0 ATP synthase has recently become the focus of anti-cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto-ATP synthase-targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto-ATP synthase-targeted cancer therapies may facilitate the development of potent anti-tumor therapies, which target this enzyme and do not exhibit clinical limitations.

Wang WJ; Shi XX; Liu YW; He YQ; Wang YZ; Yang CX; Gao F

2013-07-01

338

Effect of a nitric oxide synthase inhibitor and a CXC chemokine receptor-4 antagonist on tumor growth and metastasis in a xenotransplanted mouse model of adenoid cystic carcinoma of the oral floor.  

Science.gov (United States)

Nitric oxide (NO) is related to angiogenesis and tumor progression and chemokine receptor-4 (CXCR4) plays a central role in cell migration in metastasis and dissemination of cancer. The present study evaluated the effectiveness of a NOS inhibitor and a CXCR4 antagonist, given as single agents or in combination, in a xenotransplanted mouse model of adenoid cystic carcinoma (ACC) of the oral floor. A metastatic tumor (ACCIM) derived from a cervical metastatic lesion of human ACC that was transplantable in nude mice was used. ACCIM showed a high frequency of spontaneous metastasis to the lung when transplanted subcutaneously in nude mice. Mice with subcutaneous transplants of ACCIM were subdivided into six groups and intraperitoneally received one of the following treatments daily for 5 weeks: a) PBS (control), b) AMD3100 (CXCR4 antagonist), c) L-NAME (NOS inhibitor), d) 1400W (iNOS inhibitor), e) both AMD3100 and L-NAME (AMD3100+L-NAME) and f) both AMD3100 and 1400W (AMD3100+1400W). Tumor growth was evaluated during treatment and metastasis was assessed at 28 weeks. Single-agent treatment with AMD3100, L-NAME or 1400W inhibited tumor growth by 20.8, 26.5 and 54.5%, respectively. Combined treatment with AMD3100+L-NAME and AMD3100+1400W inhibited tumor growth remarkably by 48.0 and 50.2%, respectively. Immunohistochemical analysis revealed lower expression of CXCR4, iNOS and eNOS in tumor cells treated with AMD3100+L-NAME or AMD3100+1400W compared to control tumor cells and increased numbers of apoptotic tumor cells were demonstrated using the TUNEL method. CXCR4 expression decreased in 1400W-treated tumors using western blot analysis. When the effect of each agent on tumor-induced angiogenesis in tumor stroma was examined histologically, microvessel density was significantly lower in the groups treated with 1400W, AMD3100+L-NAME or AMD3100+1400W compared to the control, AMD3100 and L-NAME groups. Moreover, treatment with AMD3100 or 1400W markedly inhibited lung metastasis. Our results indicated that single-agent treatment with 1400W and combined treatment with AMD3100+L-NAME or AMD3100+1400W induced apoptosis and significantly inhibited tumor-induced angiogenesis and proliferation of ACCIM in vivo. Blockade of CXCR4 and iNOS was suggested to inhibit lung metastases from ACCIM. CXCR4 and iNOS may, thus, be important prognostic factors for long-term survival in ACC. PMID:23835861

Takaoka, Kazuki; Hidaka, Sayaka; Hashitani, Susumu; Segawa, Emi; Yamamura, Michiyo; Tanaka, Noriaki; Zushi, Yusuke; Noguchi, Kazuma; Kishimoto, Hiromitsu; Urade, Masahiro

2013-07-08

339

Effect of a nitric oxide synthase inhibitor and a CXC chemokine receptor-4 antagonist on tumor growth and metastasis in a xenotransplanted mouse model of adenoid cystic carcinoma of the oral floor.  

UK PubMed Central (United Kingdom)

Nitric oxide (NO) is related to angiogenesis and tumor progression and chemokine receptor-4 (CXCR4) plays a central role in cell migration in metastasis and dissemination of cancer. The present study evaluated the effectiveness of a NOS inhibitor and a CXCR4 antagonist, given as single agents or in combination, in a xenotransplanted mouse model of adenoid cystic carcinoma (ACC) of the oral floor. A metastatic tumor (ACCIM) derived from a cervical metastatic lesion of human ACC that was transplantable in nude mice was used. ACCIM showed a high frequency of spontaneous metastasis to the lung when transplanted subcutaneously in nude mice. Mice with subcutaneous transplants of ACCIM were subdivided into six groups and intraperitoneally received one of the following treatments daily for 5 weeks: a) PBS (control), b) AMD3100 (CXCR4 antagonist), c) L-NAME (NOS inhibitor), d) 1400W (iNOS inhibitor), e) both AMD3100 and L-NAME (AMD3100+L-NAME) and f) both AMD3100 and 1400W (AMD3100+1400W). Tumor growth was evaluated during treatment and metastasis was assessed at 28 weeks. Single-agent treatment with AMD3100, L-NAME or 1400W inhibited tumor growth by 20.8, 26.5 and 54.5%, respectively. Combined treatment with AMD3100+L-NAME and AMD3100+1400W inhibited tumor growth remarkably by 48.0 and 50.2%, respectively. Immunohistochemical analysis revealed lower expression of CXCR4, iNOS and eNOS in tumor cells treated with AMD3100+L-NAME or AMD3100+1400W compared to control tumor cells and increased numbers of apoptotic tumor cells were demonstrated using the TUNEL method. CXCR4 expression decreased in 1400W-treated tumors using western blot analysis. When the effect of each agent on tumor-induced angiogenesis in tumor stroma was examined histologically, microvessel density was significantly lower in the groups treated with 1400W, AMD3100+L-NAME or AMD3100+1400W compared to the control, AMD3100 and L-NAME groups. Moreover, treatment with AMD3100 or 1400W markedly inhibited lung metastasis. Our results indicated that single-agent treatment with 1400W and combined treatment with AMD3100+L-NAME or AMD3100+1400W induced apoptosis and significantly inhibited tumor-induced angiogenesis and proliferation of ACCIM in vivo. Blockade of CXCR4 and iNOS was suggested to inhibit lung metastases from ACCIM. CXCR4 and iNOS may, thus, be important prognostic factors for long-term survival in ACC.

Takaoka K; Hidaka S; Hashitani S; Segawa E; Yamamura M; Tanaka N; Zushi Y; Noguchi K; Kishimoto H; Urade M

2013-09-01

340

ATP synthase: activating versus catalytic proton transfer.  

UK PubMed Central (United Kingdom)

ATP synthase (F-ATPase) of chloroplasts, CF0CF1, is both activated and driven by transmembrane protonmotive force. We dichotomized between activating and driving proton transfer by specific inhibitors, tentoxin and venturicidin. Thylakoids membranes were submitted to voltage steps (by flashing light) superimposed to a steady pH-difference. Transient proton intake, transfer and release by CF0CF1 was monitored by spectroscopic probes. Both activities, activation and catalysis, required all three partial reactions of the proton, however, activating proton transfer rose first (monophasically, tau 1/2 approximately 15 ms) followed by another phase of equal magnitude with a time lag of about 15 ms. Both types of consecutive proton transfer reactions contribute free energy for ATP synthesis.

Groth G; Junge W

1995-01-01

 
 
 
 
341

ATP synthase: activating versus catalytic proton transfer.  

Science.gov (United States)

ATP synthase (F-ATPase) of chloroplasts, CF0CF1, is both activated and driven by transmembrane protonmotive force. We dichotomized between activating and driving proton transfer by specific inhibitors, tentoxin and venturicidin. Thylakoids membranes were submitted to voltage steps (by flashing light) superimposed to a steady pH-difference. Transient proton intake, transfer and release by CF0CF1 was monitored by spectroscopic probes. Both activities, activation and catalysis, required all three partial reactions of the proton, however, activating proton transfer rose first (monophasically, tau 1/2 approximately 15 ms) followed by another phase of equal magnitude with a time lag of about 15 ms. Both types of consecutive proton transfer reactions contribute free energy for ATP synthesis. PMID:7828724

Groth, G; Junge, W

1995-01-23

342

Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschii  

Science.gov (United States)

In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-?-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2?Å resolution. The structure revealed that MjDHDPS forms a functional homo­tetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.

Padmanabhan, Balasundaram; Strange, Richard W.; Antonyuk, Svetlana V.; Ellis, Mark J.; Hasnain, S. Samar; Iino, Hitoshi; Agari, Yoshihiro; Bessho, Yoshitaka; Yokoyama, Shigeyuki

2009-01-01

343

Thymidylate Synthase Gene and Metastasis.  

Science.gov (United States)

Thymidylate synthase (TYMS) gene amplification was observed in 23% of 31 5-FU resistant liver metastases, while no amplification was observed in metastases of patients that had not been treated with 5-FU. Patients with metastases containing TYMS amplifica...

C. Lengauer K. W. Kinzler L. Diaz T. L. Wang V. Velculescu

2004-01-01

344

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

UK PubMed Central (United Kingdom)

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

Hwang EI; Ahn BT; Lee HB; Kim YK; Lee KS; Bok SH; Kim YT; Kim SU

2001-08-01

345

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

Science.gov (United States)

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

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

2001-08-01

346

Lipophilic iminosugars : synthesis and evaluation as inhibitors of glucosylceramide metabolism  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and ?-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of...

Wennekes, Tom

347

Mechanism of Action and Inhibition of dehydrosqualene Synthase  

Energy Technology Data Exchange (ETDEWEB)

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

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

2011-12-31

348

Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts.  

UK PubMed Central (United Kingdom)

A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, glucan synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of glucan synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 degrees C and were shown to be non-competitive and non-specific inhibitors of glucan synthase.

Mackichan J; Thomsen L; Kerwin J; Latgé JP; Beauvais A

1995-10-01

349

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

350

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

Directory of Open Access Journals (Sweden)

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

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

2005-01-01

351

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

Scientific Electronic Library Online (English)

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

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

2005-01-01

352

Two distinct proton binding sites in the ATP synthase family.  

Science.gov (United States)

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

von Ballmoos, Christoph; Dimroth, Peter

2007-10-02

353

Are rod outer segment ATP-ase and ATP-synthase activity expression of the same protein?  

UK PubMed Central (United Kingdom)

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg(2+)-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F1Fo-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F1Fo-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F1Fo ATP synthase in OS. Data suggest that the OS F1Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.

Calzia D; Candiani S; Garbarino G; Caicci F; Ravera S; Bruschi M; Manni L; Morelli A; Traverso CE; Candiano G; Tacchetti C; Panfoli I

2013-07-01