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Sample records for acetolactate synthase inhibitor

  1. MULTI-ANALYTE CHEMISTRY METHODS FOR PESTICIDES WHICH ARE ACETOLACTATE SYNTHASE (ALS) INHIBITORS IN SOIL

    Science.gov (United States)

    A joint EPA/state/industry working group has developed several multi-analyte methods to analyze soils for low ppb (parts per billion) levels of herbicides (such as sulfonylureas, imidazolinones, and sulfonamides) that are acetolactate synthase (ALS) inhibitors and may cause phyto...

  2. Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.).

    Science.gov (United States)

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

    2013-09-01

    Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I50 value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene. PMID:25149235

  3. 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)

    Ribas A. Vidal

    1999-12-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Scientific Electronic Library Online (English)

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

    2014-06-01

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

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

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

    2013-02-15

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

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

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

    Scarabel, Laura; Pernin, Fanny; Délye, Christophe

    2015-09-01

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

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

    Science.gov (United States)

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

    2009-12-01

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

  12. 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)

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

    2006-07-01

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

  13. Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana.

    Science.gov (United States)

    Mourad, G; King, J

    1992-11-01

    We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5' end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3' end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K. PMID:24178380

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Scientific Electronic Library Online (English)

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

    2013-12-01

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

  16. Novel inhibitors of nitric oxide synthase with antioxidant properties.

    Science.gov (United States)

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

    2012-03-01

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

  17. 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)

    D.L.P. Gazziero

    2006-01-01

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

  18. 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)

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

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

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

    Science.gov (United States)

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

    2015-03-12

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

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

    Scientific Electronic Library Online (English)

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

    2003-09-01

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

  1. Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I.

    Science.gov (United States)

    Belenky, Inna; Steinmetz, Andrea; Vyazmensky, Maria; Barak, Ze'ev; Tittmann, Kai; Chipman, David M

    2012-06-01

    Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS. PMID:22443469

  2. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  3. 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)

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

    OpenAIRE

    Ribas A. Vidal; Aldo Merotto Jr.

    1999-01-01

    O controle contínuo de plantas daninhas através da aplicação de herbicidas que apresentam atividade em um único local de ação nas plantas favorece a seleção de biótipos resistentes a estes herbicidas, em certas espécies vegetais. Quatro experimentos foram conduzidos em condições casa-de-vegetação, na Faculdade de Agronomia da Universidade Federal do Rio Grande do Sul, com os objetivos de avaliar a ocorrência de resistência aos herbicidas inibidores da enzima acetolactato sintase (ALS) em vári...

  5. Fatty acid synthase inhibitors isolated from Punica granatum L.

    Scientific Electronic Library Online (English)

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

    2012-05-01

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

  6. 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)

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

    2013-09-01

    Full Text Available 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 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 a [...] nd susceptible to acetolactate synthase (ALS) inhibiting herbicides. Experiments were conducted in a greenhouse and in a laboratory from October 2008 to February 2010. Two resistant 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.

  7. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

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

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

    OpenAIRE

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

    2000-01-01

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

  9. Structure-guided Design of Selective Inhibitors of Neuronal Nitric Oxide Synthase

    OpenAIRE

    Huang, He; Li, Huiying; Martásek, Pavel; Roman, Linda J; Poulos, Thomas L.; Richard B. Silverman

    2013-01-01

    Nitric oxide synthases (NOSs) comprise three closely related isoforms that catalyze the oxidation of l-arginine to l-citrulline and the important second messenger nitric oxide (NO). Pharmacological selective inhibition of neuronal NOS (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. Here we present a structure-guided, selective nNOS inhibitor design based on the crystal structure of lead compound 1 in nNOS. The best inhibitor, 7, exhibited low n...

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2015-12-01

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

  12. Fumonisins and fumonisin analogs as inhibitors of ceramide synthase and inducers of apoptosis.

    Science.gov (United States)

    Desai, Kena; Sullards, M Cameron; Allegood, Jeremy; Wang, Elaine; Schmelz, Eva M; Hartl, Michaela; Humpf, Hans-Ulrich; Liotta, D C; Peng, Qiong; Merrill, Alfred H

    2002-12-30

    Sphingoid bases are growth inhibitory and pro-apoptotic for many types of cells when added to cells exogenously, and can be elevated to toxic amounts endogenously when cells are exposed to inhibitors of ceramide synthase. An important category of naturally occurring inhibitors are the fumonisins, which inhibit ceramide synthase through structural similarities with both the sphingoid base and fatty acyl-CoA co-substrates. Fumonisins cause a wide spectrum of disease (liver and renal toxicity and carcinogenesis, neurotoxicity, induction of pulmonary edema, and others), and most-possibly all-of the pathophysiologic effects of fumonisins are attributable to disruption of the sphingolipid metabolism. The products of alkaline hydrolysis of fumonisins (which occurs during the preparation of masa flour for tortillas) are aminopentols that also inhibit ceramide synthase, but more weakly. Nonetheless, the aminopentols (and other 1-deoxy analogs of sphinganine) are acylated to derivatives that inhibit ceramide synthase, perhaps as product analogs, elevate sphinganine, and kill the cells. Somewhat paradoxically, fumonisins sometimes stimulate growth and inhibit apoptosis, possibly due to elevation of sphinganine 1-phosphate, which is known to have these cellular effects. These findings underscore the complexity of sphingolipid metabolism and the difficulty of identifying the pertinent mediators unless a full profile of the potentially bioactive species is evaluated. PMID:12531553

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

    OpenAIRE

    COSTAMAGNA, Costanzo; BOSIA, Amalia; ALLOATTI, Giuseppe; GALLO, Maria Pia; GHIGO, Dario Antonio; Levi, Renzo; PENNA, Claudia

    1998-01-01

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

  14. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

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

    OpenAIRE

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

    2003-01-01

    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...

  16. Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

    Science.gov (United States)

    Bartee, David; Morris, Francine; Al-Khouja, Amer; Freel Meyers, Caren L

    2015-08-17

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis. PMID:26174207

  17. Evaluation of Improved Glycogen Synthase Kinase-3? Inhibitors in Models of Acute Myeloid Leukemia.

    Science.gov (United States)

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

    2015-11-25

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

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

    Scientific Electronic Library Online (English)

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

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

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

    Directory of Open Access Journals (Sweden)

    Po-Huang Liang

    2008-03-01

    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.

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

    OpenAIRE

    Zhang, Yonghui; Cao, Rong; Yin, Fenglin; Hudock, Michael P.; Guo, Rey-Ting; Krysiak, Kilannin; Mukherjee, Sujoy; Gao, Yi-Gui; ROBINSON, Howard; Song, Yongcheng; No, Joo Hwan; Bergan, Kyle; Leon, Annette; Cass, Lauren; Goddard, Amanda

    2009-01-01

    Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anti-cancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the ...

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

    Directory of Open Access Journals (Sweden)

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

    2011-03-01

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

  2. Inducible Nitric Oxide Synthase Inhibitor SD-3651 Reduces Proteinuria in MRL/lpr Mice Deficient in the NOS2 Gene

    OpenAIRE

    Njoku, Chinedu; Self, Sally E.; Ruiz, Philip; Hofbauer, Ann F; Gilkeson, Gary S.; Oates, Jim C.

    2008-01-01

    Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FASlpr (MRL/lpr) mice lacking a functional NOS2 (inducible NOS [iNOS]) gene (NOS2?/?) develop proliferative glomerulonephritis in a fashion similar to their wild-type (wt) littermates. This finding suggests that the effect of arginine analog NOS inhibitors is through a non-iNOS–mediated mechanism. This study wa...

  3. Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease

    OpenAIRE

    Ashe, K M; Bangari, D.; Li, L.(Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China; Department of Modern Physics, University of Science and Technology of China, Hefei, Anhui, China; Department of Physics, Nanjing University, Nanjing, Jiangsu, China; School of Physics, Shandong University, Jinan, Shandong, China; Physics Department, Shanghai Jiao Tong University, Shanghai, China); Cabrera-Salazar, M.A.; Bercury, S.D.; Nietupski, J.B.; Cooper, C.G.F.; Aerts, J.M.F.G.; Lee, E R; Copeland, D.P.; Cheng, S. H.; Scheule, R. K.; Marshall, J.

    2011-01-01

    The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which ...

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

    Science.gov (United States)

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

    2013-01-01

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

  5. Progress in the development of fatty acid synthase inhibitors as anticancer targets.

    Science.gov (United States)

    Mullen, Genevieve E; Yet, Larry

    2015-10-15

    Fatty acid synthase (E.C. 2.3.1.85; FASN) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH and plays a central role in lipid biosynthesis. Two classes of FASN exist: FASN I in animals and fungi, and FASN II in plants and prokaryotes. Animal FASN I is a homodimeric protein found in the cytosol of lipogenic tissues such as the liver and brain. Many human carcinomas exhibit elevated levels of FASN I, though the benefit to cancer cells is still unclear. Inhibition of FASN I selectively effects apoptosis in cancer cells, and the role of FASN I in chemotherapy is a growing area of research with the use of natural products and small molecule inhibitors. PMID:26364942

  6. Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

    Science.gov (United States)

    Lim, Won-Mook; Baig, Irshad Jameel; La, Im Joung; Choi, Jung-Do; Kim, Dong-Eun; Kim, Sung-Kun; Hyun, Jae-Wook; Kim, Giyoung; Kang, Chang-Ho; Kim, Young Jin; Yoon, Moon-Young

    2011-12-01

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3?M, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1?M. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds. PMID:22015678

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

    Directory of Open Access Journals (Sweden)

    Wilson Abrao Saad

    2006-01-01

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

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

    Science.gov (United States)

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

    2010-07-15

    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

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

    OpenAIRE

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

    1996-01-01

    1. The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effec...

  10. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis.

    Science.gov (United States)

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C

    2015-07-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

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

    Scientific Electronic Library Online (English)

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

    1999-11-01

    Full Text Available It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg [...] /kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 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

  14. Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

    Science.gov (United States)

    Baig, Irshad Ahmed; Moon, Ji-Young; Lee, Sang-Choon; Ryoo, Sung-Weon; Yoon, Moon-Young

    2015-10-01

    Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36?g/ml and 6.24?g/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents. PMID:25988243

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

    Science.gov (United States)

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

    2009-01-01

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

  16. Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase

    Science.gov (United States)

    Lidor, O.; Al-Quntar, A.; Pesci, E. C.; Steinberg, D.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases. PMID:26593271

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-15

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

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sprenger, Janina [Lund University, SE-221 00 Lund (Sweden); Lund University, SE-221 84 Lund (Sweden); Svensson, Bo [Lund University, SE-221 00 Lund (Sweden); SARomics Biostructures AB, Box 724, SE-220 07 Lund (Sweden); Hålander, Jenny [Lund University, SE-221 00 Lund (Sweden); Carey, Jannette [Princeton University, Princeton, New Jersey (United States); Persson, Lo [Lund University, SE-221 84 Lund (Sweden); Al-Karadaghi, Salam, E-mail: salam.al-karadaghi@biochemistry.lu.se [Lund University, SE-221 00 Lund (Sweden)

    2015-03-01

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

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

    International Nuclear Information System (INIS)

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

  2. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    Science.gov (United States)

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having ?M antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. PMID:25900220

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

    Science.gov (United States)

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

    2011-01-01

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

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

    OpenAIRE

    Shaw, K. J.; Berg, C M

    1980-01-01

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

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

    OpenAIRE

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

    2007-01-01

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

  6. 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

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

    OpenAIRE

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

    2014-01-01

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

  8. Altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats.

    OpenAIRE

    Böhme, G. A.; Bon, C; Lemaire, M.; Reibaud, M; Piot, O; Stutzmann, J M; Doble, A; Blanchard, J C

    1993-01-01

    Nitric oxide (NO) is a messenger molecule that is produced in the brain from the metabolism of L-arginine to L-citrulline. Growing evidence suggests a physiological role for NO in long-term potentiation (LTP). Since LTP is a form of synaptic plasticity thought to be involved in learning and memory, we have tested whether inhibition of endogenous NO production affects memory capacities of rats. We found that the NO synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1....

  9. ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

    OpenAIRE

    Brownlie, R. P.; Brownrigg, N. J.; Butcher, H. M.; Garcia, R.; Jessup, R.; Lee, V. J.; Tunstall, S.; Wayne, M. G.

    1993-01-01

    1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human p...

  10. 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

    OpenAIRE

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

    2002-01-01

    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 re...

  11. Acylation of naturally occurring and synthetic 1-deoxysphinganines by ceramide synthase. Formation of N-palmitoyl-aminopentol produces a toxic metabolite of hydrolyzed fumonisin, AP1, and a new category of ceramide synthase inhibitor.

    Science.gov (United States)

    Humpf, H U; Schmelz, E M; Meredith, F I; Vesper, H; Vales, T R; Wang, E; Menaldino, D S; Liotta, D C; Merrill, A H

    1998-07-24

    Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro- and threo-2-amino, 3-hydroxy- (and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with Vmax/Km of 40-125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4-6 for the threo-isomers. Ceramide synthase also acylates AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a Vmax/Km of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro- and threo-isomers are acylated with the highest apparent Vmax/Km for the erythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins. PMID:9668088

  12. Application of electron spin resonance spin-trapping technique for evaluation of substrates and inhibitors of nitric oxide synthase.

    Science.gov (United States)

    Saito, Keita; Kohno, Masahiro

    2006-02-01

    The electron spin resonance (ESR) spin-trapping technique coupled with iron-dithiocarbamate complexes is one of the most specific methods for nitric oxide (NO) detection. In this study, we applied this method for the evaluation of the substrate and the inhibitors of NO synthase (NOS). A three-line ESR signal was detected from the mixture of inducible NOS (iNOS), l-arginine (Arg), nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin, dithiothreitol, and Fe(2+)-N-(dithiocarboxy) sarcosine (DTCS-Fe), and the signal intensity increased time-dependently. The signal was not observed by excluding either Arg or NADPH, and it was decreased by the addition of hemoglobin, which is an NO scavenger, and N(G)-monomethyl-l-arginine (l-NMMA), N(G)-nitro-l-arginine (l-NAME), and aminoguanidine (AG), which are NOS inhibitors, depending on the concentration. In comparison with l-NAME and AG, l-NMMA strongly inhibited iNOS activity. By using this method, the K(m) value of Arg and the K(i) value of l-NMMA for iNOS were determined to be 12.6 and 6.1muM, respectively. These values are consistent with the reported values measured by the oxyhemoglobin and citrulline assays. These results suggest that the ESR spin-trapping technique coupled with the iron-dithiocarbamate complex can be applied for the evaluation of substrates and inhibitors of NOS, and it would be a powerful tool due to its simplicity and high specificity to NO. PMID:16360110

  13. Inducible Nitric Oxide Synthase Inhibitor SD-3651 Reduces Proteinuria in MRL/lpr Mice Deficient in the NOS2 Gene

    Science.gov (United States)

    Njoku, Chinedu; Self, Sally E.; Ruiz, Philip; Hofbauer, Ann F.; Gilkeson, Gary S.; Oates, Jim C.

    2009-01-01

    Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FASlpr (MRL/lpr) mice lacking a functional NOS2 (inducible NOS [iNOS]) gene (NOS2?/?) develop proliferative glomerulonephritis in a fashion similar to their wild-type (wt) littermates. This finding suggests that the effect of arginine analog NOS inhibitors is through a non-iNOS–mediated mechanism. This study was designed to address this hypothesis. NOS2?/? mice were given either vehicle or a NOS inhibitor (SD-3651) to determine if pharmacological NOS inhibition prevented glomerulonephritis, using wt mice as positive controls. Urine was collected fortnightly to measure albumin. At the time of full disease expression in wt mice, all mice were killed, and renal tissue was examined for light, immunofluorescence, and electron microscopic evidence of disease. Serum was analyzed for anti–double-stranded DNA antibody production. NOS2?/? mice had higher serum anti–double-stranded DNA antibody antibody levels than those of wt mice. SD-3651 therapy reduced proteinuria, glomerular immunoglobulin G deposition, and electron microscopic evidence of podocytopathy and endothelial cell swelling without affecting proliferative lesions by light microscopy. These studies confirm that genetic iNOS deficiency alone is insufficient to prevent proliferative glomerulonephritis and suggest that iNOS activity may inhibit autoantibody production. These results also suggest that SD-3651 therapy acts via a non–iNOS-mediated mechanism to prevent endothelial cell and podocyte pathology. Studies that elucidate this mechanism could provide a useful drug target for the treatment of nephritis. PMID:18797415

  14. Yeast glycogen synthase kinase-3beta pathway inhibitors from an organic extract of Streptomyces sp.

    Science.gov (United States)

    Cheenpracha, Sarot; Zhang, Hui; Mar, Annie M N; Foss, Adam P; Foo, Sek Hin; Lai, Ngit Shin; Jee, Jap Meng; Seow, Heng Fong; Ho, Coy Choke; Chang, Leng Chee

    2009-08-01

    Investigation of a microbial fermentation organic extract of Streptomyces sp. H7667 led to the isolation of three new imides, 3-[(5E)-5-methyl-4-oxo-2-hydroxy-5-octenyl]glutarimide (1), 2-amino-N-2'-(phenylacetyl)propanimide (5), and 2-amino-N-(2'-(cyclohex-2''-enyl)acetyl)acetimide (6), and one new isoflavonoid glycoside, 6-O-methyl-7-O-alpha-rhamnopyranosyldaidzein (7), along with four known compounds. Their structures were elucidated by HRESIMS, 1H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra. Compounds 1-8 were evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3beta assay. PMID:19711989

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

    OpenAIRE

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

    2008-01-01

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

  16. Endogenous Nitric Oxide Synthase Inhibitors in Sickle Cell Disease: Abnormal Levels and Correlations with Pulmonary Hypertension, Desaturation, Hemolysis, Organ Dysfunction and Death

    OpenAIRE

    Kato, Gregory J.; Wang, Zeneng; Machado, Roberto F.; Blackwelder, William C.; Taylor, James G; HAZEN, STANLEY L.

    2008-01-01

    Pulmonary hypertension (PH) in patients with sickle cell disease (SCD) is linked to intravascular hemolysis, impaired nitric oxide bioavailability, renal dysfunction, and early mortality. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases (NOS), is associated with vascular disease in other populations. We determined the plasma concentrations for several key arginine metabolites and their relationships to clinical variables in 177 patients with SCD and 29 con...

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain

    Science.gov (United States)

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15?mg/kg?1, i.p., 7 days); group treated with 7-NI (25?mg/kg?1, i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels.

  20. CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

    Science.gov (United States)

    Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-02-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  1. CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

    Science.gov (United States)

    Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-01-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

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

    Science.gov (United States)

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

    2015-04-01

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

  3. In Vitro Activity of a New Oral Glucan Synthase Inhibitor (MK-3118) Tested against Aspergillus spp. by CLSI and EUCAST Broth Microdilution Methods

    OpenAIRE

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

    2013-01-01

    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 (MEC90)/MIC90 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 fum...

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

    Science.gov (United States)

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

    2009-10-01

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

  5. Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin A, a nitrated phytotoxin produced by Streptomyces spp.

    Science.gov (United States)

    Wach, Michael J; Kers, Johan A; Krasnoff, Stuart B; Loria, Rosemary; Gibson, Donna M

    2005-02-01

    Evidence for the involvement of a bacterial nitric oxide synthase (NOS) in the biosynthesis of a phytotoxin is presented. Several species of Streptomyces bacteria produce secondary metabolites with unusual nitrogen groups, such as thaxtomin A (ThxA), which contains a nitroindole moiety. ThxA is a phytotoxin made by three pathogenic Streptomyces species that cause common scab of potato. All three species possess a gene homologous to the oxygenase domain of murine inducible NOS, and this gene, nos, is essential for normal levels of ThxA production. We grew Streptomyces turgidiscabies in the presence of several known NOS inhibitors and a nitric oxide (NO) scavenger to determine their effect on ThxA production. The NO scavenger (CPTIO) and four NOS inhibitors (NAME, NMMA, AG, and 7-NI) reduced ThxA production without affecting bacterial growth. A strain of S. turgidiscabies from which the nos gene had been deleted was grown in the presence of three NO donors (DEANO, SIN, and SNAP), and all three partially restored ThxA production. Our data suggest that bacterial nitric oxide synthases may, at least in part, produce NO for biosynthetic purposes, rather than for cellular signaling, as they do in mammals. PMID:15631947

  6. Tetrahydropyrroloquinolinone type dual inhibitors of aromatase/aldosterone synthase as a novel strategy for breast cancer patients with elevated cardiovascular risks.

    Science.gov (United States)

    Yin, Lina; Hu, Qingzhong; Hartmann, Rolf W

    2013-01-24

    The application of aromatase inhibitors to postmenopausal breast cancer patients increases the risk of cardiovascular diseases (CVD), which is believed to be caused by the abnormally high concentrations of aldosterone as a consequence of the estrogen deficiency. Dual inhibitors of aromatase (CYP19) and aldosterone synthase (CYP11B2) are therefore proposed as a novel strategy for the adjuvant therapy to reduce the CVD risk for these patients. By combining decisive structural features of CYP11B2 and CYP19 inhibitors into a common template, a series of pyridinylmethyl substituted 1,2,5,6-tetrahydro-pyrrolo[3,2,1-ij]quinolin-4-ones were designed and synthesized. Interestingly, the substituents on the methylene bridge showed strong influences on the inhibitory activities leading to opposite effects, that is, a given substituent showed an increase in inhibition of one enzyme, while it led to a decrease for the other enzyme. The compromise of this conflict led to compounds 3j, 3k, 3n, and 3p as potent and selective dual inhibitors of CYP19 and CYP11B2, especially compound 3p, which exhibited IC(50) values of 32 and 41 nM for CYP19 and CYP11B2, respectively, and a high selectivity toward CYP17 and CYP11B1. This compound is considered as a candidate for further evaluation in vivo. PMID:23281812

  7. Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2014-07-01

    Full Text Available A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS. Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS. The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration.

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

    OpenAIRE

    Blomqvist, K; Suihko, M.-L.; Knowles, J.; Penttilä, M

    1991-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

    von Leitner, E.C.; Klinke, A.; Atzler, D.; Slocum, J.L.; Lund, N.; Kielstein, J.T.; Maas, R.; Schmidt-Haupt, R.; Pekarová, Michaela; Hellwinkel, O.; Tsikas, D.; D'Alecy, L.G.; Lau, D.; Willems, S.; Kubala, Lukáš; Ehmke, H.; Meinertz, T.; Blankenberg, S.; Schwedhelm, E.; Gadegbeku, C.A.; Boger, R.H.; Baldus, S.; Sydow, K.

    2011-01-01

    Ro?. 124, ?. 4 (2011), s. 2735-U342. ISSN 0009-7322 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : arteriosclerosis * leukocytes * nitric oxide synthase Subject RIV: BO - Biophysics Impact factor: 14.739, year: 2011

  10. Novel aldosterone synthase inhibitors with extended carbocyclic skeleton by a combined ligand-based and structure-based drug design approach.

    Science.gov (United States)

    Lucas, Simon; Heim, Ralf; Negri, Matthias; Antes, Iris; Ries, Christina; Schewe, Katarzyna E; Bisi, Alessandra; Gobbi, Silvia; Hartmann, Rolf W

    2008-10-01

    Pharmacophore modeling of a series of aldosterone synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)-N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors (e.g., 17, IC50 = 2.7 nM) with pronounced selectivity toward the most important steroidogenic and hepatic CYP enzymes. PMID:18763754

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

    Science.gov (United States)

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

    2004-01-01

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

  12. Comparative evaluation of the efficacy of the cyclooxygenase pathway inhibitor and nitric oxide synthase inhibitor in the reduction of alveolar bone loss in ligature induced periodontitis in rats: An experimental study

    Directory of Open Access Journals (Sweden)

    Rekha Jagadish

    2014-01-01

    Full Text Available Background: Alveolar bone loss is the most striking feature of periodontal disease. The aim of this study was to investigate the effect of a cyclooxygenase (COX pathway inhibitor and nitric oxide synthase (NOS inhibitor in the reduction of alveolar bone loss in an experimental periodontal disease (EPD model. Materials and Methods: The study was conducted on 60 Wistar rats divided into three groups of 20 rats each and then subjected to a ligature placement around the left maxillary second molars. Group 1 rats were treated with COX inhibitor (diclofenac sodium 10 mg/kg/d, group 2 with NOS inhibitor (aminoguanidine hydrochloride 10 mg/kg/d and group 3 served as controls, receiving only saline, intraperitoneally 1h before EPD induction and daily until the sacrifice on the 11 th day. Leukogram was performed before ligation, at 6 h and at the first, seventh and 11 th days after EPD induction. After sacrifice, all the excised maxillae were subjected to morphometric and histometric analysis to measure the alveolar bone loss. Histopathological analysis was carried out to estimate cell influx, alveolar bone and cementum integrity. Results: Induction of experimental periodontitis in the rat model produced pronounced leucocytosis, which was significantly reduced by the administration of diclofenac sodium and aminoguanidine on the 11 th day. In morphometric and histometric examinations, both the test drugs significantly (P < 0.05 inhibited the alveolar bone loss as compared with the control group. Conclusion: Both COX inhibitor and NOS inhibitor are equally effective in inhibiting the inflammatory bone resorption in an experimental periodontitis model.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-15

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

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

    International Nuclear Information System (INIS)

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

  15. Dose effects of chronically infused nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester on anabolic response and arginine metabolism in rats with subacute peritonitis.

    Science.gov (United States)

    Hsiao, Chien-Chou; Lee, Chien-Hsing; Tsao, Lon-Yen; Lo, Hui-Chen

    2011-01-01

    Nitric oxide synthase (NOS) inhibitors alleviate the adverse effects of nitric oxide (NO) overproduction that occurs during peritonitis, a clinical condition that is accompanied by arginine deficiency. However, the variations in the disease severity and the dosage, route, and period of NOS inhibitor administration are debatable. Therefore, we investigated the dose effects of chronically infused NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) on the anabolism, inflammatory responses, and arginine metabolism in parenterally fed rats with cecal puncture-induced subacute peritonitis. Male Wistar rats were divided into 4 groups and were administered total parenteral nutrition solutions with 0, 5 (low dose), 25 (medium dose), or 50 (high dose) mg·kg(-1)·d(-1) of L-NAME for 7 d. Sham-operated rats administered total parenteral nutrition solution and normal healthy rats fed chow diet were also included. Our results showed that parenteral infusion significantly decreased body weight gain and plasma citrulline concentrations. In rats with subacute peritonitis, the parenteral infusion-induced increases in circulating white blood cells and NO were significantly decreased, whereas the decrease in serum albumin levels was significantly increased. Rats with subacute peritonitis that were administered chronic infusion of L-NAME had a significantly reduced nitrogen balance. In addition, rats administered the medium dose of L-NAME had significantly increased plasma arginine, ornithine, glutamate, and proline. In conclusion, chronic infusion of NOS inhibitors may not alter systemic NO homeostasis and inflammatory response but may facilitate the production of arginine-associated amino acids and nitrogen excretion in cases of subacute peritonitis. PMID:21415524

  16. Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate.

    Science.gov (United States)

    Pestel, Sabine; Nath, Annegret; Jungermann, Kurt; Schieferdecker, Henrike L

    2003-08-15

    Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver. PMID:12906929

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-29

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

  18. The effect of an specific inducible NO synthase inhibitor, S-methylisothiourea hemisulfate on cisplatin-induced nephrotoxicity; gender-related differences

    Science.gov (United States)

    Ghayyoomi, Mansooreh; Soltani, Nepton; Nematbakhsh, Mehdi; Moslemi, Fatemeh; Talebi, Ardeshir; Shirdavani, Soheila; Razmjoo, Farzaneh

    2015-01-01

    Backgrounds: It has been previously demonstrated that the increase of nitric oxide (NO) level may promote cisplatin (CP)-induced nephrotoxicity. The aim of this study was to investigate the role of inducible NO synthase (iNOS) inhibitor to prevent CP-induced nephrotoxicity. Materials and Methods: Four groups of male and four groups of female rats were treated daily with vehicle, S-methylisothiourea hemisulfate (SMT) as a selective iNOS inhibitor (5 mg/kg/twice a day), CP (2.5 mg/kg/day), and CP + SMT for 6 days. Then, all animals were sacrificed and the serum levels of creatinine (Cr), blood urea nitrogen (BUN), nitrite, and malondialdehyde (MDA) were measured. The kidney was removed immediately for histopathological study. Results: Our results showed that inhibition of iNOS by SMT could make different response in male and female animals. SMT therapy in male animals decreased serum BUN, Cr, nitrite, and MDA levels; and it also protected kidney against CP-induced nephrotoxicity. Conclusion: It is concluded that decrease in NO production by SMT has a beneficial effect on reducing CP-induced nephrotoxicity in male. However, such beneficial effect was not observed in female animals. PMID:26322278

  19. Overcoming undesirable CYP1A2 inhibition of pyridylnaphthalene-type aldosterone synthase inhibitors: influence of heteroaryl derivatization on potency and selectivity.

    Science.gov (United States)

    Heim, Ralf; Lucas, Simon; Grombein, Cornelia M; Ries, Christina; Schewe, Katarzyna E; Negri, Matthias; Müller-Vieira, Ursula; Birk, Barbara; Hartmann, Rolf W

    2008-08-28

    Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats. PMID:18672861

  20. 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)

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

    2008-12-25

    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

  1. Comparative modeling and virtual screening for the identification of novel inhibitors for myo-inositol-1-phosphate synthase.

    Science.gov (United States)

    Azam, Syed Sikander; Sarfaraz, Sara; Abro, Asma

    2014-08-01

    Myo-inositol-1-phosphate (MIP) synthase is a key enzyme in the myo-inositol biosynthesis pathway. Disruption of the inositol signaling pathway is associated with bipolar disorders. Previous work suggested that MIP synthase could be an attractive target for the development of anti-bipolar drugs. Inhibition of this enzyme could possibly help in reducing the risk of a disease in patients. With this objective, three dimensional structure of the protein was modeled followed by the active site prediction. For the first time, computational studies were carried out to obtain structural insights into the interactive behavior of this enzyme with ligands. Virtual screening was carried out using FILTER, ROCS and EON modules of the OpenEye scientific software. Natural products from the ZINC database were used for the screening process. Resulting compounds were docked into active site of the target protein using FRED (Fast Rigid Exhaustive Docking) and GOLD (Genetic Optimization for Ligand Docking) docking programs. The analysis indicated extensive hydrogen bonding network and hydrophobic interactions which play a significant role in ligand binding. Four compounds are shortlisted and their binding assay analysis is underway. PMID:24752405

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

    Directory of Open Access Journals (Sweden)

    Weiss Ingrid M

    2007-11-01

    Full Text Available Abstract Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc. The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 ?M – 10 ?M dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

  3. 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)

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

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18{_}V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 {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.

  4. Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

    Science.gov (United States)

    Cioccoloni, Giorgia; Bonmassar, Laura; Pagani, Elena; Caporali, Simona; Fuggetta, Maria Pia; Bonmassar, Enzo; D'Atri, Stefania; Aquino, Angelo

    2015-08-01

    Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis. PMID:26035182

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

    Science.gov (United States)

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

    2011-04-01

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

  6. Induction of small intestinal damage in rats following combined treatment with cyclooxygenase-2 and nitric-oxide synthase inhibitors.

    Science.gov (United States)

    Ohno, Ryoko; Yokota, Aya; Tanaka, Akiko; Takeuchi, Koji

    2004-08-01

    Nitric oxide (NO) produced by constitutively expressed NO synthase (cNOS) plays an important role in maintaining the mucosal integrity of the small intestine, in collaboration with prostaglandins produced by cyclooxygenase (COX)-1. We examined whether intestinal damage is provoked in rats under inhibition of both cNOS and COX-2. The animals were given L-NAME (N(G)-nitro-L-arginine methyl ester), aminoguanidine, or rofecoxib, either alone or in combination, and killed 24 h later. Neither L-NAME nor aminoguanidine alone provoked damage in the small intestinal mucosa within 24 h, yet L-NAME produced damage in a L-arginine-sensitive manner when administered together with rofecoxib. L-NAME up-regulated the expression of COX-2 mRNA, and the prostaglandin E(2) (PGE(2)) content following the L-NAME administration significantly increased 12 h later, in both a rofecoxib- and a L-arginine-inhibitable manner. L-NAME enhanced intestinal motility, decreased mucus secretion, and increased the number of bacteria in the mucosa. The up-regulation of COX-2 expression and PGE(2) production by L-NAME was inhibited by prior administration of atropine, at a dose that inhibited the intestinal hypermotility. The intestinal lesions induced by L-NAME plus rofecoxib were prevented by pretreatment with ampicillin and aminoguanidine as well as atropine, indicating the involvement of bacteria, inducible nitric oxide synthase, and hypermotility in the pathogenesis. These results suggest that inhibition of both cNOS and COX-2 provokes intestinal damage, similar to inhibition of both COX-1 and COX-2. Inhibition of cNOS, similar to COX-1, up-regulates COX-2 expression, the process being associated with intestinal hypermotility and bacterial invasion, and this may be a key to the occurrence of intestinal damage associated with COX-2 inhibition. PMID:15044560

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-15

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

  9. Probing the interaction between prostacyclin synthase and prostaglandin H2 analogues or inhibitors via a combination of resonance Raman spectroscopy and molecular dynamics simulation approaches.

    Science.gov (United States)

    Chao, Wei-Chih; Lu, Jyh-Feng; Wang, Jinn-Shyan; Yang, Hsiao-Ching; Chen, Hsiao-Hui; Lan, Yi-Kang; Yu, Ya-Chien; Chou, Pi-Tai; Wang, Lee-Ho

    2011-11-23

    In an aim to probe the structure-function relationship of prostacyclin synthase (PGIS), resonance Raman (RR) spectroscopy and molecular dynamic (MD) simulation approaches have been exploited to characterize the heme conformation and heme-protein matrix interactions for human PGIS (hPGIS) and zebrafish PGIS (zPGIS) in the presence and absence of ligands. The high-frequency RR (1300-1700 cm(-1)) indicates that the heme group is in the ferric, six-coordinate, low-spin state for both resting and ligand-bound hPGIS/zPGIS. The low-frequency RR (300-500 cm(-1)) and MD simulation reveal a salient difference in propionate-protein matrix interactions between hPGIS and zPGIS, as evident by a predominant propionate bending vibration at 386 cm(-1) in resting hPGIS, but two vibrations near 370 and 387 cm(-1) in resting zPGIS. Upon binding of a substrate analogue (U46619, U51605, or U44069), both hPGIS and zPGIS induce a distinctive perturbation of the propionate-protein matrix interactions, resulting in similar Raman shifts to ~381 cm(-1). On the contrary, the bending vibration remains unchanged upon binding of inhibitor/ligand (minoxidil, clotrimazole, or miconazole), indicating that these inhibitors/ligands do not interfere with the propionate-protein matrix interactions. These results, together with subtle changes in vinyl bending modes, demonstrate drastically different RR shifts with heme conformational changes in both hPGIS and zPGIS upon different ligand bindings, suggesting that PGIS exhibits a ligand-specific heme conformational change to accommodate the substrate binding. This substrate-induced modulation of the heme conformation may confer high product fidelity upon PGIS catalysis. PMID:21978190

  10. 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)

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

    Full Text Available INTRODUCTION: The role of ovarian hormones and nitric oxide in learning and memory has been widely investigated. OBJECTIVE: The present study was carried out to evaluate the effect of the nitric oxide synthase (NOS) inhibitor, N (G)-nitro-L-arginine methyl ester (L-NAME), on the ability of estradiol [...] to improve learning in OVX rats using the Morris water maze. METHODS: Forty rats were divided into five groups: (1) ovariectomized (OVX), (2) ovariectomized-estradiol (OVX-Est), (3) ovariectomized-L-NAME 10 (OVX-LN 10), (4) ovariectomized-L-NAME 50 (OVX-LN 50) and (5) ovariectomized-estradiol-L-NAME 50 (OVX-Est-LN 50). The animals in the OVX-Est group were treated with a weekly injection of estradiol valerate (2 mg/kg; i.m.). The OVX-LN 10 and OVX-LN 50 groups were treated with daily injections of 10 and 50 mg/kg L-NAME (i.p.), respectively. The animals in the OVX-Est-LN 50 group received a weekly injection of estradiol valerate and a daily injection of 50 mg/kg L-NAME. After 8 weeks, all animals were tested in the Morris water maze. RESULTS: The animals in the OVX-Est group had a significantly lower latency in the maze than the OVX group (p

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

    Directory of Open Access Journals (Sweden)

    Cho Yu

    2010-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Mona M Soliman

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-12-15

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

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Scientific Electronic Library Online (English)

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

    2010-11-01

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

  16. Valencene synthase

    OpenAIRE

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

    2011-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Suborov Evgeny V

    2012-06-01

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

  18. Effects of the combined thromboxane receptor antagonist and synthase inhibitor DTTX-30 on intestinal O2-exchange and energy metabolism during hyperdynamic porcine endotoxemia.

    Science.gov (United States)

    Matejovic, M; Radermacher, P; Zülke, C; Vlatten, A; Altherr, J; Brinkmann, A; Brückner, U B; Jauch, K W; Georgieff, M; Träger, K

    2000-01-01

    Sepsis may lead to deranged thromboxane-prostacyclin ratio with consecutive organ dysfunction. Because of the suggested role of the gut in the pathogenesis of septic shock and multiple organ failure, we investigated the effects of the novel dual thromboxane synthase inhibitor and receptor antagonist DTTX-30 (TRASI) on intestinal tissue perfusion, O2 kinetics, and energy metabolism over 24 h of hyperdynamic, normotensive porcine endotoxemia. Before, 12, 18, and 24 h after starting continuous i.v. endotoxin (LPS), we measured portal venous (PV) blood flow, intestinal oxygen extraction (iO2ER), intracapillary hemoglobin O2 saturation (HbO2%) of the ileal wall, intramucosal ileal PCO2, PV lactate-pyruvate (L-P) ratio, and plasma levels of thromboxane and prostacyclin. Treatment with TRASI (0.12 mg/kg i.v. bolus injection followed by an infusion of 0.29 mg/kg/h) initiated after 12 h of LPS infusion markedly reduced the plasma thromboxane levels and attenuated the LPS-induced fall in systemic vascular resistance, resulting in hemodynamic stabilization. TRASI did not influence the LPS-induced increase in PV blood flow nor intracapillary HbO2%, thus reflecting unchanged microcirculatory O2 availability and decreased iO2ER, possibly because of reduced O2 requirements. Nevertheless, TRASI prevented the LPS-induced increase in the PV L-P ratio, attenuated the progression of the ileal mucosal-arterial PCO2 gap, and tended to attenuate the gradual fall of PV pH. Hence, compounds like TRASI may beneficially influence LPS-related derangements of gut energy metabolism. PMID:10774620

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

    Directory of Open Access Journals (Sweden)

    J. F. Fracasso

    2009-01-01

    Full Text Available

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

  20. L-arginine-induced growth hormone secretion is not influenced by co-infusion of the nitric oxide synthase inhibitor N-monomethyl-L-arginine in healthy men

    DEFF Research Database (Denmark)

    Fisker, S; Nielsen, S

    1999-01-01

    In animals, it has been demonstrated that nitric oxide (NO) is a potent neuroregulatory substance. By intravenous infusion, L-arginine is converted to NO and citrulline, but it is unknown whether NO is responsible for the GH stimulating effect of L-arginine in humans. We investigated whether intravenous infusion of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA) influenced L-arginine stimulated GH secretion. Ten healthy men, aged 28.6 +/- 1.9 (mean +/- SEM) years were examined twice. L-arginine was infused intravenously in a dose of 0.5 g/kg, max 35 g, from 0 to 30 min, accompanied by either: (1) L-NMMA from -5 to 0 min, in a dose of 3 mg/kg, max 250 mg, and in a dose of 3.5 mg/kg, max 250 mg from 0 to 60 min; or (2) a saline infusion. Heart rate increased (P = 0.032), and diastolic blood pressure decreased (P < 0.001) in the two situations. Plasma cGMP was unchanged and identical in the two situations (P = 0.679). Urine cGMP/creatinine ratio increased during both examinations (P = 0.041). Growth hormone secretion increased significantly during L-arginine infusion (P = < 0.001) without any effect of L-NMMA (P = 0.848). We did not find evidence that NO influences GH secretion. It remains to be tested, however, whether a higher dose of L-NMMA may influence L-arginine stimulated GH secretion.

  1. 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)

    Morteza Piri

    2011-01-01

    Full Text Available Introduction: Drugs of abuse such as nicotine and morphine used systemically by addicts produce their effects via the mesolimbic dopaminergic pathway. Furthermore, evidence indicates that some behavioral effects of nicotine and morphine are mediated by nitric oxide (NO. Based on these observations, the aim of the present study was to investigate the effects of intra-nucleus accumbens (NAc injection of a nitric oxide synthase (NOS inhibitor, L-NAME, on the nicotine’s effect on the morphine-induced amnesia. Methods: As a model of memory assessment, a step-through type passive avoidance task was used. All animals were bilaterally implanted with a chronic cannulae in the NAc shell and trained by using a 1 mA foot shock. Animals were tested 24 h after training to measure step-through latency. Results: Post-training injection of morphine impaired memory performance on the test day. Pre-test administration of the same doses of morphine reversed amnesia induced by post-training administration of morphine. Moreover, administration of nicotine before the test prevented morphine amnesia. Impairment of memory because of post-training injection of morphine was also prevented by pretest administration of L-NAME. Co-administration of an ineffective dose of nicotine with ineffective doses of L-NAME synergistically improved memory that was impaired by morphine. On the other hand, pre-test intra-NAc injection of L-NAME impaired passive avoidance memory by itself. Conclusion: Considering the effects of pre-test intra-NAc injection of L-NAME alone or in combination with ineffective dose of nicotine on morphine amnesia, it may be concluded that nitric oxide system of nucleus accumbens has an important role in the improvement of morphine-induced amnesia and morphine state-dependent memory caused by nicotine.

  2. Newly developed glycogen synthase kinase-3 (GSK-3) inhibitors protect neuronal cells death in amyloid-beta induced cell model and in a transgenic mouse model of Alzheimer's disease.

    Science.gov (United States)

    Noh, Min-Young; Chun, Kwangwoo; Kang, Byung Yong; Kim, Heejaung; Park, Ji-Seon; Lee, Han-Chang; Kim, Young-Ha; Ku, Saekwang; Kim, Seung Hyun

    2013-05-31

    Glycogen synthase kinase-3 (GSK-3) is emerging as a prominent therapeutic target of Alzheimer's disease (AD). A number of studies have been undertaken to develop GSK-3 inhibitors for clinical use. We report two novel GSK-3 inhibitors (C-7a and C-7b) showing good activity and pharmacokinetic (PK) profiles. IC50 of new GSK-3 inhibitors were in the range of 120-130 nM, and they effectively reduced the A?-oligomers induced neuronal toxicity. Also, new GSK-3 inhibitors decreased the phosphorylated tau at pThr231, pSer396, pThr181, and pSer202, and inhibited the GSK-3 activity against A?-oligomers induced neuronal cell toxicity. In B6;129-Psen1(tm1Mpm) Tg(APPSwe, tauP301L)1Lfa/Mmjax model of AD, oral administration of C-7a (20 mg/kg, 50 mg/kg) showed increased total arm entries and spontaneous alteration of Y-maze which was regarded as short-term memory. In particular, 50 mg/kg C-7a treated mice significantly decreased the level of phosphorylated tau (Ser396) in brain hippocampus. We suggest that new GSK-3 inhibitor (C-7a) is potential candidates for the treatment of AD. PMID:23632329

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

    Science.gov (United States)

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

    2012-10-01

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

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

    OpenAIRE

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

    2013-01-01

    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, revea...

  5. 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

    OpenAIRE

    He, Xin; Reeve, Anne McElwee; Desai, Umesh R.; KELLOGG, GLEN E.; Reynolds, Kevin A.

    2004-01-01

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

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

    OpenAIRE

    FERRERO, Aldo; Vidotto, Francesco; BUSI, Roberto

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Park Jaeok

    2012-12-01

    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.

  8. 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)

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

    2013-09-01

    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

  9. Discovery of 4-Aryl-5,6,7,8-tetrahydroisoquinolines as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys.

    Science.gov (United States)

    Martin, Rainer E; Aebi, Johannes D; Hornsperger, Benoit; Krebs, Hans-Jakob; Kuhn, Bernd; Kuglstatter, Andreas; Alker, André M; Märki, Hans Peter; Müller, Stephan; Burger, Dominique; Ottaviani, Giorgio; Riboulet, William; Verry, Philippe; Tan, Xuefei; Amrein, Kurt; Mayweg, Alexander V

    2015-10-22

    Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg(-1). This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man. PMID:26403853

  10. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    Energy Technology Data Exchange (ETDEWEB)

    Zanotti, Franco [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Inst. of Biomembranes and Bioenergetics, CNR, Bari (Italy); Gnoni, Antonio; Mangiullo, Roberto [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Papa, Sergio, E-mail: papabchm@cimedoc.uniba.it [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Inst. of Biomembranes and Bioenergetics, CNR, Bari (Italy)

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  11. A first-in-human phase I dose-escalation, pharmacokinetic, and pharmacodynamic evaluation of intravenous LY2090314, a glycogen synthase kinase 3 inhibitor, administered in combination with pemetrexed and carboplatin.

    Science.gov (United States)

    Gray, Jhanelle E; Infante, Jeffrey R; Brail, Les H; Simon, George R; Cooksey, Jennifer F; Jones, Suzanne F; Farrington, Daphne L; Yeo, Adeline; Jackson, Kimberley A; Chow, Kay H; Zamek-Gliszczynski, Maciej J; Burris, Howard A

    2015-12-01

    Purpose LY2090314 (LY) is a glycogen synthase kinase 3 inhibitor with preclinical efficacy in xenograft models when combined with platinum regimens. A first-in-human phase 1 dose-escalation study evaluated the combination of LY with pemetrexed/carboplatin. Patients and Methods Forty-one patients with advanced solid tumors received single-dose LY monotherapy lead-in and 37 patients received LY (10-120 mg) plus pemetrexed/carboplatin (500 mg/m(2) and 5-6 AUC, respectively) across 8 dose levels every 21 days. Primary objective was maximum tolerated dose (MTD) determination; secondary endpoints included safety, antitumor activity, pharmacokinetics, and beta-catenin pharmacodynamics. Results MTD of LY with pemetrexed/carboplatin was 40 mg. Eleven dose-limiting toxicities (DLTs) occurred in ten patients. DLTs during LY monotherapy occurred at ?40 mg: grade 2 visual disturbance (n?=?1) and grade 3/4 peri-infusional thoracic pain during or shortly post infusion (n?=?4; chest, upper abdominal, and back pain). Ranitidine was added after de-escalation to 80 mg LY to minimize peri-infusional thoracic pain. Following LY with pemetrexed/carboplatin therapy, DLTs included grade 3/4 thrombocytopenia (n?=?4) and grade 4 neutropenia (n?=?1). Best overall response by RECIST included 5 confirmed partial responses (non-small cell lung cancer [n?=?3], mesothelioma, and breast cancer) and 19 patients having stable disease. Systemic LY exposure was approximately linear over dose range studied. Transient upregulation of beta-catenin measured in peripheral blood mononuclear cells (PBMCs) occurred at 40 mg LY. Conclusions The initial safety profile of LY2090314 was established. MTD LY dose with pemetrexed/carboplatin is 40 mg IV every 3 weeks plus ranitidine. Efficacy of LY plus pemetrexed/carboplatin requires confirmation in randomized trials. PMID:26403509

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

    Scientific Electronic Library Online (English)

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

    2006-04-01

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

  13. 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)

    Renato Márcio Ribeiro Viana

    2008-01-01

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

  14. Nitric Oxide Synthase Inhibitors as Antidepressants

    DEFF Research Database (Denmark)

    Wegener, Gregers; Volke, Vallo

    2010-01-01

    Affective and anxiety disorders are widely distributed disorders with severe social and economic effects. Evidence is emphatic that effective treatment helps to restore function and quality of life. Due to the action of most modern antidepressant drugs, serotonergic mechanisms have traditionally 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.

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

    Science.gov (United States)

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

    1994-02-14

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

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

    Science.gov (United States)

    Yamano, S; Tanaka, J; Inoue, T

    1994-02-14

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

  17. Modulation of the Protein Kinase C? Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE, MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES*

    OpenAIRE

    Nguyen, Tiffany T.; Ogbi, Mourad; Yu, Qilin; Fishman, Jordan B.; Thomas, Warren; Harvey, Brian J.; Fulton, David; Johnson, John A.

    2010-01-01

    The F1F0-ATP synthase provides ?90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase C? (PKC?) inhibits F1F0 activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) 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 dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits...

  18. Cell Wall Polysaccharide Synthases Are Located in Detergent-Resistant Membrane Microdomains in Oomycetes ? †

    OpenAIRE

    Briolay, Anne; Bouzenzana, Jamel; Guichardant, Michel; Deshayes, Christian; Sindt, Nicolas; Bessueille, Laurence; Bulone, Vincent

    2009-01-01

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1?3)-?-d-glucan synthases are located in detergent-resi...

  19. Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine.

    Science.gov (United States)

    Young, R J; Beams, R M; Carter, K; Clark, H A; Coe, D M; Chambers, C L; Davies, P I; Dawson, J; Drysdale, M J; Franzman, K W; French, C; Hodgson, S T; Hodson, H F; Kleanthous, S; Rider, P; Sanders, D; Sawyer, D A; Scott, K J; Shearer, B G; Stocker, R; Smith, S; Tackley, M C; Knowles, R G

    2000-03-20

    The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform. PMID:10741561

  20. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    Science.gov (United States)

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. PMID:26562543

  1. Higher plant cellulose synthases

    OpenAIRE

    Richmond, Todd

    2000-01-01

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

  2. 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)

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

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

  3. Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

    Science.gov (United States)

    Thompson, Keith; Dunford, James E; Ebetino, Frank H; Rogers, Michael J

    2002-01-18

    We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase. PMID:11785983

  4. Synthesis and evaluation of selective inhibitors of aldosterone synthase (CYP11B2) of the naphthalene and dihydronaphthalene type for the treatment of congestive heart failure and myocardial fibrosis

    OpenAIRE

    Voets, Marieke

    2006-01-01

    The aim of this work was to design and synthesize potent and highly selective CYP11B2 inhibitors, which could be used for the treatment of congestive heart failure and myocardial fibrosis. We have synthesized different heteroaryl substituted naphthalenes, dihydronaphthalenes and indenes. The compounds were tested for inhibitory activity towards human CYP11B2 and the active inhibitors were also tested towards CYP11B1 to obtain information about selectivity. Selectivity towards other steroidoge...

  5. Dimers of mitochondrial ATP synthase form the permeability transition pore.

    Science.gov (United States)

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

    2013-04-01

    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

  6. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

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

    2002-01-01

    Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-...

  7. Geranyl diphosphate synthase from mint

    Science.gov (United States)

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

    1999-01-01

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

  8. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-03-02

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

  9. Dimers of mitochondrial ATP synthase form the permeability transition pore

    OpenAIRE

    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

    2013-01-01

    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 Ca2+ like CyPD itself, and that decreasing oligomycin sens...

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

    OpenAIRE

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

    2010-01-01

    The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F1 or membrane bound F1FO E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were mori...

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

    OpenAIRE

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

    2009-01-01

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

  12. Monoterpene synthases from common sage (Salvia officinalis)

    Science.gov (United States)

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

    1999-01-01

    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.

  13. Benzo[e]isoindole-1,3-diones as potential inhibitors of glycogen synthase kinase-3 (GSK-3). Synthesis, kinase inhibitory activity, zebrafish phenotype, and modeling of binding mode.

    Science.gov (United States)

    Zou, Haixia; Zhou, Liyan; Li, Yuanzhen; Cui, Yi; Zhong, Hanbing; Pan, Zhengying; Yang, Zhen; Quan, Junmin

    2010-02-11

    Benzo[e]isoindole-1,3-dione derivatives were synthesized, and the effects on GSK-3beta activity and zebrafish embryo growth were evaluated. A series of derivatives show obvious inhibitory activity against GSK-3beta. The most potent inhibitor, 7,8-dimethoxy-5-methylbenzo[e]isoindole-1,3-dione (8a), shows nanomolar IC(50) and obvious phenotype on zebrafish embryo growth associated with the inhibition of GSK-3beta at low micromolar concentration. The interaction mode between 8a and GSK-3beta was characterized by computational modeling. PMID:20030405

  14. Structure and Function of Microsomal Prostaglandin E Synthase-1

    OpenAIRE

    Pawelzik, Sven-Christian

    2010-01-01

    The glutathione-dependent enzyme microsomal prostaglandin E synthase-1 (MPGES1) plays a pivotal role in inflammatory diseases. MPGES1 is up-regulated by pro-inflammatory cytokines in concert with cyclooxygenase (COX) -2, and the concerted action of both enzymes leads to the production of induced prostaglandin E2 (PGE2), a potent lipid mediator of inflammation, pain, and fever. Non-steroidal anti-inflammatory drugs (NSAIDs) as well as COX-2 specific inhibitors (COXIBs) are widely u...

  15. Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo

    DEFF Research Database (Denmark)

    Volke, V; Wegener, Gregers; Vasar, E; Rosenberg, R

    1999-01-01

    The aim of the present study was to investigate the effect of methylene blue, a guanylate cyclase inhibitor, on the hippocampal nitric oxide synthase activity in vivo. We used a microdialysis-based technique of measuring conversion of [3H]l-arginine to [3H]l-citrulline in freely moving rats. The administration of methylene blue (0.1 and 1 mM) via the microdialysis probe caused a dose-dependent decrease in [3H]l-citrulline efflux comparable with the effect of unselective NOS inhibitor NG-nitro-L-...

  16. Cytological and comparative proteomic analyses on male sterility in Brassica napus L. induced by the chemical hybridization agent monosulphuron ester sodium

    Science.gov (United States)

    Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility i...

  17. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids; Hansen, Frederik; Overgaard, Michael Toft; Giese, Henriette; Søndergaard, Teis

    2014-01-01

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been described, even fewer from fungi and none from Fusarium species. Multidomain proteins can be quite challenging to work with, which is why the project intends to solve the 3D-structures of single domains of P...

  18. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  19. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    MarinaKorotkova

    2011-01-01

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

  20. Calcium-Dependent Nitric Oxide Synthase Activity in Rat Thymocytes

    OpenAIRE

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

    1998-01-01

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

  1. The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

    OpenAIRE

    Pidgeon, Graham; O'Byrne, Ken; Reynolds, John

    2010-01-01

    Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be pro-tumor, an anti-carcinogenic role for thromboxane synthase has been demonstrated in a variety of...

  2. Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites.

    Science.gov (United States)

    Ozer, Ufuk; Barbour, Karen W; Clinton, Sarah A; Berger, Franklin G

    2015-12-01

    Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming dTMP for the maintenance of DNA replication and repair. Inhibitors of TYMS have been widely used in the treatment of neoplastic disease. A number of fluoropyrimidine and folate analogs have been developed that lead to inhibition of the enzyme, resulting in dTMP deficiency and cell death. In the current study, we have examined the role of oxidative stress in response to TYMS inhibitors. We observed that intracellular reactive oxygen species (ROS) concentrations are induced by these inhibitors and promote apoptosis. Activation of the enzyme NADPH oxidase (NOX), which catalyzes one-electron reduction of O2 to generate superoxide (O2 (?-)), is a significant source of increased ROS levels in drug-treated cells. However, gene expression profiling revealed a number of other redox-related genes that may contribute to ROS generation. TYMS inhibitors also induce a protective response, including activation of the transcription factor nuclear factor E2-related factor 2 (NRF2), a critical mediator of defense against oxidative and electrophilic stress. Our results show that exposure to TYMS inhibitors induces oxidative stress that leads to cell death, while simultaneously generating a protective response that may underlie resistance against such death. PMID:26443810

  3. Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes.

    Science.gov (United States)

    Briolay, Anne; Bouzenzana, Jamel; Guichardant, Michel; Deshayes, Christian; Sindt, Nicolas; Bessueille, Laurence; Bulone, Vincent

    2009-04-01

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth. PMID:19201970

  4. Chemical inhibitors of monogalactosyldiacylglycerol synthases in Arabidopsis thaliana.

    Science.gov (United States)

    Botté, Cyrille Y; Deligny, Michael; Roccia, Aymeric; Bonneau, Anne-Laure; Saïdani, Nadia; Hardré, Hélène; Aci, Samia; Yamaryo-Botté, Yoshiki; Jouhet, Juliette; Dubots, Emmanuelle; Loizeau, Karen; Bastien, Olivier; Bréhélin, Laurent; Joyard, Jacques; Cintrat, Jean-Christophe; Falconet, Denis; Block, Maryse A; Rousseau, Bernard; Lopez, Roman; Maréchal, Eric

    2011-11-01

    Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main lipids in photosynthetic membranes in plant cells. They are synthesized in the envelope surrounding plastids by MGD and DGD galactosyltransferases. These galactolipids are critical for the biogenesis of photosynthetic membranes, and they act as a source of polyunsaturated fatty acids for the whole cell and as phospholipid surrogates in phosphate shortage. Based on a high-throughput chemical screen, we have characterized a new compound, galvestine-1, that inhibits MGDs in vitro by competing with diacylglycerol binding. Consistent effects of galvestine-1 on Arabidopsis thaliana include root uptake, circulation in the xylem and mesophyll, inhibition of MGDs in vivo causing a reduction of MGDG content and impairment of chloroplast development. The effects on pollen germination shed light on the contribution of galactolipids to pollen-tube elongation. The whole-genome transcriptional response of Arabidopsis points to the potential benefits of galvestine-1 as a unique tool to study lipid homeostasis in plants. PMID:21946275

  5. Heterocyclic inhibitors of glycogen synthase kinase GSK-3

    OpenAIRE

    Martínez García, Ana; Castro Morera, Ana; Pérez Martín, María Concepción; Alonso, Mercedes; Dorronsoro Díaz, Isabel; Moreno Muñoz, Francisco José; Wandosell Jurado, Francisco

    2006-01-01

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

  6. Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo

    DEFF Research Database (Denmark)

    Volke, V; Wegener, Gregers

    1999-01-01

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

  7. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    Directory of Open Access Journals (Sweden)

    YoulaSTsantrizos

    2014-07-01

    Full Text Available The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP and geranylgeranyl pyrophosphate (GGPP are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  8. CHARACTERIZATION OF BARLEY SUCROSE PHOSPHATE SYNTHASE

    Directory of Open Access Journals (Sweden)

    Amani Abdel-Latif

    2014-08-01

    Full Text Available Sucrose phosphate synthase (SPS is one of a number of sucrose-metabolizing enzymes that regulates the sucrose synthesis pathway. SPS was assayed from green barley(HordeurnvulgareL. seedlings (GBS,from etiolated barley seedlings (DBS that were continuously grown in darkness, and barley seedlings that were grown in darkness and illuminated only for 30 minutes before returning to the dark conditions again (EBS.Except for DBS, both GBS and EBSSPS activities wereallosterically regulated by G-6-P(activator or Pi (inhibitor.Thiol reagents became sensitized to the enzyme activity, but could be restored with DTT or ?-ME. Glucose, maltose and lactose activated the enzymewhile ?-gluconolactone and mannose inhibited it. When compared to those plants which were maintained in total darkness, extractable sucrose-Psynthase activity of 30-min.illuminated seedlings increased about 4 folds by 1h .The activity remained constant for an additional two hours and then decreased to about 50% of maximal 5 h post illumination.

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

    Science.gov (United States)

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

    2013-04-01

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

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

    Science.gov (United States)

    Laughlin, Thomas F; Ahmad, Zulfiqar

    2010-04-01

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

  11. Adipocyte Mineralocorticoid Receptor Activation Leads to Metabolic Syndrome and Induction of Prostaglandin D2 Synthase.

    Science.gov (United States)

    Urbanet, Riccardo; Nguyen Dinh Cat, Aurelie; Feraco, Alessandra; Venteclef, Nicolas; El Mogrhabi, Soumaya; Sierra-Ramos, Catalina; Alvarez de la Rosa, Diego; Adler, Gail K; Quilliot, Didier; Rossignol, Patrick; Fallo, Francesco; Touyz, Rhian M; Jaisser, Frédéric

    2015-07-01

    Metabolic syndrome is a major risk factor for the development of diabetes mellitus and cardiovascular diseases. Pharmacological antagonism of the mineralocorticoid receptor (MR), a ligand-activated transcription factor, limits metabolic syndrome in preclinical models, but mechanistic studies are lacking to delineate the role of MR activation in adipose tissue. In this study, we report that MR expression is increased in visceral adipose tissue in a preclinical mouse model of metabolic syndrome and in obese patients. In vivo conditional upregulation of MR in mouse adipocytes led to increased weight and fat mass, insulin resistance, and metabolic syndrome features without affecting blood pressure. We identified prostaglandin D2 synthase as a novel MR target gene in adipocytes and AT56, a specific inhibitor of prostaglandin D2 synthase enzymatic activity, blunted adipogenic aldosterone effects. Moreover, translational studies showed that expression of MR and prostaglandin D2 synthase is strongly correlated in adipose tissues from obese patients. PMID:25966493

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

    Scientific Electronic Library Online (English)

    P.J., Christoffoleti.

    2001-04-01

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

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

    Directory of Open Access Journals (Sweden)

    P.J. Christoffoleti

    2001-04-01

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

  14. A functional cellulose synthase from ascidian epidermis

    OpenAIRE

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

    2004-01-01

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

  15. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

    Science.gov (United States)

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-11-15

    Glycosylation with O-linked ?-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit ? (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as A?-treated cells. Indeed, A? bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the A?-induced impairment in ATP production and ATPase activity. These results indicate that A?-mediated reduction of ATP synthase activity in AD pathology results from direct binding between A? and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    The role of nitric oxide (NO) in central nervous system (CNS) inflammation is uncertain. Whereas experimental autoimmune encephalomyelitis (EAE) is exacerbated in mice deficient in inducible nitric oxide synthase (iNOS), inhibitor studies have suggested a pro-inflammatory role for NO. These discrepancies may reflect balance between immunoregulatory and neurocytopathologic roles for NO. We investigated selective effects of bone marrow-derived versus CNS parenchymal sources of iNOS in EAE in chime...

  17. Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

    DEFF Research Database (Denmark)

    Graebe, Martin; Brond, Lone; Christensen, Sten; Nielsen, Soren; Olsen, Niels Vidiendal; Jonassen, Thomas E N

    2004-01-01

    The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that l-NAME treatment dose dependently deteriorated cumulative so...

  18. Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

    OpenAIRE

    Kutchera, W; Jones, D. A.; Matsunami, N.; Groden, J; McIntyre, T M; Zimmerman, G. A.; White, R. L.; Prescott, S M

    1996-01-01

    Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens ...

  19. Inhibition of hypothalamic fatty acid synthase triggers rapid activation of fatty acid oxidation in skeletal muscle

    OpenAIRE

    Cha, Seung Hun; Hu, Zhiyuan; Chohnan, Shigeru; Lane, M. Daniel

    2005-01-01

    Malonyl-CoA functions as a mediator in the hypothalamic sensing of energy balance and regulates the neural physiology that governs feeding behavior and energy expenditure. The central administration of C75, a potent inhibitor of the fatty acid synthase (FAS), increases malonyl-CoA concentration in the hypothalamus and suppresses food intake while activating fatty acid oxidation in skeletal muscle. Closely correlated with the increase in muscle fatty acid oxidation is the phosphorylation/inact...

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

    OpenAIRE

    Jing WEI; Liu, Wenhua; Yan, Zhen

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ketabchi Farzaneh

    2012-01-01

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

  2. Reuptake Inhibitor

    Directory of Open Access Journals (Sweden)

    Ângelo de Fátima

    2005-01-01

    Full Text Available (R-Fluoxetine, potent and selective serotonin reuptake inhibitor, has been synthesized in six steps, 50% overall yield and 99% ee from benzaldehyde via catalytic asymmetric allylation with Maruoka´s catalyst.

  3. Deoxyribonuclease inhibitors.

    Science.gov (United States)

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

    2014-12-17

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

  4. Producing biofuels using polyketide synthases

    Science.gov (United States)

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

    2013-04-16

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

  5. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline

    Science.gov (United States)

    Preiss, Laura; Langer, Julian D.; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E. G.; Koul, Anil; Meier, Thomas

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring’s ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens.

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

    Science.gov (United States)

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

    2010-01-01

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

  7. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    OpenAIRE

    SHI, DASHUANG; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel

    2011-01-01

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast t...

  8. Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi.

    Science.gov (United States)

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

    2010-02-01

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

  9. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-05

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

  10. Cellulose Synthase Complexes: Composition and Regulation

    OpenAIRE

    Lei, Lei; Li, Shundai; Gu, Ying

    2012-01-01

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

  11. Terpene synthases are widely distributed in bacteria

    OpenAIRE

    Yamada, Yuuki; Kuzuyama, Tomohisa; KOMATSU, MAMORU; SHIN-YA, KAZUO; OMURA, SATOSHI; CANE, DAVID E.; IKEDA, HARUO

    2014-01-01

    Terpenes are generally considered to be plant or fungal metabolites, although a small number of odoriferous terpenes of bacterial origin have been known for many years. Recently, extensive bacterial genome sequencing and bioinformatic analysis of deduced bacterial proteins using a profile based on a hidden Markov model have revealed 262 distinct predicted terpene synthases. Although many of these presumptive terpene synthase genes seem to be silent in their parent microorganisms, controlled e...

  12. Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Holden, Jeffrey K; Kang, Soosung; Beasley, Federico C; Cinelli, Maris A; Li, Huiying; Roy, Saurabh G; Dejam, Dillon; Edinger, Aimee L; Nizet, Victor; Silverman, Richard B; Poulos, Thomas L

    2015-06-18

    Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme. PMID:26091171

  13. The Structure of the L-myo-inositol-1-phosphate Synthase-NAD[superscript +]-2-deoxy-D-glucitol 6-(E)-Vinylhomophosphonate Complex Demands a Revision of the Enzyme Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Xiangshu; Foley, Kathleen M.; Geiger, James H. (MSU)

    2010-11-16

    1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of D-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD{sup +} and a high-affinity inhibitor, 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-D-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD{sup +}-2-deoxy-D-glucitol 6-phosphate. There are several other conformational changes in NAD{sup +} and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.

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

    Science.gov (United States)

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

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

  15. Arabidopsis CDS blastp result: AK242817 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242817 J090063G17 At3g48560.1 68416.m05302 acetolactate synthase, chloroplast / acetohydroxy-a ... cid synthase (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 0.0 ...

  16. Arabidopsis CDS blastp result: AK058963 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058963 001-020-C04 At3g48560.1 acetolactate synthase, chloroplast / acetohydroxy-acid synthase ... (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 2e-15 ...

  17. Arabidopsis CDS blastp result: AK109628 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109628 002-138-C02 At3g48560.1 acetolactate synthase, chloroplast / acetohydroxy-acid synthase ... (ALS ) nearly identical to SP|P17597 Acetolactate syntha ... ormerly EC 4.1.3.18) (Acetohydroxy-acid synthase) (ALS ) {Arabidopsis thaliana} 0.0 ...

  18. Nitric oxide synthase activity in Fasciola hepatica: a radiometric study.

    Science.gov (United States)

    Terenina, N B; Onufriev, M V; Gulyaeva, N V; Moiseeva, Y V; Gustafsson, M K S

    2003-06-01

    The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(omega)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica. PMID:12866797

  19. Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase. Two amino acids before the first aspartate-rich motif solely determine eukaryotic farnesyl diphosphate synthase activity.

    Science.gov (United States)

    Ohnuma, S i; Hirooka, K; Ohto, C; Nishino, T

    1997-02-21

    Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways. PMID:9030588

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

    Scientific Electronic Library Online (English)

    Marcelo Carnier, Dornelas; Paulo, Mazzafera.

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

  1. Terpene synthases are widely distributed in bacteria.

    Science.gov (United States)

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

    2015-01-20

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

  2. The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer.

    Science.gov (United States)

    Cathcart, Mary-Clare; Reynolds, John V; O'Byrne, Kenneth J; Pidgeon, Graham P

    2010-04-01

    Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI(2)/TXA(2) ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. PMID:20122998

  3. Nitric Oxide Synthase Inhibition by NG-Nitro-l-Arginine Methyl Ester Inhibits Tumor-Induced Angiogenesis in Mammary Tumors

    OpenAIRE

    Jadeski, Lorraine C.; Lala, Peeyush K.

    1999-01-01

    Using a murine breast cancer model, we earlier found a positive correlation between the expression of nitric oxide synthase (NOS) and tumor progression; treatment with inhibitors of NOS, NG-methyl-l-arginine (NMMA) and NG-nitro-l-arginine methyl ester (L-NAME), had antitumor and antimetastatic effects that were partly attributed to reduced tumor cell invasiveness. In the present study, we used a novel in vivo model of tumor angiogenesis using subcutaneous implants of tumor cells suspended in ...

  4. Structure-Function Features of a Mycoplasma Glycolipid Synthase Derived from Structural Data Integration, Molecular Simulations, and Mutational Analysis

    OpenAIRE

    Romero-García, Javier; Francisco, Carles; Biarnés, Xevi; Planas, Antoni

    2013-01-01

    Glycoglycerolipids are structural components of mycoplasma membranes with a fundamental role in membrane properties and stability. Their biosynthesis is mediated by glycosyltransferases (GT) that catalyze the transfer of glycosyl units from a sugar nucleotide donor to diacylglycerol. The essential function of glycolipid synthases in mycoplasma viability, and the absence of glycoglycerolipids in animal host cells make these GT enzymes a target for drug discovery by designing specific inhibitor...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

  7. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Fr?czyk, Tomasz; Ruman, Tomasz

    2015-01-01

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

  8. Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

    Science.gov (United States)

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

    2013-05-10

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

  9. Producing dicarboxylic acids using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-26

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

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

    Scientific Electronic Library Online (English)

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

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    L. Vargas

    2013-06-01

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

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

    Scientific Electronic Library Online (English)

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

    2013-06-01

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

  13. Geranyl diphosphate synthase large subunit, and methods of use

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

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

    Science.gov (United States)

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

    2010-01-01

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the ?-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices ?5 and ?6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100–200 ?g/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-25

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

  16. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-04-15

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

  17. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    International Nuclear Information System (INIS)

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

  18. Nitric oxide synthase in the pineal gland

    OpenAIRE

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

    1996-01-01

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

  19. Crystal Structure of the Human Prostacyclin Synthase

    OpenAIRE

    Chiang, Chia-Wang; Yeh, Hui-Chun; Wang, Lee-Ho; Chan, Nei-Li

    2006-01-01

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

  20. Mitochondrial ATP synthase: architecture, function and pathology

    OpenAIRE

    Jonckheere, An I.; Smeitink, Jan A.M.; Rodenburg, Richard J. T.

    2011-01-01

    Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to com...

  1. Tertiary model of a plant cellulose synthase

    OpenAIRE

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

    2013-01-01

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

  2. The cellulose synthase gene of Dictyostelium

    OpenAIRE

    Blanton, Richard L.; Fuller, Danny; Iranfar, Negin; Grimson, Mark J.; Loomis, William F.

    2000-01-01

    Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum. We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development. Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again. No spores or stalk ce...

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

    Directory of Open Access Journals (Sweden)

    RobertRapoport

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Üçeyler Nurcan

    2010-03-01

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

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

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

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

    2010-01-01

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

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

    OpenAIRE

    Gu, Ying; Somerville, Chris

    2010-01-01

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

  8. The cellulose synthase superfamily in fully sequenced plants and algae

    OpenAIRE

    Xu Ying; Huang Jinling; Yin Yanbin

    2009-01-01

    Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is foun...

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

    Science.gov (United States)

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

    2008-06-24

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

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Srivastava Anurag

    2010-04-01

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

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

    International Nuclear Information System (INIS)

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

  14. Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide.

    Science.gov (United States)

    Panaro, M A; Acquafredda, A; Lisi, S; Lofrumento, D D; Trotta, T; Satalino, R; Saccia, M; Mitolo, V; Brandonisio, O

    1999-01-01

    Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection. PMID:10592110

  15. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo

    OpenAIRE

    Kats, Anna; Båge, Tove; Georgsson, Pierre; Jönsson, Jörgen; Quezada, Hernán Concha; Gustafsson, Anders; Jansson, Leif; Lindberg, Claes; Näsström, Karin; Yucel-Lindberg, Tülay

    2013-01-01

    The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts st...

  16. Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from ?2-adrenoceptor stimulation

    OpenAIRE

    Ma, Weiya; Eisenach, James. C.

    2006-01-01

    ?2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to ?2-AR analgesia would also destroy nNOS and M4 receptor expression.

  17. Evidence for mediation of L-2-chloropropionic acid-induced delayed neuronal cell death by activation of a constitutive nitric oxide synthase.

    OpenAIRE

    Widdowson, P. S.; Farnworth, M.; Moore, R B; Dunn, D.; Wyatt, I

    1996-01-01

    1. Delayed neuronal cell death elicited by excess excitatory amino acid concentrations has been strongly implicated in many neurological disorders including head trauma, stroke, motor neurone disease and Huntington's disease. We have used the neurotoxin, L-2-chloropropionic acid (L-CPA) to model cellular events in vivo leading to delayed neuronal cell loss which is confined to the cerebellar cortex and can be prevented by inhibitors of nitric oxide synthase such as NG-nitro-L-arginine methyl ...

  18. Functional Dissection of N-Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N-Acetylglutamate Kinase Activity

    OpenAIRE

    Sancho-Vaello, Enea; Fernández-Murga, María L.; Rubio Zamora, Vicente

    2012-01-01

    In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. Th...

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

    Energy Technology Data Exchange (ETDEWEB)

    Leriche, Caroline; Rene, Loic; Badet, Bernard [Centre National de la Recherche Scientifique (CNRS), 91 - Gif-sur-Yvette (France). Inst. de Chimie des Substances Naturelles; Derouet, Florence; Rousseau, Bernard [CEA Centre d`Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Dept. de Biologie

    1995-12-31

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

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

    International Nuclear Information System (INIS)

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

  1. Substrate Recognition by ?-Ketoacyl-ACP Synthases

    OpenAIRE

    Borgaro, Janine G.; Chang, Andrew; Machutta, Carl A.; Zhang, Xujie; Tonge, Peter J

    2011-01-01

    ?-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ?-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII e...

  2. Inactivation of cystathionine ?-synthase with peroxynitrite

    OpenAIRE

    Celano, Laura; Gil, Magdalena; Carballal, Sebastián; Durán, Rosario; Denicola, Ana; Banerjee, Ruma; Alvarez, Beatriz

    2009-01-01

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

  3. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R.

    2014-01-01

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is m...

  4. Loop residues and catalysis in OMP synthase

    DEFF Research Database (Denmark)

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

    2012-01-01

    Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100?109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 104-fold decrease in kcat/KM for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in kcat/KM, respectively, and E101A, D104A, and G106A were s...

  5. Glycogen synthase kinase 3? regulates urine concentrating mechanism in mice

    DEFF Research Database (Denmark)

    NØrregaard, Rikke; Tao, Shixin

    2015-01-01

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3? and GSK3? isoforms. GSK3? has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3? has yet to be discerned. To investigate the role of GSK3? in urine concentration, we compared GSK3? knockout (GSK3?KO) mice with wild-type (WT) littermates. Under normal conditions, GSK3?KO mice had higher water intake and urine output. GSK3?KO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3?KO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3? in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3?KO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3? could play a crucial role in renal urine concentration and suggest that GSK3? might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus.

  6. Probing myo-inositol 1-phosphate synthase with multisubstrate adducts

    OpenAIRE

    Deranieh, Rania M.; Greenberg, Miriam L.; Le Calvez, Pierre-B.; Mooney, Maura C.; Migaud, Marie E

    2012-01-01

    The synthesis of a series of carbohydrate-nucleotide hybrids, designed to be multisubstrate adducts mimicking myo-inositol 1-phosphate synthase first oxidative transition state, is reported. Their ability to inhibit the synthase has been assessed and results have been rationalised computationally to estimate their likely binding mode.

  7. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different cellular compartments and suggest that NO may have specific actions in relation to its site of production. The localization of type I NO synthase in the vicinity of mitochondria supports a specific action of NO on mitochondrial respiration, whereas the localization of type III NO synthase in vascular endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle.

  8. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells

    OpenAIRE

    MENENDEZ, JAVIER A.; Vellon, Luciano; Mehmi, Inderjit; Oza, Bharvi P.; Ropero, Santiago; Colomer, Ramon; Lupu, Ruth

    2004-01-01

    Fatty acid synthase (FAS) activity is a potential therapeutic target to treat cancer and obesity. Here, we have identified a molecular link between FAS and HER2 (erbB-2) oncogene, a marker for poor prognosis that is overexpressed in 30% of breast and ovarian cancers. Pharmacological FAS inhibitors cerulenin and C75 were found to suppress p185HER2 oncoprotein expression and tyrosine-kinase activity in breast and ovarian HER2 overexpressors. Similarly, p185HER2 expression was dramatically down-...

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

    OpenAIRE

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

    2014-01-01

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

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

    OpenAIRE

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

    2013-01-01

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

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

    OpenAIRE

    Yang, L.; Mercke, P.; Loon, J.J.A., van; Fang, Zhiyuan; Dicke, M; Jongsma, M A

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FaNES1 linalool synthase (LIS) cDNA from strawberry with plastid targeting and a synthetic intron (LIS') was placed under the control of the wound inducible proteinase inhibitor 2 (PI2) promoter from potato. The construct pBin-PPI2-LIS' was transformed to Arabidopsis thaliana ecotype Columbia 0. Kanamycin resistant T0 seedlings were confirmed for the presence and transcription of the LIS' gene by PCR analysi...

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

    Directory of Open Access Journals (Sweden)

    Richard Scott Jope

    2011-11-01

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

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

    International Nuclear Information System (INIS)

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

  14. STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE

    Directory of Open Access Journals (Sweden)

    T. Femlin Blessia

    2012-02-01

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

  15. Activities and regulation of peptidoglycan synthases.

    Science.gov (United States)

    Egan, Alexander J F; Biboy, Jacob; Van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-10-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN. PMID:26370943

  16. Torque generation mechanism of ATP synthase

    Science.gov (United States)

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

    2010-03-01

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

  17. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    OpenAIRE

    Yuan, Guang-Jin; Zhou, Xiao-Rong; Gong, Zuo-Jiong; ZHANG, PIN; Sun, Xiao-Mei; Zheng, Shi-Hua

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-?B (NF-?B) and tumor necrosis factor-? (TNF-?) expression in the liver.

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

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes Niklas Fabian

    2014-01-01

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

  19. Inhibitors of histone demethylases

    DEFF Research Database (Denmark)

    Lohse, Brian; Kristensen, Jesper L; Kristensen, Line H; Agger, Karl; Helin, Kristian; Gajhede, Michael; Clausen, Rasmus P

    2011-01-01

    Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the inhibitors are either previously reported inhibitors of related enzymes or compounds derived from these. Development in terms of selectivity and potency is still pertinent. Several reports on the developm...

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

    Science.gov (United States)

    Schnell, Robert; Sriram, Dharmarajan; Schneider, Gunter

    2015-09-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Tyrosine nitration of prostacyclin synthase is associated with enhanced retinal cell apoptosis in diabetes.

    Science.gov (United States)

    Zou, Ming-Hui; Li, Hongliang; He, Chaoyong; Lin, Mingkai; Lyons, Timothy J; Xie, Zhonglin

    2011-12-01

    The risk of diabetic retinopathy is associated with the presence of both oxidative stress and toxic eicosanoids. Whether oxidative stress actually causes diabetic retinopathy via the generation of toxic eicosanoids, however, remains unknown. The aim of the present study was to determine whether tyrosine nitration of prostacyclin synthase (PGIS) contributes to retinal cell death in vitro and in vivo. Exposure of human retinal pericytes to heavily oxidized and glycated LDL (HOG-LDL), but not native forms of LDL (N-LDL), for 24 hours significantly increased pericyte apoptosis, accompanied by increased tyrosine nitration of PGIS and decreased PGIS activity. Inhibition of the thromboxane receptor or cyclooxygenase-2 dramatically attenuated HOG-LDL-induced apoptosis without restoring PGIS activity. Administration of superoxide dismutase (to scavenge superoxide anions) or L-N(G)-nitroarginine methyl ester (L-NAME, a nonselective nitric oxide synthase inhibitor) restored PGIS activity and attenuated pericyte apoptosis. In Akita mouse retinas, diabetes increased intraretinal levels of oxidized LDL and glycated LDL, induced PGIS nitration, enhanced apoptotic cell death, and impaired blood-retinal barrier function. Chronic administration of tempol, a superoxide scavenger, reduced intraretinal oxidized LDL and glycated LDL levels, PGIS nitration, and retina cell apoptosis, thereby preserving the integrity of blood-retinal barriers. In conclusion, oxidized LDL-mediated PGIS nitration and associated thromboxane receptor stimulation might be important in the initiation and progression of diabetic retinopathy. PMID:22015457

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

    Energy Technology Data Exchange (ETDEWEB)

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  4. Genetic deletion of microsomal prostaglandin E synthase-1 suppresses mouse mammary tumor growth and angiogenesis.

    Science.gov (United States)

    Howe, Louise R; Subbaramaiah, Kotha; Kent, Claire V; Zhou, Xi K; Chang, Sung-Hee; Hla, Timothy; Jakobsson, Per-Johan; Hudis, Clifford A; Dannenberg, Andrew J

    2013-10-01

    The cyclooxygenase/prostaglandin (COX/PG) signaling pathway is of central importance in inflammation and neoplasia. COX inhibitors are widely used for analgesia and also have demonstrated activity for cancer prophylaxis. However, cardiovascular toxicity associated with this drug class diminishes their clinical utility and motivates the development of safer approaches both for pain relief and cancer prevention. The terminal synthase microsomal PGE synthase-1 (mPGES-1) has attracted considerable attention as a potential target. Overexpression of mPGES-1 has been observed in both colorectal and breast cancers, and gene knockout and overexpression approaches have established a role for mPGES-1 in gastrointestinal carcinogenesis. Here we evaluate the contribution of mPGES-1 to mammary tumorigenesis using a gene knockout approach. Mice deficient in mPGES-1 were crossed with a strain in which breast cancer is driven by overexpression of human epidermal growth factor receptor 2 (HER2/neu). Loss of mPGES-1 was associated with a substantial reduction in intramammary PGE2 levels, aromatase activity, and angiogenesis in mammary glands from HER2/neu transgenic mice. Consistent with these findings, we observed a significant reduction in multiplicity of tumors ?1mm in diameter, suggesting that mPGES-1 contributes to mammary tumor growth. Our data identify mPGES-1 as a potential anti-breast cancer target. PMID:23624019

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

    Directory of Open Access Journals (Sweden)

    Oguz Cakir

    2013-06-01

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

  6. Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target

    DEFF Research Database (Denmark)

    Yang, Zhiping; Huang, Yuh-Chin T

    2014-01-01

    To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.

  7. Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells

    OpenAIRE

    Hamed, Hossein A; DAS, SWADESH K.; Sokhi, Upneet K.; Park, Margaret A.; Cruickshanks, Nichola; Archer, Kellie; Ogretmen, Besim; Grant, Steven; SARKAR, DEVANAND; Fisher, Paul B; Dent, Paul

    2013-01-01

    In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protecte...

  8. Monitoring the rotary motors of single F °F I-ATP synthase by synchronized multi channel TCSPC

    Science.gov (United States)

    Zarrabi, N.; Düser, M. G.; Ernst, S.; Reuter, R.; Glick, G. D.; Dunn, S. D.; Wrachtrup, J.; Börsch, M.

    2007-09-01

    Confocal time resolved single-molecule spectroscopy using pulsed laser excitation and synchronized multi channel time correlated single photon counting (TCSPC) provides detailed information about the conformational changes of a biological motor in real time. We studied the formation of adenosine triphosphate, ATP, from ADP and phosphate by F °F I-ATP synthase. The reaction is performed by a stepwise internal rotation of subunits of the lipid membrane-embedded enzyme. Using Förster-type fluorescence resonance energy transfer, FRET, we detected rotation of this biological motor by sequential changes of intramolecular distances within a single F °F I-ATP synthase. Prolonged observation times of single enzymes were achieved by functional immobilization to the glass surface. The stepwise rotary subunit movements were identified by Hidden Markov Models (HMM) which were trained with single-molecule FRET trajectories. To improve the accuracy of the HMM analysis we included the single-molecule fluorescence lifetime of the FRET donor and used alternating laser excitation to co-localize the FRET acceptor independently within a photon burst. The HMM analysis yielded the orientations and dwell times of rotary subunits during stepwise rotation. In addition, the action mode of bactericidal drugs, i.e. inhibitors of F °F I-ATP synthase like aurovertin, could be investigated by the time resolved single-molecule FRET approach.

  9. Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

    International Nuclear Information System (INIS)

    3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

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

    International Nuclear Information System (INIS)

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3?, 5?-adenosine diphosphate (3?, 5?-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3?, 5?-ADP is bound as the 3?, 5?-ADP part of CoA in the known structures of the CoA–AcpS and 3?, 5?-ADP–AcpS binary complexes. The position of the second 3?, 5?-ADP has never been described before. It is in close proximity to the first 3?, 5?-ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP

  11. Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.

    OpenAIRE

    Ohnuma, S; Nakazawa, T.; Hemmi, H.; Hallberg, A. M.; Koyama, T.; Ogura, K; Nishino, T

    1996-01-01

    Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatme...

  12. Fluorescent Assays for Ceramide Synthase Activity.

    Science.gov (United States)

    Couttas, Timothy A; Don, Anthony S

    2016-01-01

    Ceramides are the central lipid metabolite of the sphingolipid family, and exert a potent influence over cell polarity, differentiation, and survival through their biophysical properties and their specific interactions with cell signaling proteins. Literature on the importance of ceramides in physiology and pathological conditions continues to grow, with ceramides having been identified as central effectors in major human pathologies such as diabetes and neurodegenerative conditions. In mammals, ceramide synthesis from a sphingoid base and a variable length fatty acid is catalyzed by a family of six ceramide synthases (CERS1-6), whose active sites exhibit differential specificity for different length fatty acids. CERS activity has traditionally been measured using radioactive substrates. More recently mass spectrometry has been used. In this chapter, we describe a fluorescent CERS assay, the results of which can be quantified using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Methods for quantification with either TLC or HPLC are described. PMID:26552672

  13. Mitochondrial ATP synthase: architecture, function and pathology.

    Science.gov (United States)

    Jonckheere, An I; Smeitink, Jan A M; Rodenburg, Richard J T

    2012-03-01

    Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F(1), situated in the mitochondrial matrix, and F(o), located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions. PMID:21874297

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

    OpenAIRE

    Stahlberg, Henning; Müller, Daniel J; Suda, Kitaru; Fotiadis, Dimitrios; Engel, Andreas; Meier, Thomas; Matthey, Ulrich; Dimroth, Peter

    2001-01-01

    Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F0 domain, which drives the extra-membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9–14 ?-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic ...

  15. Hypoxia-induced relaxation of porcine retinal arterioles in vitro depends on inducible NO synthase and EP4 receptor stimulation in the perivascular retina

    DEFF Research Database (Denmark)

    Hansen, Pernille OversØ; Kringelholt, Sidse

    2015-01-01

    PURPOSE: Hypoxia-induced relaxation of porcine retinal arterioles has been shown to be reduced during inhibition of prostaglandin synthesis and nitric oxide synthase (NOS). The purpose of this study was to identity the specific prostaglandin receptor(s) and source(s) of NO mediating this effect. METHODS: Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a myograph and were exposed to hypoxia in the presence of one of the following: the general NO synthase inhibitor L-NAME, the selective iNOS inhibitor 1400W, the selective nNOS inhibitor 7-nitroindazole, the general cyclooxygenase (COX) inhibitor ibuprofen or an antagonist to the FP- (AL 8810), DP- (BWA868C), EP1 - (SC-19220), EP2 - (PF-044189) or EP4 receptors (GW627368X). The experiments were repeated after removal of the perivascular retinal tissue. RESULTS: Hypoxia induced relaxation of retinal arterioles with preserved perivascular retinal tissue. This relaxation was significantly reduced in the presence of L-NAME, 1400W, ibuprofen and the EP4 receptor antagonist GW627368X. The simultaneous addition of L-NAME or 1400W in combination with ibuprofen, but not GW627368X, reduced hypoxia-induced vasorelaxation additively as compared to the effect of the compounds individually. CONCLUSION: Hypoxia-induced vasorelaxation of porcine retinal arterioles is mediated by inducible NOS and stimulation of EP4 receptors acting through separate pathways, but mechanisms unrelated to the studied prostaglandin receptors and NOS products are also involved.

  16. Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.

    Science.gov (United States)

    Ohnuma, S; Nakazawa, T; Hemmi, H; Hallberg, A M; Koyama, T; Ogura, K; Nishino, T

    1996-04-26

    Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP. PMID:8626566

  17. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    OpenAIRE

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-?-bisabolene synthase from ginger rhizome, and ?-humulene synthase and ?-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identificat...

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

    Indian Academy of Sciences (India)

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

    2010-06-01

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

  19. Subcellular Targeting Domains of Sphingomyelin Synthase 1 and 2

    OpenAIRE

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

    2011-01-01

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

  20. delta-Aminolevulinic acid synthase from Euglena gracilis.

    OpenAIRE

    Beale, S I; FOLEY, T; Dzelzkalns, V

    1981-01-01

    delta-Aminolevulinic acid (ALA) synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] activity was detected in cell extracts of the unicellular green flagellate alga Euglena gracilis. The enzyme was identified by substrate and cofactor requirements, and activity was proportional to number of cells extracted and duration of incubation. The incubation product was spectrophotometrically and chromatographically identical to ALA. ALA synthase activity is present in t...

  1. ATP Synthase - The Structure of the Stator Stalk

    OpenAIRE

    Weber, Joachim

    2007-01-01

    ATP synthase synthesizes ATP from ADP and inorganic phosphate by a unique rotary mechanism where two subcomplexes move relative to each other, powered by a proton or sodium gradient. The non-rotating parts of the machinery are held together by the “stator stalk”. Significant progress towards a structural model for the holoenzyme was made recently, when the structure of a major portion of the stator stalk of mitochondrial ATP synthase was resolved.

  2. INHIBITION OF NITRIC OXIDE SYNTHASE BY COBALAMINS AND COBINAMIDES*

    OpenAIRE

    Weinberg, J Brice; Chen, Youwei; Jiang, Ning; Beasley, Bethany E.; Salerno, John C.; Ghosh, Dipak K.

    2009-01-01

    Cobalamins (Cbl) are important co-factors for methionine synthase and methylmalonyl-coA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on 14-C-L-arginine-to-14-C-L-citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2-Cbi) potently inhibited all isoforms, whfile cyanocobalamin, methylcobalamin, and adenosylcobalami...

  3. New developments in cancer treatment with the novel thymidylate synthase inhibitor raltitrexed ('Tomudex').

    OpenAIRE

    Blackledge, G

    1998-01-01

    Following the demonstration of efficacy, tolerability and quality-of-life benefits of raltitrexed ('Tomudex'), principally in advanced colorectal but also in other cancers, an extensive evaluation of combination therapy with other agents in patients with colorectal and other tumour types is being undertaken. This work has been prompted by preclinical observations of enhanced activity of raltitrexed when coadministered with other cytotoxic agents or radiotherapy and by preliminary results show...

  4. Inhibitors of nitric oxide synthase selectively reduce flow in tumor-associated neovasculature.

    OpenAIRE

    Andrade, S.P.; Hart, I. R.; Piper, P. J.

    1992-01-01

    1. The effects of L-arginine analogues, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA) and methylene blue on blood flow in a murine adenocarcinoma and melanoma have been investigated. 2. Sponge implants in Balb/c and C57/BL mice were used to host proliferating tumour cells while the washout of 133Xe was employed to assess local blood flow in the implanted sponges. 3. Pharmacological inhibition of nitric oxide (NO) reduced blood flow in both tumours but this ef...

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

    Czech Academy of Sciences Publication Activity Database

    Broží?ková, Carole; Mikulecká, Anna; Otáhal, Jakub

    2014-01-01

    Ro?. 63, ?. 5 (2014), s. 637-648. ISSN 0862-8408 R&D Projects: GA ?R(CZ) GAP303/10/0999; GA ?R(CZ) GPP304/11/P386; GA ?R(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : 7-nitroindazole * open field test * ladder rung walking test * brain excitability * blood gas analysis * rat Subject RIV: FH - Neurology Impact factor: 1.293, year: 2014

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

    OpenAIRE

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

    2010-01-01

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

  7. PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

    Science.gov (United States)

    BOOTH, LAURENCE; ROBERTS, JANE L.; CRUICKSHANKS, NICHOLA; TAVALLAI, SEYEDMEHRAD; WEBB, TIMOTHY; SAMUEL, PETER; CONLEY, ADAM; BINION, BRITTANY; YOUNG, HAROLD F.; POKLEPOVIC, ANDREW; SPIEGEL, SARAH; DENT, PAUL

    2015-01-01

    The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1?/XBP1 enhanced killing whereas knock down of eIF2?/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

  8. Mikimopine synthase (mis) gene on pRi1724.

    Science.gov (United States)

    Suzuki, K; Tanaka, N; Kamada, H; Yamashita, I

    2001-01-24

    By determination of the nucleotide sequence adjacent to the right border of T-DNA of the mikimopine-type Ri plasmid (pRi1724) in Agrobacterium rhizogenes, a new open reading frame (ORF) encoding 318 amino acids was found. A transcript of 1.35 kb derived from this ORF was observed in hairy roots of Ajuga reptans by northern blotting analysis. Including its own promoter and terminator, this ORF was isolated from the pRi1724 T-DNA and introduced into tobacco plants by the Agrobacterium-binary vector system. Since mikimopine, an opine and a stereoisomer of cucumopine, was accumulated in all organs of the transgenic tobacco plants, the new ORF was deduced to be the mikimopine synthase gene. For comparison, the nucleotide sequence of cucumopine synthase encoded on pRi2659 was also determined. No homology was found between mikimopine synthase and cucumopine synthase at the nucleotide, but partial homology was found at the amino acid level. Mikimopine synthase and cucumopine synthase produced by a protein expression system using E. coli catalyzed the synthesis of mikimopine and cucumopine from L-histidine and alpha-ketoglutaric acid, requiring NADH as a cofactor. These synthesized opines were identified by paper electrophoresis, TLC and HPLC analyses. The synthesized mikimopine or cucumopine could be degraded by A. rhizogenes strains harboring Ri plasmids encoding the respective catabolic enzyme. PMID:11223242

  9. Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

    Science.gov (United States)

    Lu, Heshu; Yang, Mingfeng; Liu, Chunmei; Lu, Ping; Cang, Huaixing; Ma, Lanqing

    2013-08-01

    The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P2?2?2?, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution. PMID:23908031

  10. Inhibition of glycogen synthase kinase-3 reduces L-DOPA-induced neurotoxicity

    International Nuclear Information System (INIS)

    The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), used for the treatment of Parkinson's disease, remains controversial. Although there are many reports suggesting that long-term treatment of L-DOPA causes neuronal death, an increasing body of recent evidence has proposed that L-DOPA might be neuroprotective rather than neurotoxic. We investigated the effect of L-DOPA on neuronally differentiated PC12 (nPC12) cells by treating cells with various concentrations of L-DOPA for 24 h. We also studied whether glycogen synthase kinase (GSK)-3 activation is related to L-DOPA-induced neurotoxicity by simultaneously treating cells with several concentrations of L-DOPA and a GSK-3 inhibitor for 24 h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, cell counting kit-8, and DAPI staining all showed that L-DOPA decreased nPC12 cell viability at high concentrations. In addition, 100 ?M L-DOPA treatment significantly increased the activity of GSK-3 and death signals including cytochrome c, activated caspase-3 and cleaved PARP, and decreased survival signals including heat shock transcription factor-1 in a concentration-dependent manner. Treatment with GSK-3 inhibitor VIII or lithium chloride prevented L-DOPA-induced cell death. Together, these results suggest that L-DOPA induces neuronal cell death at high concentrations and that the neurotoxic effect of L-DOPA might be mediated in part by GSK-3 activation

  11. Calcium/calmodulin dependence of nitric oxide synthase from Viviparus ater

    Directory of Open Access Journals (Sweden)

    D Tagliazucchi

    2005-04-01

    Full Text Available The calcium ion dependence of soluble and particulate nitric oxyde synthase (NOS activity fromViviparus ater immunocytes was investigated. At a calcium ion concentration of 2 nM, the NOS activitymeasured by citrulline formation was 27.1 ± 2.2 and 9.3 ± 0.8 pmol/min/106cell for soluble andparticulate NOS, respectively. The increase in free calcium ion concentration to 300 nM increasesenzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 pmol/min/106cell, respectively. The 50 % activation of thecalcium-dependent activity is 91 and 97 nM Ca2+ for soluble and particulate enzymes. Trifluoperazine,an inhibitor of the calmodulin-dependent enzyme, partially inhibits both activities. Soluble NOS is fivetimes more sensitive than particulate NOS. The behaviour of both activities with three NOS inhibitors(7-nitroindazole, S-methylisothiourea sulphate, diphenyleneiodonium is very similar, with IC50 valuesthat are not significantly different. The calcium ion dependence of NOS activities, in a range of freecalcium ion variations, which are transiently observed in receptor-stimulated cells, suggests that nitricoxyde in V. ater immunocytes not only has a defensive role but also signalling relevance in crosstalkingbetween immunocytes and other cells.

  12. Human methionine synthase reductase is a molecular chaperone for human methionine synthase

    OpenAIRE

    Yamada, Kazuhiro; Gravel, Roy A; Toraya, Tetsuo; Matthews, Rowena G

    2006-01-01

    Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a Kact value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the...

  13. Inactivation of cystathionine ?-synthase with peroxynitrite

    Science.gov (United States)

    Celano, Laura; Gil, Magdalena; Carballal, Sebastián; Durán, Rosario; Denicola, Ana; Banerjee, Ruma; Alvarez, Beatriz

    2009-01-01

    Cystathionine ?-synthase (CBS) is a homocysteine metabolizing enzyme that contains pyridoxal phosphate (PLP) and a six-coordinate heme cofactor of unknown function. CBS was inactivated by peroxynitrite, the product of nitric oxide and superoxide radicals. The IC50 was ~150 ?M for 5 ?M ferric CBS. Stopped-flow kinetics and competition experiments showed a direct reaction with a second-order rate constant of (2.4–5.0) × 104 M?1 s?1 (pH 7.4, 37 °C). The radicals derived from peroxynitrite, nitrogen dioxide and carbonate radical, also inactivated CBS. Exposure to peroxynitrite did not modify bound PLP but led to nitration of Trp208, Trp43 and Tyr223 and alterations in the heme environment including loss of thiolate coordination, conversion to high spin and bleaching, with no detectable formation of oxo-ferryl compounds nor promotion of one-electron processes. This study demonstrates the susceptibility of CBS to reactive oxygen/nitrogen species, with potential relevance to hyperhomocysteinemia, a risk factor for cardiovascular diseases. PMID:19733148

  14. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan?s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  15. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.

    2014-01-01

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

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

    International Nuclear Information System (INIS)

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca2+ ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg2+ ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures

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

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  18. Small-molecule caspase inhibitors

    International Nuclear Information System (INIS)

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

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

    Science.gov (United States)

    Tezuka, Daisuke; Ito, Akira; Mitsuhashi, Wataru; Toyomasu, Tomonobu; Imai, Ryozo

    2015-05-01

    The rice genome contains a family of kaurene synthase-like (OsKSL) genes that are responsible for the biosynthesis of various diterpenoids, including gibberellins and phytoalexins. While many OsKSL genes have been functionally characterized, the functionality of OsKSL2 is still unclear and it has been proposed to be a pseudogene. Here, we found that OsKSL2 is drastically induced in roots by methyl jasmonate treatment and we successfully isolated a full-length cDNA for OsKSL2. Sequence analysis of the OsKSL2 cDNA revealed that the open reading frame of OsKSL2 is mispredicted in the two major rice genome databases, IRGSP-RAP and MSU-RGAP. In vitro conversion assay indicated that recombinant OsKSL2 catalyzes the cyclization of ent-CDP into ent-beyerene as a major and ent-kaurene as a minor product. ent-Beyerene is an antimicrobial compound and OsKSL2 is induced by methyl jasmonate; these data suggest that OsKSL2 is a functional ent-beyerene synthase that is involved in defense mechanisms in rice roots. PMID:25824047

  20. PDE5 inhibitors enhance the lethality of standard of care chemotherapy in pediatric CNS tumor cells

    Science.gov (United States)

    Roberts, Jane L; Booth, Laurence; Conley, Adam; Cruickshanks, Nichola; Malkin, Mark; Kukreja, Rakesh C; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease. PMID:24651037

  1. Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site.

    Science.gov (United States)

    Liu, Yi-Liang; Lindert, Steffen; Zhu, Wei; Wang, Ke; McCammon, J Andrew; Oldfield, Eric

    2014-06-24

    We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC50 versus TbFPPS ? 20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC50 values in the range of ? 1-3 µM (as compared with ? 0.5 µM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg(2+) to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (Tm), quite different to the large increases in ?Tm seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads. PMID:24927548

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

    Directory of Open Access Journals (Sweden)

    Colpo Anna

    2010-10-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK-3 ? and ? are two serine-threonine kinases controlling insulin, Wnt/?-catenin, NF-?B signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3? and GSK-3? function in multiple myeloma (MM. Methods GSK-3 ? and ? expression and cellular localization were investigated by Western blot (WB and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 ? and ? isoforms. Survival signaling pathways were studied with WB analysis. Results GSK-3? and GSK-3? were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3? knock down decreased MM cell viability, while GSK-3? knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of ?-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3? knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. Conclusions These data suggest that in MM cells GSK-3? and ? i play distinct roles in cell survival and ii modulate the sensitivity to proteasome inhibitors.

  3. Inhibitory effects of four plants flavonoids extracts on fatty acid synthase.

    Science.gov (United States)

    Chen, Jun; Zhuang, Donghong; Cai, Weijia; Xu, Liyan; Li, Enmin; Wu, Yunying; Sugiyama, Kazuo

    2009-01-01

    Fatty acid synthase (FAS) had been found overexpress and hyperactive in most cancers. Pharmacological inhibitors of FAS activity preferentially repress cancer cell proliferation and induce cancer cell apoptosis without affecting nonmalignant fibroblasts. These made FAS an excellent drug target for cancer therapy. The activity of FAS in 11 different kinds of cancer cells, including esophageal carcinoma (EC109, EC8712, H5E973), gastric carcinoma (N87, BGC823), lung carcinoma (A549, 95-D), hepatoma (HepG2), uterine cervix cancer (HeLa) and leukaemia (K562, U937) were compared using spectrophotometric method. We selected the cell line with the highest FAS activity as cell model to study the inhibitory effect of the flavonoids extracts on FAS. Four plants including Canavium album Raeuseh leaves, Bombax ceiba Linn, Brassica albograbra Bailey, and Citrus reticulata Blanco were selected for extracting flavonoids. The results showed significantly different FAS activity among different cancer cells. The FAS activity is the lowest in gastric cancer cell N87 (15.91 ± 3.61 U/mg protein) and the highest in lung cancer cell A549 (127.36 ± 10.14 U/mg protein). The cancer cell A549 was used as cell model to test the inhibitory effort of flavonoids extracts on FAS. Results showed that the flavonoids extracts of Citrus reticulata Blanco and Canavium album Raeuseh leaves have higher inhibitory effect on FAS activity compared with the universally used FAS inhibitor C75 and both extracts inhibit cancer cell proliferation when added to cultured cancer cells. These studies provided a good cell model for testing the inhibitory effect on FAS activity and suggested that Citrus reticulata Blanco rind and Canavium album Raeuseh leaves are good biomaterials for separating and purifying natural flavonoids FAS inhibitors and exploring their inhibitory mechanisms. PMID:25084411

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

    Directory of Open Access Journals (Sweden)

    Asuncion Valenzuela MM

    2015-02-01

    Full Text Available Malyn M Asuncion Valenzuela, Imilce Castro, Amber Gonda, Carlos J Diaz Osterman, Jessica M Jutzy, Jonathan R Aspe, Salma Khan, Jonathan W Neidigh, Nathan R Wall Center for Health Disparities and Molecular Medicine, Division of Biochemistry, Department of Basic Sciences, Loma Linda University, Loma Linda, CA, USA Abstract: New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor and 5-fluorouracil (thymidylate synthase inhibitor were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma. Keywords: IAPs, exosomes, pancreatic cancer, antimetabolites, gemcitabine, cladribine, hydroxyurea, 5-fluorodeoxyuridine, 5-fluorouracil

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

    International Nuclear Information System (INIS)

    Glycogen Synthase Kinase-3 (GSK-3) ? and ? are two serine-threonine kinases controlling insulin, Wnt/?-catenin, NF-?B signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3? and GSK-3? function in multiple myeloma (MM). GSK-3 ? and ? expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 ? and ? isoforms. Survival signaling pathways were studied with WB analysis. GSK-3? and GSK-3? were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3? knock down decreased MM cell viability, while GSK-3? knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of ?-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3? knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. These data suggest that in MM cells GSK-3? and ? i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

  6. Caspofungin Inhibits Rhizopus oryzae 1,3-?-d-Glucan Synthase, Lowers Burden in Brain Measured by Quantitative PCR, and Improves Survival at a Low but Not a High Dose during Murine Disseminated Zygomycosis

    OpenAIRE

    Ibrahim, Ashraf S; Bowman, Joel C.; Avanessian, Valentina; Brown, Keturah; Spellberg, Brad; Edwards, John E.; Douglas, Cameron M.

    2005-01-01

    Rhizopus oryzae is the most common cause of zygomycosis, a life-threatening infection that usually occurs in patients with diabetic ketoacidosis. Despite standard therapy, the overall rate of mortality from zygomycosis remains >50%, and new strategies for treatment are urgently needed. The activities of caspofungin acetate (CAS) and other echinocandins (antifungal inhibitors of the synthesis of 1,3-?-d-glucan synthase [GS]) against the agents of zygomycosis have remained relatively unexplored...

  7. Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis

    OpenAIRE

    Havrylyuk D. Ya.; Fomenko I. S.; Panasyuk N. B.; Lesyk R. B.; Sklyarov A. Ya.

    2011-01-01

    Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor) and conventional non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO) system and intensity of oxidative stress in the mucous membrane of the large intestine (MMLI) in rats with experimental ulcerative colitis. Methods. Ulcerative colitis was induced by administration of acetic acid. The activity of NO-synthases, content of NO, and parameters of lipoperoxidation processes were measured in MML...

  8. [DNA methyltransferase inhibitors * histone deacetylase inhibitors].

    Science.gov (United States)

    Kikuchi, Jiro; Furukawa, Yusuke

    2014-06-01

    Epigenetics is a cell intrinsic mechanism to maintain genomic integrity by modifying chromatin architecture independently of changes in heritable DNA sequences namely genetic code. Chromatin is composed of nucleosome cores, in which DNA(147bp) is wrapped around a core histone octamer(two each of histones H2A, H2B, H3 and H4), arranged in a "beads-on-a-string array" with linker histones and non-histone nuclear proteins. The chromatin structure could be altered by chemical modifications of DNA and histones, including methylation and acetylation, without affecting genetic codes. In mammals, DNA methylation is mediated via DNA methyltransferases (Dnmt) at CpG dinucleotides. Histones are modified by numerous enzymes, such as histone acetyltransferases (HATs), deacetylases (HDACs), methyltransferases and demethylases, in spatio-temporarily distinct manners. These modifications could alter chromatin structures to regulate a wide variety of biological processes such as gene expression, cell cycle progression and DNA repair. Given the biological importance of epigenetic modifications, it is easy to speculate that the abnormalities of chromatin modifying enzymes and reader proteins underlie several human diseases such as cancer, inflammation and metabolic disorders. Because epigenetic states are reversible and could be modified in response to extrinsic signals, including small molecular compounds, an increased understanding of their molecular framework would allow us to treat pathological conditions caused by epigenetic alterations. Indeed, Dnmt inhibitors and HDAC inhibitors have already applied to the treatment of hematological malignancies with considerable success. PMID:25016817

  9. Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm.

    Science.gov (United States)

    Stubbe, Joanne; Tian, Jiamin; He, Aimin; Sinskey, Anthony J; Lawrence, Adam G; Liu, Pinghua

    2005-01-01

    This review focuses on nontemplate-dependent polymerases that use water-soluble substrates and convert them into water-insoluble polymers that form granules or inclusions within the cell. The initial part of the review summarizes briefly the current knowledge of polymer formation catalyzed by starch and glycogen synthases, polyphosphate kinase (a polymerase), cyanophycin synthetases, and rubber synthases. Specifically, our current understanding of their mechanisms of initiation, elongation (including granule formation), termination, remodeling, and polymer reutilization will be presented. General underlying principles that govern these types of polymerization reactions will be enumerated as a paradigm for all nontemplate-dependent polymerizations. The bulk of the review then focuses on polyhydroxyalkanoate (PHA) synthases that generate polyoxoesters. These enzymes are of interest as they generate biodegradable polymers. Our current knowledge of PHA production and utilization in vitro and in vivo as well as the contribution of many proteins to these processes will be reviewed. PMID:15952894

  10. Carbonic anhydrase inhibitors.

    Science.gov (United States)

    Supuran, Claudiu T; Scozzafava, Andrea; Casini, Angela

    2003-03-01

    At least 14 different carbonic anhydrase (CA, EC 4.2.1.1) isoforms were isolated in higher vertebrates, where these zinc enzymes play crucial physiological roles. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII), others are membrane-bound (CA IV, CA IX, CA XII, and CA XIV), CA V is mitochondrial and CA VI is secreted in saliva. Three acatalytic forms are also known, which are denominated CA related proteins (CARP), CARP VIII, CARP X, and CARP XI. Several important physiological and physio-pathological functions are played by many CA isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides as well as inorganic, metal complexing anions. The catalytic and inhibition mechanisms of these enzymes are understood in detail, and this helped the design of potent inhibitors, some of which possess important clinical applications. The use of such enzyme inhibitors as antiglaucoma drugs will be discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: several potent sulfonamide inhibitors inhibited the growth of a multitude of tumor cells in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Furthermore, some other classes of compounds that interact with CAs have recently been discovered, some of which possess modified sulfonamide or hydroxamate moieties. Some sulfonamides have also applications as diagnostic tools, in PET and MRI or as antiepileptics or for the treatment of other neurological disorders. Future prospects for drug design applications for inhibitors of these ubiquitous enzymes are also discussed. PMID:12500287

  11. Resistance to Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Mathieu Métifiot

    2010-06-01

    Full Text Available Integrase (IN is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance.

  12. Cathepsin D inhibitors

    Directory of Open Access Journals (Sweden)

    M. Gacko

    2007-11-01

    Full Text Available Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirectproduct, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes withcathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme.Methods used to determine the activity and concentration of these inhibitors and their analytical, preparative and therapeuticapplications are discussed.

  13. Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death.

    Science.gov (United States)

    Yang, C-S; Matsuura, K; Huang, N-J; Robeson, A C; Huang, B; Zhang, L; Kornbluth, S

    2015-06-01

    Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer. PMID:25151963

  14. Concentration Gradient Effects of Sodium and Lithium Ions and Deuterium Isotope Effects on the Activities of H+-ATP Synthase from Chloroplasts

    OpenAIRE

    Chen, M.-F.; Wang, J.-D.; Su, T.-M.

    2009-01-01

    We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H+-ATP synthase from chloroplasts (CF0F1). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF0F1. In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF0. In the presence of sodium or lithium ions and with the application of a membrane pot...

  15. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    OpenAIRE

    Abir U. Igamberdiev; Kleczkowski, Leszek A.

    2015-01-01

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, ...

  16. Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

    OpenAIRE

    Ro, Y T; Eom, C Y; Song, T.; Cho, J W; Kim, Y.M.

    1997-01-01

    Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. Th...

  17. Molecular Evolution and Functional Divergence of Soluble Starch Synthase Genes in Cassava (Manihot Esculenta Crantz)

    OpenAIRE

    Zefeng Yang; Yifan Wang; Shuhui Xu; Chenwu Xu; Changjie Yan

    2013-01-01

    Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in plants. Cassava starch has many remarkable characteristics, which should be influenced by the evolution of SS genes in this starchy root crop. In this work, we performed a comprehensive phylogenetic and evolutionary analysis of the soluble starch synthases in cassava. Genome-wide identification showed that there are 9 genes encoding soluble starch synthases in cassava. All of the soluble starch synthases encod...

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

    OpenAIRE

    Laughlin, Thomas F.; Ahmad, Zulfiqar

    2010-01-01

    Previously melittin, the ?-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the ?-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic ?-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  19. Identification of poly G bound to thymidylate synthase.

    Science.gov (United States)

    Thorndike, J; Kisliuk, R L

    1986-09-14

    Thymidylate synthase activity is increased in some methotrexate-resistant strains of Streptococcus faecium. The purified enzyme is associated with a polynucleotide which is not removed by dialysis. This polynucleotide contains one mole each of purine ribose and phosphate per mole base. Phosphate analyses after incubation with digestive enzymes indicate a tetranucleotide with one terminal phosphate. The constituent nucleosides are recovered quantitatively in a specific assay for guanosine. On HPLC, they are inseparable from authentic guanosine and the UV spectrum after HPLC is identical to that of guanosine. We conclude that poly G (GpGpGpGp) is bound to thymidylate synthase. PMID:3094514

  20. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

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

    2015-01-01

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

  1. Substrate Recognition by ?-Ketoacyl-ACP Synthases

    Science.gov (United States)

    Borgaro, Janine G.; Chang, Andrew; Machutta, Carl A.; Zhang, Xujie; Tonge, Peter J.

    2011-01-01

    ?-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ?-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14 residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the kcat/Km for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  2. Loop residues and catalysis in OMP synthase

    DEFF Research Database (Denmark)

    Wang, Gary P.; Hansen, Michael Riis

    2012-01-01

    Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100?109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 104-fold decrease in kcat/KM for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in kcat/KM, respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of kcat, with minor effects on kcat/KM. Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. ?102?106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 104-fold reduction of kcat but almost unaltered KD values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1?-3H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1?-3H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p)]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3?, 5?-adenosine diphosphate (3?, 5?-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3?, 5?-ADP is bound as the 3?, 5?-ADP part of CoA in the known structures of the CoA–AcpS and 3?, 5?-ADP–AcpS binary complexes. The position of the second 3?, 5?-ADP has never been described before. It is in close proximity to the first 3?, 5?-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  4. Thymidine Phosphorylase Inhibitors.

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim

    Karachi : Bentham Science Publishers, 2011 - (Atta-ur-Rahman, F.; Choudhary, M.), s. 116-147 ISBN 978-1-60805-162-5 R&D Projects: GA MŠk 1M0508; GA AV ?R 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors * angiogenesis * cancer chemotherapy Subject RIV: CC - Organic Chemistry

  5. Proteinaceous alpha-araylase inhibitors

    DEFF Research Database (Denmark)

    Svensson, Birte; Fukuda, Kenji; Nielsen, P.K.; Bønsager, Birgit Christine

    2004-01-01

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous a-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors act...

  6. Homology modeling and Molecular docking studies of AS1 (Anthranilate synthase component I (TrpE model of Mycobacterium tuberculos

    Directory of Open Access Journals (Sweden)

    Naresh Kumar K

    2013-07-01

    Full Text Available The emergence of multi-drug resistant (MDR strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE, whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. Anthranilate synthase is an interesting target enzyme for antimicrobial activity due to its presence in microorganisms for the synthesis of the essential amino acid tryptophan.  In the present study three compounds Cannabigerolic acid, cannabinolic acid and adhumulone from Cannabis sativa have been used for insilio docking studies.  Inhibitory studies (invitro of these compounds against Microorganism have reported earlier.  Our approach is to find out the compounds inhibiting the AS1 of MTB by insilico docking and also find out compounds having similar pharmacophore characters from ZINC database so that those compounds can be procured of synthesized in laboratory and used for AS1 inhibitor studies.  This study shows that AS can be used as a target enzyme to investigate the mode of action of our compounds in MTB.

  7. Dysregulation of FURIN by prostaglandin-endoperoxide synthase 2 in lung epithelial NCI-H292 cells.

    Science.gov (United States)

    Brant, Kelly A; Leikauf, George D

    2014-03-01

    Because proprotein convertases (PCSKs) activate growth factors and matrix metalloproteinase, these enzymes have been implicated in non-small cell lung cancer tumor progression and aggressiveness. Previous studies indicate that one PCSK member, FURIN is overexpressed in NSCLC, but little is known regarding the mechanisms driving PCSKs expression during malignant change. We sought to determine whether prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) (PTGS2) (aka COX2), whose expression is also frequently increased in NSCLC, differentially regulates PCSK expression and activity between normal (NHBE) and NSCLC epithelial cells (NCI-H292, NCI-H441, A549). NSCLC cells exhibit significantly greater cell-associated and secreted PCSK activity as compared with NHBE. The heightened activity is consistent with increased FURIN, PCSK4, and PCSK6 protein in the NCSLC cells. Inhibition of PTGS2 activity using NS-398 and siRNA decreased FURIN mRNA, protein, activity along with cell proliferation in NCI-H292 cells but not NHBE cells. NSCLC also expressed elevated levels of the transcription factor E2F1. When NCI-H292 cells were transfected with E2F1 siRNA, both PTGS2 expression and PCSK activity were attenuated, arguing a pivotal role for E2F1 in the differential regulation of PCSKs by PTGS2. Our results highlight a novel role for PTGS2 in NSCLC and may provide a mechanism, whereby PTGS2 inhibitors suppress lung cancer cell growth. PMID:23065687

  8. Crystallization and preliminary crystallographic studies of dihydrofolate reductase-thymidylate synthase from Trypanosoma cruzi, the Chagas disease pathogen

    International Nuclear Information System (INIS)

    Crystals of complexes of the T. cruzi dihydrofolate reductase-thymidylate synthase enzyme with three antifolates in two space groups have been obtained that diffracted to 2.1–2.8 Å resolution. The antifolates used for cocrystallization were dihydrotriazine-based and quinazoline-based antifolates. Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase (TcDHFR-TS) was crystallized in complexes with the dihydrotriazine-based or quinazoline-based antifolates C-448, cycloguanil (CYC) and Q-8 in order to gain insight into the interactions of this DHFR enzyme with classical and novel inhibitors. The TcDHFR-TS–C-448–NDP–dUMP crystal belonged to space group C2221 with two molecules per asymmetric unit and diffracted to 2.37 Å resolution. The TcDHFR-TS–CYC, TcDHFR-TS–CYC–NDP and TcDHFR-TS–Q-8–NDP crystals belonged to space group P21 with four molecules per asymmetric unit and diffracted to 2.1, 2.6 and 2.8 Å resolution, respectively. Crystals belonging to the two different space groups were suitable for structure determination

  9. Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

    OpenAIRE

    Jones, Christopher G.; Moniodis, Jessie; Zulak, Katherine G.; Scaffidi, Adrian; Plummer, Julie A.; Ghisalberti, Emilio L.; Barbour, Elizabeth L.; Bohlmann, Jo?rg

    2012-01-01

    Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of ?-, ?-, epi-?-santalene, and ?-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). ...

  10. Intrinsic uncoupling in the ATP synthase of Escherichia coli.

    Science.gov (United States)

    D'Alessandro, Manuela; Turina, Paola; Melandri, B Andrea

    2008-12-01

    The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi. PMID:18952048

  11. Crystallization of ?1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    International Nuclear Information System (INIS)

    ?1-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. ?1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å3 Da?1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

  12. Spermidine synthase as affected by osmotic stress in oat leaves

    International Nuclear Information System (INIS)

    Osmotically-induced putrescine (Put) accumulation in cereals could result not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of spermidine synthase, the enzyme which catalyzes the transformation of Put to spermidine (Spd). To test the latter possibility, they evaluated Spd synthase activity in oat leaves as affected by osmotic stress. They developed a new assay for Spd synthase activity by adding S-adenosylmethionine, C14-Put and pyridoxal phosphate to the assay mixture. Incorporation of the C14-label into Spd can be detected after 45 min of incubation at 370C. Labelled Spd is separated from labelled Put or spermine by elution with HCl in Dowex 50 W-H+ columns. In peeled oat leaves floated in the dark over 0.6 M sorbitol in 1mM PO4 buffer (pH 5.8) for 6 and 136 h. Spd synthase activity is reduced by 24 and 53%, respectively, as compared with controls. The results suggest that the activity of this enzyme is inhibited by osmotic stress, and could partially account for the accumulation of Put

  13. A Common Genetic Basis for Cross-Sensitivity to Mesotrione and Nicosulfuron in Sweet Corn Hybrid Cultivars and Inbreds Grown Throughout North America

    Science.gov (United States)

    In previous research, the sweet corn inbred line Cr1 was observed to be sensitive to multiple postemergence herbicides, including four acetolactate synthase (ALS)-inhibiting herbicides, three 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides, a growth regulator herbicide combination, ...

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

    Science.gov (United States)

    Using whole genome Affymetrix ATH1 GeneChips we characterized the transcriptional response of Arabidopsis thaliana Columbia 24 hours after treatment with five different herbicides. Four of them (chloransulam, imazapyr, primisulfuron, sulfometuron) inhibit acetolactate synthase (A...

  15. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel (Maryland); (GWU); (Georgia)

    2012-05-24

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

  16. Role of nitric oxide-synthase and cyclooxygenase/lipooxygenase systems in development of experimental ulcerative colitis.

    Science.gov (United States)

    Sklyarov, A Ya; Panasyuk, N B; Fomenko, I S

    2011-02-01

    Development of ulcerative colitis was accompanied by the activation of iNOS/COX-2/5-LOX and increased contents of nitric oxide (NO), prostaglandin E? (PGE?), and leukotriene B? (LTB?). The following information was assessed: morphological changes, activity of nitric oxide-synthase, content of nitric oxide, and indexes of lipoperoxidation processes in the mucous membrane of the large intestine (MMLI). Colitis was induced in rats by intrarectal administration of 1 ml of 4% acetic acid. Aminoguanidine--selective inducible nitric oxide-synthase (iNOS) blocker, celecoxib--cyclooxygenase-2 (COX-2) inhibitor, indomethacin--non-selective COX inhibitor and AA-861--5-lipooxygenase (5-LOX) blocker were administered in 1 ml volumes per os 1 hour before and 24 hours after the intrarectal application of acetic acid. It was noticed that blockage of iNOS by aminoguanidine caused enhancement of cytoprotective mechanisms, reduction of iNOS activity and oxidative stress, and an increase in blood L-arginine level as compared to their respective indexes in colitis. Combined blockage of iNOS and COX-2 displayed additive character of their effect on the processes of lipoperoxidation and activity of iNOS. Combined blockage of iNOS, COX-2 and 5-LOX had a manifested cytoprotective effect under condition of ulcerative colitis and was accompanied by a sharp decline in NOS activity and oxidative stress. If each of these systems, iNOS/NO, COX-2/PGE? and 5-LOX/LTB? are simultaneously activated due to inflammation, they contribute to the destructive damage of the MMLI, development of oxidative stress, and affect components of the antioxidant protection system. The obtained results substantiate the relevance of treatment of the inflammatory processes with the use of medication capable of combined blockage of iNOS, COX-2, and 5-LOX. PMID:21451211

  17. Novel type III polyketide synthases from Aloe arborescens.

    Science.gov (United States)

    Mizuuchi, Yuusuke; Shi, She-Po; Wanibuchi, Kiyofumi; Kojima, Akiko; Morita, Hiroyuki; Noguchi, Hiroshi; Abe, Ikuro

    2009-04-01

    Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85-96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl-CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild-type PKS obtained from A. arborescens. Interestingly, the aloesone-forming activity was maximum at 50 degrees C, and the novel heptaketide pyrone was non-enzymatically converted to aloesone. In PKS3, the active-site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site-directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone. PMID:19348024

  18. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1?3),(1?4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  19. Benzoylurea Chitin Synthesis Inhibitors.

    Science.gov (United States)

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs. PMID:26168369

  20. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    LENUS (Irish Health Repository)

    Cathcart, Mary-Clare

    2012-02-01

    BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced\\/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC.

  1. Neuronal nitric oxide synthase supports Renin release during sodium restriction through inhibition of phosphodiesterase 3

    DEFF Research Database (Denmark)

    Sällström, Johan; Jensen, Boye L

    2010-01-01

    BACKGROUND: Mice with targeted deletion of neuronal nitric oxide (NO) synthase (nNOS?(/)?) display inability to increase plasma renin concentration (PRC) in response to sodium restriction. nNOS has a distinct expression at the macula densa (MD), and in the present study, it was tested whether nNOS supports renin release by cyclic guanosine monophosphate (cGMP)-mediated inhibition of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase 3 (PDE3) in juxtaglomerular (JG) cells. METHODS: The experiments were performed in conscious nNOS?(/)? and wild types after 10 days on a low-sodium diet by acute treatment with the PDE3-inhibitor milrinone, the PDE5 inhibitor zaprinast, or vehicle, using a crossover study protocol. PRC was measured with the antibody-trapping technique and blood pressure with telemetry. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were estimated by measurements of inulin- and para-amino hippuric acid (PAH) clearances, respectively. RESULTS: The basal PRC was reduced innNOS?(/)? compared to the wild types. Administration of milrinone caused a more pronounced PRC increase in nNOS?(/)?, resulting in normalized renin levels, whereas PDE5 inhibition did not affect PRC in any genotype. The blood pressure was similar in both genotypes, and milrinone did not affect blood pressure compared to vehicle. GFR and RPF were similar at baseline and were reduced by milrinone. CONCLUSIONS: The present study provides in vivo evidence supporting the view that NO, selectively derived from nNOS, mediates renin release during sodium restriction by inhibiting PDE3, which would increase renin release by elevating cAMP levels in the JG cells.

  2. Sequencing of aromatase inhibitors

    OpenAIRE

    Bertelli, G

    2005-01-01

    Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain p...

  3. Osteocompatibility of Biofilm Inhibitors

    OpenAIRE

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), f...

  4. Cathepsin D inhibitors.

    OpenAIRE

    ?ukasz Minarowski; Alicja Karwowska; Alina Minarowska; Marek Gacko

    2008-01-01

    Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231 residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirect product, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes with cathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme. Methods used to dete...

  5. NK cell function triggered by multiple activating receptors is negatively regulated by glycogen synthase kinase-3?.

    Science.gov (United States)

    Kwon, Hyung-Joon; Kwon, Soon Jae; Lee, Heejae; Park, Hye-Ran; Choi, Go-Eun; Kang, Sang-Wook; Kwon, Seog Woon; Kim, Nacksung; Lee, Soo Young; Ryu, Sangryeol; Kim, Sun Chang; Kim, Hun Sik

    2015-09-01

    Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3? as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3? or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3? isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3?. Importantly, the regulation of NK cell function by GSK-3? was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3? negatively regulates NK cell activation and that modulation of GSK-3? function could be used to enhance NK cell activation. PMID:26022178

  6. Neuronal nitric oxide synthase-dependent amelioration of diastolic dysfunction in rats with chronic renocardiac syndrome.

    Science.gov (United States)

    Bongartz, Lennart G; Soni, Siddarth; Cramer, Maarten-Jan; Steendijk, Paul; Gaillard, Carlo A J M; Verhaar, Marianne C; Doevendans, Pieter A; van Veen, Toon A; Joles, Jaap A; Braam, Branko

    2015-02-01

    We have recently described the chronic renocardiac syndrome (CRCS) in rats with renal failure, cardiac dysfunction and low nitric oxide (NO) availability by combining subtotal nephrectomy and transient low-dose NO synthase (NOS) inhibition. Cardiac gene expression of the neuronal isoform of NOS (nNOS) was induced. Hence, we studied the role of nNOS, in vivo cardiac function and ?-adrenergic response in our CRCS model by micromanometer/conductance catheter. Left ventricular (LV) hemodynamics were studied during administration of dobutamine (dobu), the highly specific irreversible inhibitor of nNOS L-VNIO [L-N5-(1-Imino-3-butenyl)-ornithine], or both at steady state and during preload reduction. Rats with CRCS showed LV systolic dysfunction at baseline, together with prolonged diastolic relaxation and rightward shift of the end-systolic pressure-volume relationships. After L-VNIO infusion, diastolic relaxation of CRCS rats further prolonged. The time constant of active relaxation (tau) increased by 25 ± 6% from baseline (p < 0.05), and the maximal rate of pressure decrease was 36 ± 7% slower (p < 0.001). These variables did not change in controls. In our CRCS model, nNOS did not seem to affect systolic dysfunction. In summary, in this model of CRCS, blockade of nNOS further worsens diastolic dysfunction and L-VNIO does not influence inherent contractility and the response to dobu stress. PMID:25759702

  7. Potent Inhibitory Effect of Chinese Dietary Spices on Fatty Acid Synthase.

    Science.gov (United States)

    Jiang, Bing; Liang, Yan; Sun, Xuebing; Liu, Xiaoxin; Tian, Weixi; Ma, Xiaofeng

    2015-09-01

    Dietary spices have been adopted in cooking since ancient times to enhance flavor and also as food preservatives and disease remedies. In China, the use of spices and other aromatic plants as food flavoring is an integral part of dietary behavior, but relatively little is known about their functions. Fatty acid synthase (FAS) has been recognized as a remedy target, and its inhibitors might be applied in disease treatment. The present work was designed to assess the inhibitory activities on FAS of spices extracts in Chinese menu. The in vitro inhibitory activities on FAS of 22 extracts of spices were assessed by spectrophotometrically monitoring oxidation of NADPH at 340 nm. Results showed that 20 spices extracts (90.9 %) exhibited inhibitory activities on FAS, with half inhibition concentration (IC50) values ranging from 1.72 to 810.7 ?g/ml. Among them, seven spices showed strong inhibitory effect with IC50 values lower than 10 ?g/ml. These findings suggest that a large proportion of the dietary spices studied possess promising inhibitory activities on FAS, and subsequently might be applied in the treatment of obesity and obesity-related human diseases. PMID:25924694

  8. Inhibitory effects of sea buckthorn procyanidins on fatty acid synthase and MDA-MB-231 cells.

    Science.gov (United States)

    Wang, Yi; Nie, Fangyuan; Ouyang, Jian; Wang, Xiaoyan; Ma, Xiaofeng

    2014-10-01

    Fatty acid synthase (FAS) is overexpressed in many human cancers including breast cancer and is considered to be a promising target for therapy. Sea buckthorn has long been used to treat a variety of maladies. Here, we investigated the inhibitory effect of sea buckthorn procyanidins (SBPs) isolated from the seeds of sea buckthorn on FAS and FAS overexpressed human breast cancer MDA-MB-231 cells. The FAS activity and FAS inhibition were measured by a spectrophotometer at 340 nm of nicotinamide adenine dinucleotide phosphate (NADPH) absorption. We found that SBP potently inhibited the activity of FAS with a half-inhibitory concentration (IC50) value of 0.087 ?g/ml. 3-4,5-Dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide (MTT) assay was used to test the cell viability. SBP reduced MDA-MB-231 cell viability with an IC50 value of 37.5 ?g/ml. Hoechst 33258/propidium iodide dual staining and flow cytometric analysis showed that SBP induced MDA-MB-231 cell apoptosis. SBP inhibited intracellular FAS activity with a dose-dependent manner. In addition, sodium palmitate could rescue the cell apoptosis induced by SBP. These results showed that SBP was a promising FAS inhibitor which could induce the apoptosis of MDA-MB-231 cells via inhibiting FAS. These findings suggested that SBP might be useful for preventing or treating breast cancer. PMID:24957042

  9. Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury

    International Nuclear Information System (INIS)

    The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process

  10. Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress

    Directory of Open Access Journals (Sweden)

    Roberta Venè

    2014-09-01

    Full Text Available Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenylretinamide (4HPR, the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11-dien-28-oic acid methyl ester, arsenic trioxide (As2O3, and phenylethyl isothiocyanate (PEITC, while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3? (GSK3?. The antioxidant N-acetylcysteine decreased GSK3? phosphorylation and poly(ADP-ribose polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3? phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1, and transient elevation of intracellular glutathione (GSH in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3? or transfection with the non-phosphorylatable GSK3?-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3? phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3? phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance.

  11. Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.

    Science.gov (United States)

    Bianchi-Smiraglia, A; Wawrzyniak, J A; Bagati, A; Marvin, E K; Ackroyd, J; Moparthy, S; Bshara, W; Fink, E E; Foley, C E; Morozevich, G E; Berman, A E; Shewach, D S; Nikiforov, M A

    2015-11-01

    Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent. PMID:25909885

  12. Proton transport coupled ATP synthesis by the purified yeast H+ -ATP synthase in proteoliposomes.

    Science.gov (United States)

    Förster, Kathrin; Turina, Paola; Drepper, Friedel; Haehnel, Wolfgang; Fischer, Susanne; Gräber, Peter; Petersen, Jan

    2010-11-01

    The H(+)/ATP synthase from yeast mitochondria, MF?F?, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF?F? liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s?¹ at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MF?F?, was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H?PO??. PMID:20691145

  13. Study of aldosterone synthase inhibition as an add-on therapy in resistant hypertension.

    Science.gov (United States)

    Karns, Adam D; Bral, Jacqueline M; Hartman, Daniel; Peppard, Thomas; Schumacher, Christoph

    2013-03-01

    Aldosterone inhibition with mineralcorticoid receptor antagonists (MRAs) is an effective treatment for resistant hypertension. Aldosterone synthase inhibitors (ASIs) are currently being investigated as a new therapeutic strategy to reduce aldosterone secretion from the adrenal gland. In this study, the efficacy and safety of the first-generation ASI LCI699 (0.25 mg twice daily, 1 mg 4 once daily, and 0.5 mg/1 mg twice daily) was compared with placebo and eplerenone (50 mg twice daily), in patients with resistant hypertension. Placebo-adjusted decreases in systolic blood pressure (BP) with LCI699 ranged from 2.6 mm Hg to 4.3 mm Hg at week 8; changes in diastolic BP ranged from +0.3 mm Hg to -1.2 mm Hg. However, reductions were smaller than observed with eplerenone 50 mg twice daily (9.9 mm Hg and 2.9 mm Hg for systolic and diastolic BP, respectively) and not statistically significant vs placebo. LCI699 suppressed plasma aldosterone levels in a dose-related manner with corresponding dose-dependent increases in plasma renin activity and plasma 11-deoxycorticosterone. LCI699 and eplerenone were well tolerated. These data demonstrate that aldosterone synthesis inhibition with LCI699 lowers BP modestly in patients with resistant hypertension. Aldosterone synthesis inhibition might offer an attractive adjunct to aldosterone receptor blockade, although the potential of a combination MRA/ASI has not yet been tested. PMID:23458591

  14. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use

    Science.gov (United States)

    Pizza, F. X.; Hernandez, I. J.; Tidball, J. G.

    1998-01-01

    The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water-treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for L-NAME-treated animals compared with water-treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between L-NAME-treated and control animals, suggesting that L-NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells.

  15. Glycogen Synthase Kinase 3? Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

    Directory of Open Access Journals (Sweden)

    Liang Ma

    2013-08-01

    Full Text Available Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3? (GSK3?, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3? inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3? by either lithium chloride (LiCl or specific GSK3? inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952 and type II (ARK1 endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3? activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  16. Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

    DEFF Research Database (Denmark)

    Graebe, Martin; Brond, Lone

    2004-01-01

    The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that l-NAME treatment dose dependently deteriorated cumulative sodium balance by reducing urinary sodium excretion. Five weeks after CBL, renal clearance studies were performed, followed by Western blotting of the electroneutral type 3 sodium/proton exchanger (NHE3) and the Na-K-ATPase present in proximal tubules. Untreated CBL rats showed a decreased proximal reabsorption with a concomitant reduction of NHE3 and Na-K-ATPase levels, indicating that tubular segments distal to the proximal tubules were responsible for the increased sodium reabsorption. l-NAME-treated CBL rats showed an increased proximal reabsorption measured by the lithium clearance method and showed a marked increase in NHE3 and Na-K-ATPase protein levels. Our results show that chronic l-NAME treatment exacerbates the sodium retention found in CBL rats by a significant increase in proximal tubular reabsorption.

  17. Role of Glycogen Synthase Kinase-3? in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Xanthi Antoniou

    2010-01-01

    Full Text Available The phosphorylation of Amyloid Precursor Protein (APP at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK, glycogen synthase kinase-3? (GSK-3? and cyclin-dependent kinase 5 (Cdk5 can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 ?M for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3? activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3? reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3? signaling pathway.

  18. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    Energy Technology Data Exchange (ETDEWEB)

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: tmiller2@utep.edu [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  19. Inhibitory effect of organotin compounds on rat neuronal nitric oxide synthase through interaction with calmodulin

    International Nuclear Information System (INIS)

    Organotin compounds, triphenyltin (TPT), tributyltin, dibutyltin, and monobutyltin (MBT), showed potent inhibitory effects on both L-arginine oxidation to nitric oxide and L-citrulline, and cytochrome c reduction catalyzed by recombinant rat neuronal nitric oxide synthase (nNOS). The two inhibitory effects were almost parallel. MBT and TPT showed the highest inhibitory effects, followed by tributyltin and dibutyltin; TPT and MBT showed inhibition constant (IC50) values of around 10 ?M. Cytochrome c reduction activity was markedly decreased by removal of calmodulin (CaM) from the complete mixture, and the decrease was similar to the extent of inhibition by TPT and MBT. The inhibitory effect of MBT on the cytochrome c reducing activity was rapidly attenuated upon dilution of the inhibitor, and addition of a high concentration of CaM reactivated the cytochrome c reduction activity inhibited by MBT. However, other cofactors such as FAD, FMN or tetrahydrobiopterin had no such ability. The inhibitory effect of organotin compounds (100 ?M) on L-arginine oxidation of nNOS almost vanished when the amount of CaM was sufficiently increased (150-300 ?M). It was confirmed by CaM-agarose column chromatography that the dissociation of nNOS-CaM complex was induced by organotin compounds. These results indicate that organotin compounds disturb the interaction between CaM and nNOS, thereby inhibiting electron transfer from the reductase domain to cytochrome c and the oxygenase domain

  20. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    International Nuclear Information System (INIS)

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [14C]-L-arginine to [14C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [14C]-L-arginine to [14C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  1. Expression of nitric oxide synthases in rat adrenal zona fasciculata cells.

    Science.gov (United States)

    Cymeryng, Cora B; Lotito, Sebastián P; Colonna, Cecilia; Finkielstein, Carla; Pomeraniec, Yael; Grión, Natalia; Gadda, Luciana; Maloberti, Paula; Podestá, Ernesto J

    2002-04-01

    Nitric oxide (NO) synthase (NOS) expression was analyzed in rat adrenal zona fasciculata. Both neuronal NOS and endothelial NOS mRNAs were detected by RT-PCR, immunohistochemistry, and immunoblot analysis. The biochemical characterization of adrenal zona fasciculata NOS enzymatic activity confirmed the presence of a constitutive isoform. In a cell line derived from mouse adrenal cortex, only endothelial NOS expression was detected by both RT-PCR and immunoblot analysis. Nitrate plus nitrite levels in Y1 cell incubation medium were increased in the presence of L-arginine and the calcium ionophore A23187, but not D-arginine, indicating enzymatic activity. Moreover, a low, but significant, conversion of Larginine to L-citrulline, abolished by the NOS inhibitor, N(G)-nitro-L-arginine, was detected in Y1 cells. The effect of L-arginine on pregnenolone production was examined. L-Arginine decreased both basal and ACTH-stimulated pregnenolone production in Y1 cells. The inhibitory effect of L-arginine could be attributed to endogenously generated NO, because it was blocked by N(G)-nitro-L-arginine, and it was mimicked by the addition of a NO donor, diethylenetriamine-NO. An inhibitory effect of NO on pregnenolone production from 22Rhydroxycholesterol and on steroidogenic acute regulatory protein expression was also determined. Taken together, these results suggest that at least part of the adrenal NO could derive from steroidogenic cells and modulate their function. PMID:11897679

  2. Comparative computational analysis of active and inactive cofactors of nitric oxide synthase.

    Science.gov (United States)

    Menyhárd, Dóra K

    2009-03-12

    Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide from L-Arg in the presence of oxygen. Of the two electrons required for the first step of the reaction, the second is primarily donated by the tetrahydrobiopterin (H4B) cofactor bound adjacent to the heme, which is eventually reduced back to resting state by the ultimate electron source of the reaction, the flavins of the NOS reductase domain. Density functional theory calculations were carried out to identify those protonation states of different cofactor molecules that best support radicalization of the cofactor and the coupled increase in the electron density of the heme-bound oxygen molecule. Three cofactor molecules were studied, native H4B, an active analogue, 5-methyl-H4B, and the inactive 4-amino-H4B. Findings support the emerging model where H4B and 5-methyl-H4B are coupled proton/electron sources of NOS catalysis, while 4-amino-H4B is an inhibitor due to its inability to donate the catalytically required proton. PMID:19708267

  3. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

    OpenAIRE

    Kida, Y; Esposito-Del Puente, A; Bogardus, C; Mott, D M

    1990-01-01

    Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatas...

  4. Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Havrylyuk D. Ya.

    2011-04-01

    Full Text Available Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor and conventional non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO system and intensity of oxidative stress in the mucous membrane of the large intestine (MMLI in rats with experimental ulcerative colitis. Methods. Ulcerative colitis was induced by administration of acetic acid. The activity of NO-synthases, content of NO, and parameters of lipoperoxidation processes were measured in MMLI. Results. COX-2/5-LOX inhibition by 2A5DHT compound did not cause considerable destructive changes of the MMLI of rats. The activity of inducible nitric oxide synthase (iNOS declined more than 2 fold as compared to their activity in colitis. The intensity of lipoperoxidation processes was found to be much lower than under the separate effect of celecoxib or indomethacine. Conclusions. Dual COX-2/5-LOX inhibition by 2A5DHT has a significant cytoprotective effect in MMLI that is accompanied by reduction of oxidative stress and activity of NO-synthases. The substance 2A5DHT significantly overexceeds the cytoprotective effects of both selective and non-selective COX/LOX inhibitors and can be used in the treatment of inflammatory bowel disease

  5. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Science.gov (United States)

    Koo, Hyun Jo; Gang, David R

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-?-bisabolene synthase from ginger rhizome, and ?-humulene synthase and ?-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (-)-caryolan-1-ol synthase and ?-zingiberene/?-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-?-turmerone and (+)-?-turmerone, are produced from (-)-?-zingiberene and (-)-?-sesquiphellandrene, respectively, via ?-zingiberene/?-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

  6. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    Science.gov (United States)

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Padvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research. PMID:26638491

  7. Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents.

    Science.gov (United States)

    Piano, Valentina; Benjamin, Daniel I; Valente, Sergio; Nenci, Simone; Marrocco, Biagina; Mai, Antonello; Aliverti, Alessandro; Nomura, Daniel K; Mattevi, Andrea

    2015-11-20

    Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy. PMID:26322624

  8. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    Science.gov (United States)

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls. PMID:26450210

  9. HIV-1 Entry Inhibitor

    Science.gov (United States)

    Soluble forms (sCD4) of human CD4, the HIV-1 primary receptor, are potent HIV-1 entry inhibitors. Both four-domain (D1-4) and two-domain (D1D2) sCD4 and their fusion proteins have been tested as candidate therapeutics in animal models and in human clinical trials and were well tolerated by patients with no significant clinical or immunologic toxicities and exhibited significant inhibitory activities. However, their activities were transient and the virus rapidly rebound.

  10. Deficiency of mitochondrial ATP synthase of nuclear genetic origin.

    Czech Academy of Sciences Publication Activity Database

    Sperl, W.; Ješina, Pavel; Zeman, J.; Mayr, J. A.; DeMeirleir, L.; VanCoster, R.; Pícková, Andrea; Hansíková, H.; Houš?ková, H.; Krej?ík, Zden?k; Koch, J.; Smet, J.; Muss, W.; Holme, E.; Houšt?k, Josef

    2006-01-01

    Ro?. 16, ?. 11 (2006), s. 821-829. ISSN 0960-8966 R&D Projects: GA MZd(CZ) NR7790; GA MŠk(CZ) 1M0520 Grant ostatní: CZ-AT(CZ) 6-06-3 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondria * ATP synthase * disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.615, year: 2006

  11. Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

    OpenAIRE

    Banerjee, Kamalika; Gupta, Utkarsh; Gupta, Sanjay; Wadhwa, Gulshan; Gabrani, Reema; Sharma, Sanjeev Kumar; Jain, Chakresh Kumar

    2011-01-01

    Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB struc...

  12. The Cellulose Synthase Complex: A Polymerization Driven Supramolecular Motor

    OpenAIRE

    Diotallevi, Fabiana; Mulder, Bela

    2007-01-01

    We present a biophysical model for the propulsion of the cellulose synthase complex, the motile transmembrane protein complex responsible for the biosynthesis of cellulose microfibrils, the dominant architectural component of the cell walls of higher plants. Our model identifies the polymerization and the crystallization of the cellulose chains as the combined driving forces and elucidates the role of polymer flexibility and membrane elasticity as force transducers. The model is elaborated us...

  13. Metabolism of aromatic amines by prostaglandin H synthase.

    OpenAIRE

    Boyd, J A; Eling, T. E.

    1985-01-01

    The metabolism of aromatic amines by the peroxidase activity of prostaglandin H synthase (PHS) has been studied in this laboratory by use of two model compounds, the carcinogenic primary amine 2-aminofluorene (2-AF) and the substituted amine aminopyrine (AP). 2-AF is oxidized by PHS to 2, 2-azobisfluorene, 2-aminodifluorenylamine, 2-nitrofluorene, polymeric material, and products covalently bound to macromolecules. In the presence of phenolic compounds, 2-AF oxidation results in the formation...

  14. Regulation of phenoxazinone synthase expression in Streptomyces antibioticus.

    OpenAIRE

    G. H. Jones

    1985-01-01

    The cloned gene for the subunit of phenoxazinone synthase (PHS), an enzyme implicated in the biosynthesis of actinomycin in Streptomyces antibioticus, was used as a probe to study the regulation of the enzyme. The direction of transcription of the PHS gene was determined with end-labeled restriction fragments derived from the gene. Low-resolution S1 mapping revealed that transcription was initiated at a position which may lie within the SphI restriction site, which represents the limit of the...

  15. Mitochondrial diseases and genetic defects of ATP synthase.

    Czech Academy of Sciences Publication Activity Database

    Houšt?k, Josef; Pícková, Andrea; Vojtíšková, Alena; Mrá?ek, Tomáš; Pecina, Petr; Ješina, Pavel

    2006-01-01

    Ro?. 1757, ?. 9-10 (2006), s. 1400-1405. ISSN 0005-2728 R&D Projects: GA MZd(CZ) NR7790; GA MŠk(CZ) 1M0520 Grant ostatní: CZ-AT(CZ) 6-06-3 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondrial diseases * ATP synthase * ROS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.237, year: 2006

  16. Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

    OpenAIRE

    Barney, Brett M.; Wahlen, Bradley D.; Garner, EmmaLee; Wei, Jiashi; Seefeldt, Lance C.

    2012-01-01

    Wax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four differe...

  17. Identification of a family of animal sphingomyelin synthases

    OpenAIRE

    Huitema, Klazien; van den Dikkenberg, Joep; Brouwers, Jos F. H. M.; Holthuis, Joost C. M.

    2003-01-01

    Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM syntha...

  18. Isoflavone synthase genes in legumes and non-leguminous plants.

    Czech Academy of Sciences Publication Activity Database

    Pi?manová, Martina; Koblovská, R.; Lap?ík, O.; Honys, David

    Washington, D.C : IEEE Computer Society, 2012 - (Sloan, K.), s. 344-347 ISBN 978-0-7695-4706-0. [International Conference on Biomedical Engineering and Biotechnology /2012/. Macau (CN), 28.05.2012-30.05.2012] R&D Projects: GA ?R GA525/09/0994; GA ?R(CZ) GAP501/11/1462; GA MŠk(CZ) OC10054 Institutional support: RVO:61389030 Keywords : legumes * non-leguminous plants * isoflavone synthase Subject RIV: EF - Botanics

  19. Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

    OpenAIRE

    Jope, Richard S.; Yuskaitis, Christopher J.; Beurel, Eléonore

    2006-01-01

    Deciphering what governs inflammation and its effects on tissues is vital for understanding many pathologies. The recent discovery that glycogen synthase kinase-3 (GSK3) promotes inflammation reveals a new component of its well-documented actions in several prevalent diseases which involve inflammation, including mood disorders, Alzheimer’s disease, diabetes, and cancer. Involvement in such disparate conditions stems from the widespread influences of GSK3 on many cellular functions, with this...

  20. Organization of the thymidylate synthase gene of herpesvirus saimiri.

    OpenAIRE

    Bodemer, W.; Niller, H H; Nitsche, N; Scholz, B; Fleckenstein, B.

    1986-01-01

    Herpesvirus saimiri codes, unlike most other herpesviruses, for a thymidylate synthase (TS). The TS gene of herpesvirus saimiri is unusual in structure and regulation of expression. It is transcribed into a nonspliced mRNA of 2,190 nucleotides. The single open reading frame of the viral TS gene, instructing a polypeptide of 33.5 kilodaltons, has extensive sequence homology with the corresponding TS coding sequences of human cells and of various procaryotes; the putative polypeptide derived fr...

  1. trans activation of the thymidylate synthase promoter of herpesvirus saimiri.

    OpenAIRE

    Lang, G; Fleckenstein, B.

    1990-01-01

    Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expr...

  2. ATP Synthase: Subunit-Subunit Interactions in the Stator Stalk

    OpenAIRE

    Weber, Joachim

    2006-01-01

    In ATP synthase, proton translocation through the Fo subcomplex and ATP synthesis/hydrolysis in the F1 subcomplex are coupled by subunit rotation. The static, non-rotating portions of F1 and Fo are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b2?; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measure...

  3. Modulation of nitric oxide synthase activity in macrophages

    OpenAIRE

    Jorens, P.G.; K. E. Matthys; Bult, H

    1995-01-01

    L-Arginine is converted to the highly reactive and unstable nitric oxide (NO) and L-citrulline by an enzyme named nitric oxide synthase (NOS). NO decomposes into other nitrogen oxides such as nitrite (NO2-) and nitrate (NO2-), and in the presence of superoxide anion to the potent oxidizing agent peroxynitrite (ONOO?). Activated rodent macrophages are capable of expressing an inducible form of this enzyme (iNOS) in response to appropriate stimuli, i.e., lipopolysaccharide (LPS) and...

  4. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  5. Inducible Nitric Oxide Synthase Genetic Polymorphism and Risk of Asbestosis

    OpenAIRE

    Alenka Franko; Metoda Dodi?-Fikfak; Niko Arneri?; Vita Dolžan

    2011-01-01

    Asbestos, a known occupational pollutant, may upregulate the activity of inducible nitric oxide synthase (iNOS) and thus the production of nitric oxide (NO). This study investigated whether iNOS?(CCTTT)n polymorphism is associated with an increased asbestosis risk in exposed workers. The study cohort consisted of 262 cases with asbestosis and 265 controls with no asbestos-related disease. For each subject the cumulative asbestos exposure data were available. The number of CCTTT repeats wa...

  6. The cardiovascular biology of microsomal prostaglandin E synthase-1

    OpenAIRE

    Wang, Miao; FitzGerald, Garret A.

    2010-01-01

    Both traditional and purpose designed nonsteroidal anti-inflammatory drugs (NSAIDs), selective for inhibition of cyclooxygenase (COX) -2 alleviate pain and inflammation but confer a cardiovascular hazard, attributable to inhibition of COX-2 derived prostacyclin (PGI2). Deletion of microsomal PGE synthase–1 (mPGES-1), the dominant enzyme that converts the COX derived intermediate product, PGH2, to form PGE2, modulates inflammatory pain in rodents. By contrast with COX-2 deletion or inhibition,...

  7. Use of linalool synthase in genetic engineering of scent production

    Science.gov (United States)

    Pichersky, Eran (Chelsea, MI)

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  8. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion.

    Czech Academy of Sciences Publication Activity Database

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Ro?. 183, ?. 2 (2012), s. 189-192. ISSN 0166-6851 R&D Projects: GA ?R(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * Sleeping sickness * Ubiquinone * Solanesyl-diphosphate synthase * Digitonin permeabilization * In situ tagging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112000539

  9. Synthesis of Lysine Methyltransferase Inhibitors

    Science.gov (United States)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  10. ACE INHIBITORS: A COMPREHENSIVE REVIEW

    Directory of Open Access Journals (Sweden)

    Pradeep Kumar Arora* and Ashish Chauhan

    2013-02-01

    Full Text Available Hypertension is a chronic increase in blood pressure, characterized as primary and secondary hypertension. The disorder is associated with various risk factors like obesity, diabetes, age, lack of exercise etc. Hypertension is being treated since ancient times by Ayurvedic, Chinese and Unani medicine. Now various allopathic drugs are available which include diuretics, calcium channel blockers, ?-blockers, ?-blockers, vasodilators, central sympatholytics and ACE-inhibitors. Non-pharmacological treatments include weight reduction, dietary sodium reduction, increased potassium intake and reduction in alcohol consumption. ACE-inhibitors are widely used in the treatment of hypertension by inhibiting the angiotensin converting enzyme responsible for the conversion of angiotensin I to angiotensin II (responsible for vasoconstriction. Various structure activity relationship studies led to the synthesis of ACE-inhibitors, some are under clinical development. This comprehensive review gives various guidelines on classification of hypertension, hypertension therapy including ancient, pharmacological, non-pharmacological therapies, pharmacoeconomics, historical perspectives of ACE, renin, renin angiotensin system (circulating vs local RAS, mechanism of ACE inhibitors, and development of ACE inhibitors. Review also emphasizes on the recent advancements on ACE inhibitors including drugs in clinical trials, computational studies on ACE-inhibitors, peptidomimetics, dual, natural, multi-functional ACE inhibitors, and conformational requirements for ACE-inhibitors.

  11. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  12. PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.

    Science.gov (United States)

    Nomura, Christopher T; Taguchi, Seiichi

    2007-01-01

    Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases. PMID:17123079

  13. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis.

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant iNOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age and sex showed no such changes. Our data support the hypothesis that NO is a pathogenic factor in MS, and that NOS IR is strongly expressed in brain regions appearing normal by MRI

  14. From bacterial to human dihydrouridine synthase: automated structure determination

    International Nuclear Information System (INIS)

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel ?-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer

  15. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel ?-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  16. 3-Pyridyl Substituted Aliphatic Cycles as CYP11B2 Inhibitors: Aromaticity Abolishment of the Core Significantly Increased Selectivity over CYP1A2

    OpenAIRE

    Yin, Lina; Hu, Qingzhong; Hartmann, Rolf W.

    2012-01-01

    Aldosterone synthase (CYP11B2) is a promising therapeutic target for the treatment of cardiovascular diseases related to abnormally high aldosterone levels. On the basis of our previously identified lead compounds I–III, a series of 3-pyridinyl substituted aliphatic cycles were designed, synthesized and tested as CYP11B2 inhibitors. Aromaticity abolishment of the core was successfully applied to overcome the undesired CYP1A2 inhibition. This study resulted in a series of potent and selective ...

  17. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    Science.gov (United States)

    Singh, Shailendra P.; Tao, Shixin; Fields, Timothy A.; Webb, Sydney; Harris, Raymond C.; Rao, Reena

    2015-01-01

    ABSTRACT Glycogen synthase kinase-3? (GSK3?) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3? expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-?1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-?1 treatment increased GSK3? expression and GSK3 inhibition abolished TGF-?1-induced SMAD3 activation and ?-smooth muscle actin (?-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3? stimulated ?-SMA expression even in the absence of TGF-?1 treatment. These results suggest that TGF-? regulates GSK3?, which in turn is important for TGF-?–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-? signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury. PMID:26092126

  18. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario (UIUC); (JHU-MED)

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  19. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    Energy Technology Data Exchange (ETDEWEB)

    Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  20. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    International Nuclear Information System (INIS)

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  1. [Parameters of NO synthase system of gastric mucosa in rats under stress conditions and inhibition of cyclooxygenase].

    Science.gov (United States)

    Fomenko, I S; Bondarchuk, T I; Bilets'ka, L P; Panasiuk, N B; Skliarov, O Ia

    2014-01-01

    In experiments on rats with modeled water-restrained stress, the influence of nonsteroidal anti-inflammatory drugs of different genesis on morphological status of gastric mucosa and changes of NO-synthase system parameters have been studied Administration of nonselective cyclooxygenese inhibitor naproxen in the water-restrained stress model in rats potentiated the increase of severity of damage of gastric mucosa. At the same time, the activity of both inducible and constitutive isoforms ofNO-sythase decreased. The parameters of lipoperoxidation remained at the level observed during water-restrained stress. It was shown the advantages of the use of H2S-releasinfg nonsteroidal anti-inflammatory drug ATB-346, which are associated with its cytoprotective effect of the drug manifested by a decreased total area of gastric damage. However, parameters of lipoperoxidation and NO-syntase system did not differ substantially from those in the group treated with napoxen, indicating the prevalence of parent molecule (naproxen) in regulation of function of NO-system Administration of dual COX/LOX inhibitor, the compound 2A5DHT, caused a decrease of gastric damage as compared to the effect ofnaproxen. The activity of iNOS remained much higher than under condition of the naproxen action. PMID:25007521

  2. Preventing Fusarium head blight of wheat and cob rot of maize by inhibition of fungal deoxyhypusine synthase.

    Science.gov (United States)

    Woriedh, Mayada; Hauber, Ilona; Martinez-Rocha, Ana Lilia; Voigt, Christian; Maier, Frank J; Schröder, Marcus; Meier, Chris; Hauber, Joachim; Schäfer, Wilhelm

    2011-05-01

    Upon posttranslational activation, the eukaryotic initiation factor-5A (eIF-5A) transports a subset of mRNAs out of the nucleus to the ribosomes for translation. Activation of the protein is an evolutionary highly conserved process that is unique to eIF-5A, the conversion of a lysine to a hypusine. Instrumental for the synthesis of hypusine is the first of two enzymatic reactions mediated by deoxyhypusine synthase (DHS). We show that DHS of wheat and the pathogenic fungus Fusarium graminearum, which causes one of the most destructive crop diseases worldwide, are transcriptionally upregulated during their pathogenic interaction. Although DHS of wheat, fungus, and human can be equally inhibited by the inhibitor CNI-1493 in vitro, application during infection of wheat and maize flowers results in strong inhibition of the pathogen without interference with kernel development. Our studies provide a novel strategy to selectively inhibit fungal growth without affecting plant growth. We identified fungal DHS as a target for the development of new inhibitors, for which CNI-1493 may serve as a lead substance. PMID:21463208

  3. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice.

    Science.gov (United States)

    Singh, Shailendra P; Tao, Shixin; Fields, Timothy A; Webb, Sydney; Harris, Raymond C; Rao, Reena

    2015-08-01

    Glycogen synthase kinase-3? (GSK3?) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3? expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-?1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-?1 treatment increased GSK3? expression and GSK3 inhibition abolished TGF-?1-induced SMAD3 activation and ?-smooth muscle actin (?-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3? stimulated ?-SMA expression even in the absence of TGF-?1 treatment. These results suggest that TGF-? regulates GSK3?, which in turn is important for TGF-?-SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-? signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury. PMID:26092126

  4. TNF-? expression in neutrophils and its regulation by glycogen synthase kinase-3: a potentiating role for lithium.

    Science.gov (United States)

    Giambelluca, Miriam S; Bertheau-Mailhot, Geneviève; Laflamme, Cynthia; Rollet-Labelle, Emmanuelle; Servant, Marc J; Pouliot, Marc

    2014-08-01

    Glycogen synthase kinase 3 (GSK-3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK-3 inhibitor. GSK-3? is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK-3 inhibition on the production of TNF-? by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF-? release. In lipopolysaccharide-stimulated human neutrophils, GSK-3 inhibitors mimicked the effect of LiCl, each potentiating TNF-? release after 4 h, in a concentration-dependent fashion, by up to a 3-fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK-3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK-3 inhibition markedly increases (by more than 3-fold) de novo TNF-? protein synthesis. Our findings shed light on a novel mechanism of control of TNF-? expression in neutrophils with GSK-3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases. PMID:24803542

  5. Structure-based design of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3? inhibitors to activate Wnt/?-catenin pathway.

    Science.gov (United States)

    Yue, Hong; Lu, Feng; Shen, Chen; Quan, Jun-Min

    2015-08-01

    Deregulation of Wnt/?-catenin pathway is closely related to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), and glycogen synthase kinase 3? (GSK-3?), the central negative regulator of Wnt pathway, is regarded as an important target for these diseases. Here, we report a series of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3? inhibitors by rational-design and synthesis, which show high selectivity against GSK-3? over Cyclin-dependent kinase 2 (CDK2), and significantly activate the cellular Wnt/?-catenin pathway. The structure-activity relationship of these GSK-3? inhibitors was also explored by in silico molecular docking. PMID:26057861

  6. Inhibitors of Fatty Acid Synthesis Induce PPAR?-Regulated Fatty Acid ?-Oxidative Genes: Synergistic Roles of L-FABP and Glucose

    OpenAIRE

    Huan Huang; Avery L. McIntosh; Martin, Gregory G.; Petrescu, Anca D.; Kerstin K. Landrock; Danilo Landrock; Ann B. Kier; Friedhelm Schroeder

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor-? (PPAR?) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity whil...

  7. 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3?– acts as an insulin-sensitizer avoiding downstream effects of activated ?-catenin

    OpenAIRE

    Gebhardt, Rolf; Lerche, Katja S; Götschel, Frank; Günther, Robert; Kolander, Jens; Teich, Lars; Zellmer, Sebastian; Hofmann, Hans-Jörg; Eger, Kurt; Hecht, Andreas; Gaunitz, Frank

    2009-01-01

    Glycogen synthase kinase-3? (GSK-3?) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3? with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3? inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular A...

  8. Identification and Characterization of a Re-Citrate Synthase in Dehalococcoides Strain CBDB1?‡

    OpenAIRE

    Marco-Urrea, Ernest; Paul, Steffanie; Khodaverdi, Viola; Seifert, Jana; von Bergen, Martin; Kretzschmar, Utta; Adrian, Lorenz

    2011-01-01

    The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and ...

  9. Homodimeric Hexaprenyl Pyrophosphate Synthase from the Thermoacidophilic Crenarchaeon Sulfolobus solfataricus Displays Asymmetric Subunit Structures†

    OpenAIRE

    Sun, Han-Yu; Ko, Tzu-Ping; Kuo, Chih-Jung; Guo, Rey-Ting; Chou, Chia-Cheng; Liang, Po-Huang; Wang, Andrew H. -J.

    2005-01-01

    Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C30-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C30-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central act...

  10. Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function*

    OpenAIRE

    OGAWA, HIROYASU; Shionyu, Masafumi; Sugiura, Nobuo; Hatano, Sonoko; Nagai, Naoko; Kubota, Yukihiko; Nishiwaki, Kiyoji; SATO, TAKASHI; Gotoh, Masanori; Narimatsu, Hisashi; Shimizu, Katsuji; Kimata, Koji; Watanabe, Hideto

    2010-01-01

    Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis....

  11. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-01-01

    The enzyme 5-phosphoribosyl-1-?-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sul...

  12. Comparison of PGH2 binding site in prostaglandin synthases

    OpenAIRE

    Doble Mukesh; Paragi-Vedanthi Padmapriya

    2010-01-01

    Abstract Background Prostaglandin H2 (PGH2) is a common precursor for the synthesis of five different Prostanoids via specific Prostanoid Synthases. The binding of this substrate with these Synthases is not properly understood. Moreover, currently no crystal structure of complexes bound with PGH2 has been reported. Hence, understanding the interactions of PGH2 and characterizing its binding sites in these synthases is crucial for developing novel therapeutics based on these proteins as target...

  13. Affinity comparison of different THCA synthase to CBGA using modeling computational approaches

    OpenAIRE

    Alaoui, Moulay Abdelaziz El; Ibrahimi, Azeddine; Semlali, Oussama; Tarhda, Zineb; Marouane, Melloul; Najwa, Alaoui; Soulaymani, Abdelmajid; Fahime, Elmostafa El

    2014-01-01

    The ?9-Tetrahydrocannabinol (THCA) is the primary psychoactive compound of Cannabis Sativa. It is produced by ?1- Tetrahydrocannabinolic acid synthase (THCA) which catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) the precursor of the THCA. In this study, we were interested by the three dimensional structure of THCA synthase protein. Generation of models were done by MODELLER v9.11 and homology modeling with ?1-tetrahydrocannabinolic acid (THCA) synthase X ray stru...

  14. Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

    OpenAIRE

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

    1980-01-01

    An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defect...

  15. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis.

    Science.gov (United States)

    Lei, Lei; Singh, Abhishek; Bashline, Logan; Li, Shundai; Yingling, Yaroslava G; Gu, Ying

    2015-10-01

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress. PMID:26443667

  16. Corrosion inhibitors. Manufacture and technology

    International Nuclear Information System (INIS)

    Detailed information is presented relating to corrosion inhibitors. Areas covered include: cooling water, boilers and water supply plants; oil well and refinery operations; fuel and lubricant additives for automotive use; hydraulic fluids and machine tool lubes; grease compositions; metal surface treatments and coatings; and general processes for corrosion inhibitors

  17. Kinase dysfunction and kinase inhibitors.

    Science.gov (United States)

    London, Cheryl A

    2013-02-01

    With recent advances in molecular biology, abnormalities in cancer cells that contribute to dysregulation of cell survival and proliferation are being characterized with greater precision. Through this process, key abnormalities in cancer cells involving proteins that regulate signal transduction, migration, mitosis and other critical processes have been identified. Such abnormalities often involve a class of proteins called kinases that act to phosphorylate other proteins in the cell, resulting in activation of these proteins in the absence of appropriate stimulation/regulation. Given their role in tumour biology, substantial effort has been directed at blocking the function of these proteins. Several approaches have been used, including monoclonal antibodies and small molecule inhibitors. While antibodies are primarily directed at cell surface proteins, small molecule inhibitors, also known as kinase inhibitors, target proteins throughout the cell. A variety of kinase inhibitors have been approved for the treatment of human cancers. In some instances, these inhibitors have exhibited significant clinical efficacy, and it is likely that their biological activity will be further enhanced as combination regimens with standard treatment modalities are explored. The use of kinase inhibitors in dogs and cats is relatively recent, although two inhibitors, toceranib (Palladia; Pfizer Animal Health, Madison, NJ, USA) and masitinib (Kinavet; Catalent Pharma Solutions, Somerset, NJ, USA) have been approved by the Federal Drug Administration (USA) for use in dogs. This article reviews the biology of protein kinase dysfunction in human and animal cancers, and the application of specific kinase inhibitors to veterinary cancer patients. PMID:23331696

  18. Anthranilamide inhibitors of factor Xa.

    Science.gov (United States)

    Mendel, David; Marquart, Angela L; Joseph, Sajan; Waid, Philip; Yee, Ying K; Tebbe, Anne Louise; Ratz, Andrew M; Herron, David K; Goodson, Theodore; Masters, John J; Franciskovich, Jeffry B; Tinsley, Jennifer M; Wiley, Michael R; Weir, Leonard C; Kyle, Jeffrey A; Klimkowski, Valentine J; Smith, Gerald F; Towner, Richard D; Froelich, Larry L; Buben, John; Craft, Trelia J

    2007-09-01

    SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays. PMID:17624775

  19. Molecular size estimation of plasma membrane ?-glucan synthase from red beet root

    International Nuclear Information System (INIS)

    Cellulose and cell wall ?-D-glucans in higher plants are thought to be synthesized by the plasma membrane enzyme, ?-glucan synthase. This enzyme has never been purified to homogeneity, hence its subunit composition is unknown. Partial purification of red beet root glucan synthase by glycerol density gradient centrifugation followed by SDS-PAGE yielded a highly enriched subunit of 68 kDa. Radiation inactivation of plasma membranes gave a molecular size the 450 kDa for the holoenzyme complex. This suggests that glucan synthase consists of 6 to 7 subunits and confirms electron microscope studies showing that glucan synthases exist as multi-subunit complexes embedded within the membrane

  20. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    OpenAIRE

    Del Campo, M.; Y KAYA; Ofengand, J

    2001-01-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines the...

  1. Proteinaceous alpha-araylase inhibitors.

    DEFF Research Database (Denmark)

    Svensson, Birte; Fukuda, Kenji

    2004-01-01

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous a-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function.

  2. Kinetic inhibitor of hydrate crystallization.

    Science.gov (United States)

    Storr, Mark T; Taylor, Paul C; Monfort, Jean-Pierre; Rodger, P Mark

    2004-02-11

    We present the results of a combined theoretical/experimental study into a new class of kinetic inhibitor of gas hydrate formation. The inhibitors are based on quaternary ammonium zwitterions, and were identified from a computational screen. Molecular dynamics simulations were used to characterize the effect of the inhibitor on the interface between a type II hydrate and natural gas. These simulations show that the inhibitor is bifunctional, with the hydrophobic end being compatible with the water structure present at the hydrate interface, while the negatively charged functional group promotes a long ranged water structure that is inconsistent with the hydrate phase; the sulfonate-induced structure was found to propagate strongly over several solvation shells. The compound was subsequently synthesized and used in an experimental study of both THF and ethane hydrate formation, and was shown to have an activity that was comparable with an existing commercial kinetic inhibitor: PVP. PMID:14759217

  3. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    Directory of Open Access Journals (Sweden)

    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  4. Potential Role of Glycogen Synthase Kinase-3? in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Zhou, Yi-Xia; Shi, Zhan; Singh, Pavneet; Yin, Hao; Yu, Yan-Ni; Li, Long; Walsh, Michael P; Gui, Yu; Zheng, Xi-Long

    2016-02-01

    Glycogen synthase kinase (GSK)-3?, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3? results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3? regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3? inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM ?-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3? inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3? inhibitors were mimicked by transfection with GSK-3? siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3? mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM ?-actin. As expected, overexpression of constitutively active or wild-type GSK-3? in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3? reduces myocardin transcriptional activity, suggesting a role for GSK-3? in myocardin transcriptional activity and smooth muscle differentiation. J. Cell. Physiol. 231: 393-402, 2016. © 2015 Wiley Periodicals, Inc. PMID:26129946

  5. Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy.

    OpenAIRE

    S. Ahmad; Rightmire, B; Jensen, R.A.

    1986-01-01

    The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-P...

  6. Selective inhibition of prostacyclin synthase activity by rofecoxib

    OpenAIRE

    Griffoni, Cristiana; Spisni, Enzo; Strillacci, Antonio; Toni, Mattia; Bachschmid, Markus Michael; Tomasi, Vittorio

    2007-01-01

    The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibit...

  7. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    International Nuclear Information System (INIS)

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS

  8. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    Energy Technology Data Exchange (ETDEWEB)

    Serer, María I.; Bonomi, Hernán R. [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina); Guimarães, Beatriz G. [Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin BP 48, 91192 Gif-sur-Yvette CEDEX (France); Rossi, Rolando C. [Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires (Argentina); Goldbaum, Fernando A.; Klinke, Sebastián, E-mail: sklinke@leloir.org.ar [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina)

    2014-05-01

    This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

  9. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu [Purdue University, 175 South University Street, West Lafayette, IN 47907-2063 (United States)

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  10. Regulation of endothelial nitric oxide synthase by phosphorylation

    OpenAIRE

    Mohamed, Annisuddin

    2007-01-01

    Since its recognition as an endothelium-derived relaxing factor, the control and consequences of nitric oxide (NO) production have been investigated intensely. We know now that NO is not simply a vasodilator or regulator of smooth muscle tone but is a potent anti-platelet agent, neuromodulator and regulator of gene expression. NO is synthesized from the amino acid Larginine by a family of enzymes termed NO synthases (NOS). The ‘endothelial’ (eNOS or NOS III) and ‘neuronal’ (nNOS, NOS I or bNO...

  11. Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses

    OpenAIRE

    Zhou, Jie; YANG Yang; YU, JUAN; Wang, Like; Yu, Xiang; Ohtani, Misato; Kusano, Miyako; Saito, Kazuki; Demura, Taku; Zhuge, Qiang

    2013-01-01

    Galactinol synthase (GolS; EC 2.4.1.123) is a member of the glycosyltransferase eight family that catalyzes the first step in the biosynthesis pathway of the raffinose family of oligosaccharides (RFOs). The accumulation of RFOs in response to abiotic stress indicates a role for RFOs in stress adaptation. To obtain information on the roles of RFOs in abiotic stress adaptation in trees, we investigated the expression patterns of nine Populus trichocarpaGolS (PtrGolS) genes with special referenc...

  12. Substrate Recognition by the Human Fatty-acid Synthase*

    OpenAIRE

    Carlisle-Moore, Loretha; Gordon, Chris R.; Machutta, Carl A.; MILLER, W. TODD; Tonge, Peter J

    2005-01-01

    The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5?-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced kcat and kcat...

  13. Kinetic Mechanism of OMP Synthase:  A Slow Physical Step Following

    DEFF Research Database (Denmark)

    Wang, G.P.; Lundegaard, Claus; Jensen, Kaj Frank; Grubmeyer, C.

    1999-01-01

    Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5‘-monophophate (OMP) from orotate and a-d-5-phosphoribosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partitioning, and chemical quench studies were used to determine rate and equilibrium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a KD of 33 µM. Binding of OMP and orotate also occurred to a single class of two sites per dimer, with KD values of 3 and 2...

  14. Biological effects of deuteronation: ATP synthase as an example

    OpenAIRE

    Olgun Abdullah

    2007-01-01

    Abstract Background In nature, deuterium/hydrogen ratio is ~1/6600, therefore one of ~3300 water (H2O) molecules is deuterated (HOD + D2O). In body fluids the ratio of deuterons to protons is ~1/15000 because of the lower ionization constant of heavy water. The probability of deuteronation rather than protonation of Asp 61 on the subunit c of F0 part of ATP synthase is also ~1/15000. The contribution of deuteronation to the pKa of Asp 61 is 0.35. Theory and Discussion In mitochondria, the rel...

  15. ATP synthase: from single molecule to human bioenergetics

    OpenAIRE

    KAGAWA, Yasuo

    2010-01-01

    ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (?3?3???) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simult...

  16. Activation of Methyltetrahydrofolate by Cobalamin-Independent Methionine Synthase

    OpenAIRE

    Taurog, Rebecca E.; Matthews, Rowena G

    2006-01-01

    Cobalamin-independent methionine synthase (MetE) catalyzes the final step of de novo methionine synthesis using the triglutamate derivative of methyltetrahydrofolate (CH3-H4PteGlu3) as methyl donor and homocysteine (Hcy) as methyl acceptor. This reaction is challenging because at physiological pH the Hcy thiol is not a strong nucleophile and CH3-H4PteGlu3 provides a very poor leaving group. Our laboratory has previously established that Hcy is ligated to a tightly bound zinc ion in the MetE a...

  17. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish (UAB)

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  18. Copper Corrosion Inhibitors. A review

    Directory of Open Access Journals (Sweden)

    M. M. Antonijevic and M. B. Petrovic

    2008-01-01

    Full Text Available The literature dealing with the electrochemical corrosion of copper and possibility of its prevention using inhibitors is examined. Inorganic compounds are investigated as well, but organic compounds and their derivatives in much greater numbers. Researches are directed to influence of compounds structure, concentration, method of application as well as media that inhibitor is used in on inhibition efficiency. Moreover, action mechanisms are studied. The attempts to find models, which can enable prediction of possibilities of newly synthesized compounds to act as corrosion inhibitors, combining theory and practical investigations of substances with similar structure are also significant.

  19. Nuclear glycogen synthase kinase-3 {beta} (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Mentzingen, Leticia; Andrade, Josiana G. de; Logullo, Carlos [Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ (Brazil). Centro de Biociencias e Biotecnologia. Lab. de Quimica e Funcao de Proteinas e Peptideos (LQFPP); Andrade, Caroline P. de; Vaz Junior, Itabajara [Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS (Brazil). Centro de Biotecnologia

    2008-07-01

    Full text: Glycogen synthase kinase-3 (GSK3) is recognized as a key component of a large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. Recent observations point to functions for phosphorylases several transcription factors in the nucleus. Also, GSK3b participate of the canonical W nt signalling pathway, which has been studied intensively in embryonic and cancer cells. Like in many other signaling pathways, most components in W nt signal transduction were highly conserved during the evolution. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Although GSK3 is traditionally considered a cytosolic protein, it is also present in nuclei. Nuclear GSK3 is particularly interesting because of the many transcription factors that it regulates enabling GSK3 to influence many signaling pathways that converge on these transcription factors, thereby regulating the expression of many genes. Our group identified that GSK-3 {beta} could be detected in different stage eggs of R. micro plus. In this work we detected the GSK-3 in isolated nuclear fraction from the egg homogenates of R. micro plus by western-blot analysis, using anti-GSK- 3 {beta} antibodies. The enzyme activity was also detected radiochemically throughout embryogenesis in same fraction. The GSK-3 activity was inhibiting by using SB 216763 (selective molecule inhibitors of GSK-3). Taken together our results suggest that GSK-3 {beta} isoform probably is involved in gene transcription factors during R. micro plus embryo development.

  20. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

    Directory of Open Access Journals (Sweden)

    M. Chacur

    2010-04-01

    Full Text Available Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO. In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT. Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test and allodynia (von Frey hair test. Control animals did not present any alteration (sham-animals. The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL, blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30 in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%. Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%, reaching the greatest increase (60% 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

  1. Metabolism of phenol and hydroquinone to reactive products by macrophage peroxidase or purified prostaglandin H synthase

    International Nuclear Information System (INIS)

    Macrophages, an important cell-type of the bone marrow stroma, are possible targets of benzene toxicity because they contain relatively large amounts of prostaglandin H synthase (PHS), which is capable of metabolizing phenolic compounds to reactive species. PHS also catalyzes the production of prostaglandins, negative regulators of myelopoiesis. Studies indicate that the phenolic metabolites of benzene are oxidized in bone marrow to reactive products via peroxidases. With respect to macrophages, PHS peroxidase is implicated, as in vivo benzene-induced myelotoxicity is prevented by low doses of nonsteroidal anti-inflammatory agents, drugs that inhibit PHS. Incubations of either 14C-phenol or 14C-hydroquinone with a lysate of macrophages collected from mouse peritoneum (greater than 95% macrophages), resulted in an irreversible binding to protein that was dependent upon H2O2, incubation time, and concentration of radiolabel. Production of protein-bound metabolites from phenol or hydroquinone was inhibited by the peroxidase inhibitor aminotriazole. Protein binding from 14C-phenol also was inhibited by 8 microM hydroquinone, whereas binding from 14C-hydroquinone was stimulated by 5 mM phenol. The nucleophile cysteine inhibited protein binding of both phenol and hydroquinone and increased the formation of radiolabeled water-soluble metabolites. Similar to the macrophage lysate, purified PHS also catalyzed the conversion of phenol to metabolites that bound to protein and DNA; this activation was both H2O2- and arachidonic acid-dependent. These results indicate a role for macrophage peroxidase, possibly PHS peroxidase, in the conversion of phenol and hydroquinone to reactive metabolites and suggest that the macrophage should be considered when assessing the hematopoietic toxicity of benzene

  2. Nuclear glycogen synthase kinase-3 ? (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    International Nuclear Information System (INIS)

    Full text: Glycogen synthase kinase-3 (GSK3) is recognized as a key component of a large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. Recent observations point to functions for phosphorylases several transcription factors in the nucleus. Also, GSK3b participate of the canonical W nt signalling pathway, which has been studied intensively in embryonic and cancer cells. Like in many other signaling pathways, most components in W nt signal transduction were highly conserved during the evolution. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Although GSK3 is traditionally considered a cytosolic protein, it is also present in nuclei. Nuclear GSK3 is particularly interesting because of the many transcription factors that it regulates enabling GSK3 to influence many signaling pathways that converge on these transcription factors, thereby regulating the expression of many genes. Our group identified that GSK-3 ? could be detected in different stage eggs of R. micro plus. In this work we detected the GSK-3 in isolated nuclear fraction from the egg homogenates of R. micro plus by western-blot analysis, using anti-GSK- 3 ? antibodies. The enzyme activity was also detected radiochemically throughout embryogenesis in same fraction. The GSK-3 activity was inhibiting by using SB 216763 (selective molecule inhibitors of GSK-3). Taken together our results suggest that GSK-3 ? isoform probably is involved in gene transcription factors during R. micro plus embryo development

  3. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

    Scientific Electronic Library Online (English)

    M., Chacur; R.J.B., Matos; A.S., Alves; A.C., Rodrigues; V., Gutierrez; Y., Cury; L.R.G., Britto.

    2010-04-01

    Full Text Available Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of [...] 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

  4. Modulation of baroreceptor activity by gene transfer of nitric oxide synthase to carotid sinus adventitia.

    Science.gov (United States)

    Meyrelles, Silvana S; Sharma, Ram V; Mao, Hui Z; Abboud, Francois M; Chapleau, Mark W

    2003-05-01

    Administration of nitric oxide (NO) or NO donors to isolated carotid sinus and carotid bodies inhibits the activity of baroreceptor and chemoreceptor afferent nerves. Furthermore, NO synthase (NOS) is present in endothelial cells and in sensory nerves innervating the carotid sinus region. The major goal of this study was to determine whether overexpression of NOS in carotid sinus modulates baroreceptor activity. Rabbits were anesthetized, and adenoviral vectors (5 x 10(8) plaque-forming units) encoding genes for either beta-galactosidase (beta-Gal) or endothelial type III NOS (eNOS) were applied topically to the adventitial surface of one carotid sinus. In some experiments, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) was applied to the carotid sinus immediately after the vector. Four to five days later, baroreceptor activity and carotid sinus diameter were measured from the vascularly isolated carotid sinus of the anesthetized rabbits. Transgene expression was confirmed by X-Gal staining of beta-Gal and measurement of NOS activity by citrulline assay. The expression was restricted to the carotid sinus adventitia. Baroreceptor activity was decreased significantly, and the pressure-activity curve was shifted to higher pressures in eNOS-transduced (n = 5) compared with beta-Gal-transduced (n = 5) carotid sinuses. The pressure corresponding to 50% of maximum activity averaged 55 +/- 6 and 76 +/- 7 mmHg in beta-Gal- and eNOS-transduced carotid sinuses, respectively (P < 0.05). Decreased baroreceptor activity was accompanied by a significant increase in carotid diameter in the eNOS-transduced carotid sinuses (n = 5). l-NAME prevented the inhibition of baroreceptor activity and the increase in carotid diameter in eNOS-transduced carotid sinuses (n = 5). We conclude that adenoviral-mediated gene transfer of eNOS to carotid sinus adventitia causes sustained, NO-dependent inhibition of baroreceptor activity and resetting of the baroreceptor function curve to higher pressures. PMID:12676743

  5. Role of endothelial nitric oxide synthase in the regulation of SREBP activation by oxidized phospholipids.

    Science.gov (United States)

    Gharavi, Nima M; Baker, Nancy A; Mouillesseaux, Kevin P; Yeung, Winnie; Honda, Henry M; Hsieh, Xavier; Yeh, Michael; Smart, Eric J; Berliner, Judith A

    2006-03-31

    Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, such as interleukin (IL)-8. Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC was mediated through the activation of sterol regulatory element-binding protein (SREBP). We now present evidence for the role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment of EC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine 1177, dephosphorylation of threonine 495, and the conversion of L-arginine to L-citrulline. Activation of eNOS by Ox-PAPC was regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated that pretreatment of EC with NOS inhibitor, Nomega-nitro-L-arginine-methyl ester (L-NAME), significantly inhibited Ox-PAPC-induced IL-8 synthesis. Because SREBP activation had been previously shown to regulate IL-8 transcription by Ox-PAPC, we examined the effects of L-NAME on Ox-PAPC-induced SREBP activation. Our data demonstrated that Ox-PAPC-induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatment with L-NAME. Interestingly, treatment of EC with NO donor, S-nitroso-N-acetylpenicillamine, did not activate SREBP, suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2*-), in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2*- through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBP activation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP and SREBP-mediated inflammatory processes. PMID:16497987

  6. Altered nitric oxide synthase 3 distribution in mesenteric arteries of hypertensive rats.

    Science.gov (United States)

    Sullivan, Jennifer C; Pollock, David M; Pollock, Jennifer S

    2002-02-01

    A high-salt (HS) diet and angiotensin II (Ang II) are both associated with the development of hypertension and impaired endothelial function. We hypothesize that alterations in nitric oxide synthase (NOS) activity or subcellular localization of NOS 3 protein may contribute to endothelial dysfunction in salt-dependent hypertension. To test this hypothesis, two models of salt-dependent hypertension were studied: DOCA-salt and Ang II. For Ang II hypertension, rats were divided into 4 groups: control on normal or HS diet, Ang II-infused on normal or HS diet. The mesenteric arterial bed was isolated and frozen in liquid nitrogen. Frozen arteries were homogenized and separated into cytosolic and particulate fractions. NOS activity was assayed by determining the conversion of (3)H-arginine to (3)H-citrulline in the absence and presence of the NOS inhibitor, Nomega-nitro-L-arginine. NOS 3 protein expression was significantly increased in the cytosol of arteries from DOCA-salt compared with placebo rats and in Ang II-infused and Ang HS rats compared with control. NOS 3 expression in the particulate fraction was comparable among all groups. NOS activity (pmol/30 min/total protein) was significantly increased in the cytosolic fraction of arteries from DOCA-salt rats compared with placebo and in Ang HS rats compared with control. NOS activity was comparable in the particulate fraction in all rat groups. In conclusion, there is an altered subcellular distribution of NOS 3 in salt-dependent hypertension that may contribute to the development of hypertension and endothelial dysfunction. PMID:11882615

  7. Reduction of nilutamide by NO synthases: implications for the adverse effects of this nitroaromatic antiandrogen drug.

    Science.gov (United States)

    Ask, K; Dijols, S; Giroud, C; Casse, L; Frapart, Y-M; Sari, M-A; Kim, K-S; Stuehr, D J; Mansuy, D; Camus, P; Boucher, J-L

    2003-12-01

    Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of l-arginine to l-citrulline with formation of the widespread signal molecule NO. Beside their fundamental role in NO biosynthesis, these enzymes are also involved in the formation of reactive oxygen species and in the interactions with some xenobiotic compounds. Nilutamide is a nonsteroidal antiandrogen that behaves as a competitive antagonist of the androgen receptors and is proposed in the treatment of metastatic prostatic carcinoma. However, therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions mediated by toxic mechanism(s), which remain(s) poorly investigated. Here, we studied the interaction of NOSs with nilutamide. Our results show that the purified recombinant neuronal NOS reduced the nitroaromatic nilutamide to the corresponding hydroxylamine. The reduction of nilutamide catalyzed by neuronal NOS proceeded with intermediate formation of a nitro anion free radical easily observed by EPR, was insensitive to the addition of the usual heme ligands and l-arginine analogues, but strongly inhibited by O(2) and a flavin/NADPH binding inhibitor. Involvement of the reductase domain of nNOS in the reduction of nilutamide was confirmed by (i) the ability of the isolated reductase domain of nNOS to catalyze the reaction and (ii) the stimulating effect of Ca(2+)/calmodulin on the accumulation of hydroxylamine and nitro anion radical. In a similar manner, the recombinant inducible and endothelial NOS isoforms also displayed nitroreductase activity, albeit with lower yields. The selective reduction of nilutamide to its hydroxylamino derivative by the NOSs could explain some of the toxic effects of this drug. PMID:14680368

  8. Glycogen synthase kinase 3 is a potential drug target for African trypanosomiasis therapy.

    Science.gov (United States)

    Ojo, Kayode K; Gillespie, J Robert; Riechers, Aaron J; Napuli, Alberto J; Verlinde, Christophe L M J; Buckner, Frederick S; Gelb, Michael H; Domostoj, Mathias M; Wells, Susan J; Scheer, Alexander; Wells, Timothy N C; Van Voorhis, Wesley C

    2008-10-01

    Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 "short" (Tb10.161.3140) than for T. brucei GSK-3 "long" (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found. PMID:18644955

  9. Glycogen Synthase Kinase 3 Is a Potential Drug Target for African Trypanosomiasis Therapy?

    Science.gov (United States)

    Ojo, Kayode K.; Gillespie, J. Robert; Riechers, Aaron J.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Buckner, Frederick S.; Gelb, Michael H.; Domostoj, Mathias M.; Wells, Susan J.; Scheer, Alexander; Wells, Timothy N. C.; Van Voorhis, Wesley C.

    2008-01-01

    Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 “short” (Tb10.161.3140) than for T. brucei GSK-3 “long” (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found. PMID:18644955

  10. In silico designing and molecular docking of a potent analog against Staphylococcus aureus porphobilinogen synthase

    Science.gov (United States)

    Kumar, Pasupuleti Santhosh; Kumar, Yellapu Nanda; Prasad, Uppu Venkateswara; Yeswanth, Sthanikam; Swarupa, Vimjam; Sowjenya, Gopal; Venkatesh, Katari; Srikanth, Lokanathan; Rao, Valasani Koteswara; Sarma, Potukuchi Venkata Gurunatha Krishna

    2014-01-01

    Background: The emergence of multidrug-resistant strains of Staphylococcus aureus, there is an urgent need for the development of new antimicrobials which are narrow and pathogen specific. Aim: In this context, the present study is aimed to have a control on the staphylococcal infections by targeting the unique and essential enzyme; porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of ?-aminolevulinic acid, an essential step in the tetrapyrrole biosynthesis. Hence developing therapeutics targeting PBGS will be the promising choice to control and manage the staphylococcal infections. 4,5-dioxovalerate (DV) is known to inhibit PBGS. Materials and Methods: In view of this, in this study, novel dioxovalerate derivatives (DVDs) molecules were designed so as to inhibit PBGS, a potential target of S. aureus and their inhibitory activity was predicted using molecular docking studies by molecular operating environment. The 3D model of PBGS was constructed using Chlorobium vibrioform (Protein Data Bank 1W1Z) as a template by homology modeling method. Results: The built structure was close to the crystal structure with Z score ? 8.97. Molecular docking of DVDs into the S. aureus PBGS active site revealed that they are showing strong interaction forming H-bonds with the active sites of K248 and R217. The ligand–receptor complex of DVD13 showed a best docking score of ? 14.4555 kcal/mol among DV and all its analogs while the substrate showed docking score of ? 13.0392 kcal/mol showing interactions with S199, K217 indicating that DVD13 can influence structural variations on the enzyme and thereby inhibiting the enzyme. Conclusion: The substrate analog DVD13 is showing significant interactions with active site of PBGS and it may be used as a potent inhibitor to control S. aureus infections. PMID:25035635

  11. Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase

    International Nuclear Information System (INIS)

    Expression, crystallization and preliminary X-ray diffraction studies of a novel bifunctional N-acetylglutamate synthase/kinase from X. campestris homologous to vertebrate N-acetylglutamate synthase are reported. A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6222, with unit-cell parameters a = b = 134.60, c = 192.11 Å, and diffract to about 3.0 Å resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method

  12. Acetohydroxy acid synthase is a target for leucine containing peptide toxicity in Escherichia coli.

    OpenAIRE

    Gollop, N.; Tavori, H; Barak, Z

    1982-01-01

    Acetohydroxy acid synthase from a mutant resistant to leucine-containing peptides was insensitive to leucine inhibition. It is concluded that acetohydroxy acid synthase is a target for the toxicity of the high concentrations of leucine brought into Escherichia coli K-12 by leucine-containing peptides.

  13. Attachment of fatty acid substrate fragments to prostaglandin (PG) H synthase during reaction with arachidonate

    International Nuclear Information System (INIS)

    Pure ovine synthase was incubated aerobically with 14C-arachidonate to inactivate the cyclooxygenase. After solvent extraction to remove the bulk of the lipid, the inactive protein was analyzed by polyacrylamide gel electrophoresis. In SDS-PAGE radioactive label was associated with protein that comigrated with the 70 K Da synthase subunit, as well as with protein that accumulated at the upper edge of the resolving gel. In HPLC radioactivity was found in two peaks eluting in the region of unreacted synthase. SDS-PAGE analysis of pooled material from these HPLC peaks gave a distribution of radioactivity similar to that obtained with the unfractionated material. The radioactivity and protein content of inactivated synthase purified by HPLC indicated that 0.3-1.0 mole of substrate fragment were bound per mole of synthase subunit. Incubation of a mixture of the synthase and ovalbumin with arachidonate resulted in 5-fold more labelling of synthase than ovalbumin. Thus, a substrate fragment appears to become selectively attached to the synthase during reaction, and may represent the product of a self-inactivation event

  14. Neuronal nitric oxide synthase-dependent afferent arteriolar function in angiotensin II-induced hypertension.

    Science.gov (United States)

    Ichihara, A; Imig, J D; Navar, L G

    1999-01-01

    This study was designed to determine the influence of neuronal nitric oxide synthase (nNOS) in tubular flow-dependent regulation of afferent arteriolar diameter in hypertensive Sprague-Dawley rats that received 60 ng/min angiotensin II (Ang II) subcutaneously for 13 days. Systolic blood pressure of control and Ang II-infused rats averaged 122+/-2 (n=23) and 194+/-2 mm Hg (n=24). Afferent arteriolar responses to the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC; 0.1 to 10 micromol/L) and the nonselective NOS inhibitor Nomega-nitro-L-arginine (L-NNA; 1 to 100 micromol/L) were assessed in vitro using the blood-perfused juxtamedullary nephron preparation. At a perfusion pressure of 160 mm Hg, afferent arteriolar diameters from control and Ang II-infused rats averaged 18.7+/-1.1 microm (n=8) and 18.1+/-1.1 microm (n=9), respectively, and decreased by 19. 9+/-1.5% and 11.8+/-1.1%, respectively, in response to 10 micromol/L L-SMTC. The L-SMTC-induced afferent arteriolar constriction was significantly greater in control than in Ang II-infused rats. In contrast, 100 micromol/L L-NNA constricted afferent arterioles similarly in both control (n=8) and Ang II-infused (n=7) rats. After transection of the loops of Henle to interrupt flow to the macula densa, the vasoconstrictor responses to L-SMTC but not to L-NNA were reversed. Increasing distal volume delivery by addition of 10 mmol/L acetazolamide to the blood perfusate significantly enhanced the afferent arteriolar constrictor responses to 10 micromol/L L-SMTC (34.5+/-4.8%, n=7) in normotensive rats. In contrast, in Ang II-infused rats, acetazolamide treatment did not enhance the responses to L-SMTC (n=8). These results indicate that chronic Ang II infusion reduces the ability of nNOS-derived nitric oxide to counteract the afferent arteriolar response to increased distal tubular flow. PMID:9931148

  15. Identification of a satellite fatty acid ethyl ester synthase from human myocardium as a glutathione S-transferase.

    OpenAIRE

    Bora, P.S.; Spilburg, C. A.; Lange, L G

    1989-01-01

    Nonoxidative alcohol metabolism catalyzed by fatty acid ethyl ester (FAEE) synthases may contribute to extrahepatic injury resulting from alcohol abuse. Unlike rabbit myocardial FAEE synthase, that from human heart has a satellite minor synthase (I) eluting from DEAE cellulose at a conductivity of 5 mS. Synthase I was purified 1,118-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and Superose-12 fast-protein liquid chromatographies. SDS-PAGE showed a single polypept...

  16. Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Vegetable Soybean During Fruit Development

    OpenAIRE

    Kassinee Sitthiwong; Toshiyuki Matsui; Nobuyuki Okuda; Haruo Suzuki

    2005-01-01

    This study investigated the changes in carbohydrate content and activities of acid invertase, sucrose synthase (SS) and sucrose phosphate synthase (SPS) in two vegetable soybean cultivars (Glycine max (L.) Merr. vars. Ajigen and Fuuki) during fruit development ranging from 28 to 63 days after anthesis. In both cultivars, sucrose was the predominant sugar while fructose and glucose were found in trace amounts. Sucrose accumulation was highest at 35 and 42 days after anthesis in Fuuki and Ajige...

  17. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

    Science.gov (United States)

    Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

    1992-04-01

    Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

  18. Aromatase Inhibitors and Bone Loss

    OpenAIRE

    Perez, Edith A.; M., Serene; Durling, Frances C.; WEILBAECHER, KATHERINE

    2006-01-01

    The aromatase inhibitors (AIs) anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin) are significantly more effective than the selective estrogen-receptor modulator (SERM) tamoxifen in preventing recurrence in estrogen receptor–positive early breast cancer. Aromatase inhibitors are likely to replace SERMs as first-line adjuvant therapy for many patients. However, AIs are associated with significantly more osteoporotic fractures and greater bone mineral loss. As antiresorptive ...

  19. STAT inhibitors for cancer therapy

    OpenAIRE

    Furqan, Muhammad; Akinleye, Akintunde; Mukhi, Nikhil; Mittal, Varun; Chen, Yamei; Liu, Delong

    2013-01-01

    Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review s...

  20. Subcellular Targeting Domains of Sphingomyelin Synthase 1 and 2

    Directory of Open Access Journals (Sweden)

    Yeang Calvin

    2011-12-01

    Full Text Available Abstract Sphingomyelin synthase (SMS sits at the crossroads of sphingomyelin (SM, ceramide, diacylglycerol (DAG metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

  1. Biological effects of deuteronation: ATP synthase as an example

    Directory of Open Access Journals (Sweden)

    Olgun Abdullah

    2007-02-01

    Full Text Available Abstract Background In nature, deuterium/hydrogen ratio is ~1/6600, therefore one of ~3300 water (H2O molecules is deuterated (HOD + D2O. In body fluids the ratio of deuterons to protons is ~1/15000 because of the lower ionization constant of heavy water. The probability of deuteronation rather than protonation of Asp 61 on the subunit c of F0 part of ATP synthase is also ~1/15000. The contribution of deuteronation to the pKa of Asp 61 is 0.35. Theory and Discussion In mitochondria, the release of a deuteron into the matrix side half-channel of F0 is likely to be slower than that of a proton. As another example, deuteronation may slow down electron transfer in the electron transport chain (ETC by interfering with proton coupled electron transport reactions (PCET, and increase free radical production through the leakage of temporarily accumulated electrons at the downstream complexes. Conclusion Deuteronation, as exemplified by ATP synthase and the ETC, may interfere with the conformations and functions of many macromolecules and contribute to some pathologies like heavy water toxicity and aging.

  2. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  3. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    Science.gov (United States)

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  4. Nicotinamide phosphoribosyltransferase inhibitors, design, preparation and SAR

    DEFF Research Database (Denmark)

    Christensen, Mette Knak; Erichsen, Kamille Dumong; Olesen, Uffe Hogh; Tjørnelund, Jette; Fristrup, Peter; Thougaard, Annemette; Nielsen, Søren Jensby; Sehested, Maxwell; Jensen, Peter B.; Loza, Einars; Kalvinsh, Ivars; Garten, Antje; Kiess, Wieland; Bjorkling, Fredrik

    2013-01-01

    Existing pharmacological inhibitors for nicotinamide phosphoribosyltransferase (NAMPT) are promising therapeutics for treating cancer. Using medicinal and computational chemistry methods, the structure-activity relationship for novel classes of NAMPT inhibitors is described and compounds optimized. Compounds are designed inspired by the NAMPT inhibitor APO866 and cyanoguanidine inhibitor scaffolds. In comparison with recently published derivatives the new analogues exhibit an equally potent anti...

  5. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Energy Technology Data Exchange (ETDEWEB)

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer able to degrade p53 which in consequence accumulates in the nucleus. (orig.)

  6. NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines

    OpenAIRE

    Yang, Y.; Ikezoe, T; Nishioka, C; Bandobashi, K; Takeuchi, T; Adachi, Y.; Kobayashi, M.; Takeuchi, S.; Koeffler, H P; Taguchi, H.

    2006-01-01

    HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27 kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3?/? (GSK-3?/?) as a substrate. To explore the role of Akt signalling i...

  7. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5?-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  8. Characterization of botryococcene synthase enzyme activity, a squalene synthase-like activity from the green microalga Botryococcus braunii, Race B.

    Science.gov (United States)

    Okada, Shigeru; Devarenne, Timothy P; Murakami, Masahiro; Abe, Hiroki; Chappell, Joseph

    2004-02-01

    The extracellular matrix of the alga Botryococcus braunii, Race B, consists mainly of botryococcenes, which have potential as a hydrocarbon fuel. Botryococcenes are structurally similar to squalene raising the possibility of a common enzyme for the biosynthesis of both. While B. braunii squalene synthase (SS) enzyme activity has been documented, botryococcene synthase (BS) enzyme activity has not been. In the current study, an assay for BS activity has been developed and used to show that many of the assay conditions for BS enzyme activity are similar to those of SS. However, SS enzyme activity is stimulated by Tween 80 while BS enzyme activity is inhibited. Moreover, BS enzyme activity was correlated with the accumulation of botryococcenes during a B. braunii culture growth cycle, which was distinctly different from the profile of SS enzyme activity. While the current results indicate a conservation of enzymological features amongst the BS and SS enzymes, raising the possibility of one enzyme capable of catalyzing both activities, they are also consistent with these two activities arising from separate and distinct enzymes. PMID:14725863

  9. Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

    DEFF Research Database (Denmark)

    You, J P; Wang, Qian

    1994-01-01

    The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of L-NOARG was completely reversed by L-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. L-NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 M HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO2. In vessel segments in which the endothelium had been removed beforehand 15% CO2 induced relaxation that was not different from that seen in vessels with intact endothelium. L-NOARG had no affect in endothelium denuded vessels. The results suggest that high CO2 elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by L-NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall.

  10. Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

    Science.gov (United States)

    Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W V; Sivaraman, J

    2015-11-01

    ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ?20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

  11. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W. (SJCH)

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  12. Nitric oxide synthase in tissues around failed hip prostheses.

    Science.gov (United States)

    Stea, S; Visentin, M; Donati, M E; Granchi, D; Ciapetti, G; Sudanese, A; Toni, A

    2002-12-01

    Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening. PMID:12361623

  13. The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms

    Directory of Open Access Journals (Sweden)

    Dashuang Shi

    2015-06-01

    Full Text Available N-acetylglutamate synthase (NAGS catalyzes the production of N-acetylglutamate (NAG from acetyl-CoA and l-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1 in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups.

  14. Discovery of new acylaminopyridines as GSK-3 inhibitors by a structure guided in-depth exploration of chemical space around a pyrrolopyridinone core.

    Science.gov (United States)

    Sivaprakasam, Prasanna; Han, Xiaojun; Civiello, Rita L; Jacutin-Porte, Swanee; Kish, Kevin; Pokross, Matt; Lewis, Hal A; Ahmed, Nazia; Szapiel, Nicolas; Newitt, John A; Baldwin, Eric T; Xiao, Hong; Krause, Carol M; Park, Hyunsoo; Nophsker, Michelle; Lippy, Jonathan S; Burton, Catherine R; Langley, David R; Macor, John E; Dubowchik, Gene M

    2015-05-01

    Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3?. We identified several series of promising new GSK-3? inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3? inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses. PMID:25845281

  15. Inhibition of farnesyl pyrophosphate synthase attenuates angiotensin II-induced fibrotic responses in vascular smooth muscle cells.

    Science.gov (United States)

    Du, Chang-Qing; Liu, Xiao-Wei; Zeng, Guang-Zhong; Jin, Hong-Feng; Tang, Li-Jiang

    2015-06-01

    Through the regulation of the RhoA/Rho kinase (ROCK) pathway, angiotensin II (Ang II)-induced fibrotic responses contribute to vascular remodeling. Farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiovascular remodeling through the modulation of the above-mentioned pathway. However, the role of FPPS in Ang II-induced fibrotic responses and the related molecular mechanisms have not yet been elucidated. In the present study, vascular smooth muscle cells (VSMCs) from Sprague-Dawley (SD) rats were stimulated with Ang II. Cell proliferation was measusred usin the cell counting kit-8 (CCK-8). The levels of connective tissue growth factor (CTGF), FPPS, and those of phosphorylated and total extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK) were determined by western blot analysis. RhoA activity was determined using a pull-down assay. The results revealed that stimulation with Ang II enhanced cell proliferation, and increased the protein expression levels of FPPS and CTGF in the VSMCs. The inhibition of FPPS with ibandronate sodium attenuated the Ang II-induced increase in cell proliferation, CTGF expresison and RhoA activity; these effects were partially reversed by treatment with geranylgeraniol and were mimicked by GGTI-286. Furthermore, both SB203580 (a specific inhibitor of p38) and SP600125 (JNK1, JNK2 and JNK3 inhibitor) diminished the Ang II-induced production of CTGF; however, the inhibition of FPPS reduced the Ang II-induced activation of p38 mitogen-activated protein kinase (MAPK) and JNK. In conclusion, our data indicate that FPPS may play an important role in Ang II-induced fibrotic responses in VSMCs, and the underlying mechanisms at least partly involve the modulation of RhoA activity, and the p38 and JNK pathways. PMID:25847782

  16. Nitric oxide enhances MPP(+)-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum.

    Science.gov (United States)

    Obata, T; Yamanaka, Y

    2001-06-01

    We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis. PMID:11384616

  17. Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats

    Science.gov (United States)

    Lu, Qing-Bo; Feng, Xue-Mei; Tong, Ning; Sun, Hai-Jian; Ding, Lei; Wang, Yu-Jiao; Wang, Xuan; Zhou, Ye-Bo

    2015-01-01

    A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or N?-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR. PMID:26485682

  18. Neuronal and endothelial nitric oxide synthase gene knockout mice

    Scientific Electronic Library Online (English)

    P.L., Huang.

    1999-11-01

    Full Text Available Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarg [...] ed stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

  19. Cell-free expression and assembly of ATP synthase.

    Science.gov (United States)

    Matthies, Doreen; Haberstock, Stefan; Joos, Friederike; Dötsch, Volker; Vonck, Janet; Bernhard, Frank; Meier, Thomas

    2011-10-28

    Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F(1)F(o)-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry ?(3)?(3)???ab(2)c(13). Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems. PMID:21925509

  20. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning

    OpenAIRE

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J.; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M.

    2014-01-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H+-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H+-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H+-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the a...

  1. IRAK-4 inhibitors for inflammation.

    Science.gov (United States)

    Wang, Zhulun; Wesche, Holger; Stevens, Tracey; Walker, Nigel; Yeh, Wen-Chen

    2009-01-01

    Interleukin-1 receptor-associated kinases (IRAKs) are key components in the signal transduction pathways utilized by interleukin-1 receptor (IL-1R), interleukin-18 receptor (IL-18R), and Toll-like receptors (TLRs). Out of four members in the mammalian IRAK family, IRAK-4 is considered to be the "master IRAK", the only family member indispensable for IL-1R/TLR signaling. In humans, mutations resulting in IRAK-4 deficiency have been linked to susceptibility to bacterial infections, especially recurrent pyogenic bacterial infections. Furthermore, knock-in experiments by several groups have clearly demonstrated that IRAK-4 requires its kinase activity for its function. Given the critical role of IRAK-4 in inflammatory processes, modulation of IRAK-4 kinase activity presents an attractive therapeutic approach for the treatment of immune and inflammatory diseases. The recent success in the determination of the 3-dimensional structure of the IRAK-4 kinase domain in complex with inhibitors has facilitated the understanding of the mechanistic role of IRAK-4 in immunity and inflammation as well as the development of specific IRAK-4 kinase inhibitors. In this article, we review the biological function of IRAK-4, the structural characteristics of the kinase domain, and the development of small molecule inhibitors targeting the kinase activity. We also review the key pharmacophores required for several classes of inhibitors as well as important features for optimal protein/inhibitor interactions. Lastly, we summarize how these insights can be translated into strategies to develop potent IRAK-4 inhibitors with desired properties as new anti-inflammatory therapeutic agents. PMID:19689377

  2. Theanaphthoquinone inhibits fatty acid synthase expression in EGF-stimulated human breast cancer cells via the regulation of EGFR/ErbB-2 signaling

    International Nuclear Information System (INIS)

    Fatty acid synthase (FAS) is a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids. Most breast cancers require lipogenesis for growth. Here, we demonstrated the effects of theanaphthoquinone (TNQ), a member of the thearubigins generated by the oxidation of theaflavin (TF-1), on the expression of FAS in human breast cancer cells. TNQ was found to suppress the EGF-induced expression of FAS mRNA and FAS protein in MDA-MB-231 cells. Expression of FAS has previously been shown to be regulated by the SREBP family of transcription factors. In this study, we demonstrated that the EGF-induced nuclear translocation of SREBP-1 was blocked by TNQ. Moreover, TNQ also modulated EGF-induced ERK1/2 and Akt phosphorylation. Treatment of MDA-MB-231 cells with PI 3-kinase inhibitors, LY294002 and Wortmannin, inhibited the EGF-induced expression of FAS and nuclear translocation of SREBP-1. Treatment with TNQ inhibited EGF-induced EGFR/ErbB-2 phosphorylation and dimerization. Furthermore, treatment with kinase inhibitors of EGFR and ErbB-2 suggested that EGFR/ErbB-2 activation was involved in EGF-induced FAS expression. In constitutive FAS expression, TNQ inhibited FAS expression and Akt autophosphorylation in BT-474 cells. The PI 3-kinase inhibitors and tyrosine kinase inhibitors of EGFR and ErbB-2 also reduced constitutive FAS expression. In addition, pharmacological blockade of FAS by TNQ decreased cell viability and induced cell death in BT-474 cells. In summary, our findings suggest that TNQ modulates FAS expression by the regulation of EGFR/ErbB-2 pathways and induces cell death in breast cancer cells

  3. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Science.gov (United States)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-01-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2?Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  4. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    Energy Technology Data Exchange (ETDEWEB)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A. [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States); Nusrat, Asma, E-mail: anusrat@emory.edu [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States)

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  5. Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

    Indian Academy of Sciences (India)

    A Rus; Ma Peinado; S Blanco; Ml Del Moral

    2011-03-01

    Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury.Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that L-NIO would not be useful in alleviating the adverse effects of cardiac H/R.

  6. The redox sensitive glycogen synthase kinase 3? suppresses the self-protective antioxidant response in podocytes upon oxidative glomerular injury.

    Science.gov (United States)

    Li, Changbin; Ge, Yan; Peng, Ai; Gong, Rujun

    2015-11-24

    The redox sensitive glycogen synthase kinase (GSK) 3 has been recently implicated in the pathogenesis of proteinuric glomerulopathy. However, prior studies are less conclusive because they relied solely on chemical inhibitors of GSK3, which provide poor discrimination between the isoforms of GSK3 apart from potential off target activities. In murine kidneys, the ? rather than the ? isoform of GSK3 was predominantly expressed in glomeruli and distributed intensely in podocytes. By employing the doxycycline-activated Cre-loxP site specific gene targeting system, GSK3? was successfully knocked out (KO) selectively in podocytes in adult mice, resulting in a phenotype no different from control littermates. Electron microscopy of glomeruli in KO mice demonstrated more glycogen accumulation in podocytes but otherwise normal ultrastructures. Upon oxidative glomerular injury induced by protein overload, KO mice excreted significantly less albuminuria and had much attenuated podocytopathy and glomerular damage. The anti-proteinuric and glomerular protective effect was concomitant with diminished accumulation of reactive oxygen species in glomeruli in KO mice, which was likely secondary to a reinforced Nrf2 antioxidant response in podocytes. Collectively, our data suggest that GSK3? is dispensable for glomerular function and histology under normal circumstances but may serve as a therapeutic target for protecting from oxidative glomerular injuries. PMID:26567873

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

    Science.gov (United States)

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

    2013-01-01

    Objective. Nitric oxide (NO) has numerous important functions in the kidney. The role of NO in cisplatin (CP)-induced nephrotoxicity is not completely understood. This study was designed to determine the role of NO synthase inhibitor (L-NAME) on the severity of CP-induced nephrotoxicity in rats. Methods. Sixty four male (M) and female (F) Wistar rats were randomly divided into eight groups. The sham groups (group 1, male, n = 6 and group 2, female, n = 6) received saline. Groups 3 (male, n = 8) and 4 (female, n = 8) were treated with L-NAME (4?mg/kg, i.p.), and groups 5 (male, n = 8) and 6 (female, n = 8) received CP (3?mg/kg) for 7 days. Groups 7 (male, n = 8) and 8 (female, n = 8) were treated with L-NAME and CP for 7 days. Results. The CP-alone treated rats showed weight loss and increase in serum levels of blood urea nitrogen (BUN) and creatinine (Cr). Coadministration of L-NAME and CP did not improve weight loss, and it increased the levels of BUN and Cr in male but not in female rats (P < 0.05). CP alone increased kidney damage significantly (P < 0.05 ), however, the damage induced by combination of CP and L-NAME was gender-related. Conclusion. NOS inhibition by L-NAME increased CP-induced nephrotoxicity, which was gender-related. PMID:24167747

  8. Ginsenoside R(e) increases fertile and asthenozoospermic infertile human sperm motility by induction of nitric oxide synthase.

    Science.gov (United States)

    Zhang, Hong; Zhou, Qing-Ming; Li, Xiao-Da; Xie, Yi; Duan, Xin; Min, Feng-Ling; Liu, Bing; Yuan, Zhi-Gang

    2006-02-01

    We investigated the effects of Ginsenoside R(e) on human sperm motility in fertile and asthenozoospermic infertile individuals in vitro and the mechanism by which the Ginsenosides play their roles. The semen samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Spermatozoa were separated by Percoll and incubated with 0, 1, 10 or 100 microM of Ginsenoside R(e). Total sperm motility and progressive motility were measured by computer-aided sperm analyzer (CASA). Nitric oxide synthase (NOS) activity was determined by the 3H-arginine to 3H-citrulline conversion assay, and the NOS protein was examined by the Western blot analysis. The production of sperm nitric oxide (NO) was detected using the Griess reaction. The results showed that Ginsenoside R(e) significantly enhanced both fertile and infertile sperm motility, NOS activity and NO production in a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside R(e). And pretreatment with a NOS inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-Acetyl-L-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside R(e). Data suggested that Ginsenoside R(e) is beneficial to sperm motility, and that induction of NOS to increase NO production may be involved in this benefit. PMID:16526279

  9. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    Science.gov (United States)

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction. PMID:25595727

  10. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    International Nuclear Information System (INIS)

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  11. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating occludin, claudin-1 and E-cadherin expression.

    Science.gov (United States)

    Severson, Eric A; Kwon, Mike; Hilgarth, Roland S; Parkos, Charles A; Nusrat, Asma

    2010-07-01

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin. PMID:20617560

  12. Acetylglutamate synthase in Neurospora crassa: characterization, localization, and genetic behavior of a regulatory enzyme of arginine biosynthesis

    International Nuclear Information System (INIS)

    This study describes the characterization and localization of the first enzyme of arginine biosynthesis in Neurospora crassa. A radioactive assay was developed to detect this enzyme whereby radioactive substrate and product molecules could be separated by ion-exchange chromatography. The enzyme was found to have a pH optimum of 9.0 and K/sub m/ values for glutamate and acetyl-CoA of approximately 4.7 and 0.45 mM, respectively. The enzyme was shown to be feedback inhibited by arginine. Half-maximal inhibition was observed at 0.13 mM arginine, a concentration which is similar to be in vivo cytosolic concentration of 0.2 mM. Arginine was found to act as a competitive inhibitor with respect to acetyl-CoA. Acetylglutamate synthase was localized to the mitochondrion. However, in contrast to the mitochondrial matrix location of the other ornithine biosynthetic enzymes, this enzyme was found to reside on the mitochondrial inner membrane

  13. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    Science.gov (United States)

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2?Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  14. Nitric Oxide Synthase Activation as a Trigger of N-methyl-N-nitrosourea-Induced Photoreceptor Cell Death.

    Science.gov (United States)

    Hisano, Suguru; Koriyama, Yoshiki; Ogai, Kazuhiro; Sugitani, Kayo; Kato, Satoru

    2016-01-01

    Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death. PMID:26427435

  15. Reduced expression of prostacyclin synthase and nitric oxide synthase in subcutaneous arteries of type 2 diabetic patients.

    Science.gov (United States)

    Safiah Mokhtar, Siti; M Vanhoutte, Paul; W S Leung, Susan; Imran Yusof, Mohd; Wan Sulaiman, Wan Azman; Zaharil Mat Saad, Arman; Suppian, Rapeah; Ghulam Rasool, Aida Hanum

    2013-01-01

    Diabetic endothelial dysfunction is characterized by impaired endothelium-dependent relaxation. In this study, we measured the expression of endothelial nitric oxide synthase (eNOS), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS), and prostacyclin receptor (IP) in subcutaneous arteries of type-2 diabetic and non-diabetic patients. Subcutaneous arteries were dissected from tissues from seven diabetics (4 males and 3 females) and seven non-diabetics (5 males and 2 females) aged between 18 to 65 years, who underwent lower limb surgical procedures. Diabetics had higher fasting blood glucose compared to non-diabetics, but there were no differences in blood pressure, body mass index and age. Patients were excluded if they had uncontrolled hypertension, previous myocardial infarction, coronary heart disease, renal or hepatic failure and tumor. The relative expression levels of eNOS, COX-1, COX-2, PGIS and IP receptor were determined by Western blotting analysis, normalized with the ?-actin level. Increased expression of COX-2 was observed in subcutaneous arteries of diabetics compared to non-diabetics, whereas the expression levels of eNOS and PGIS were significantly lower in diabetics. There were no significant differences in expression levels of COX-1 and IP receptor between the two groups. Immunohistochemical study of subcutaneous arteries showed that the intensities of eNOS and PGIS staining were lower in diabetics, with higher COX-2 staining. In conclusion, type-2 diabetes is associated with higher COX-2 expression, but lower eNOS and PGIS expression in subcutaneous arteries. These alterations may lead to impaired endothelium-dependent vasodilatation, and thus these proteins may be potential targets for protection against the microvascular complications of diabetes. PMID:24225501

  16. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase.

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe B

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C(16) and C(18) products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C(6) and C(10)-C(12)) substrate preferences leading to the C(8) lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding. Udgivelsesdato: 2007-Feb

  17. Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1.

    Science.gov (United States)

    Tommasi, Sara; Zanato, Chiara; Lewis, Benjamin C; Nair, Pramod C; Dall'Angelo, Sergio; Zanda, Matteo; Mangoni, Arduino A

    2015-12-14

    Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme involved in the metabolism of asymmetric dimethylarginine (ADMA) and N-monomethyl arginine (NMMA), which are endogenous inhibitors of the nitric oxide synthase (NOS) family of enzymes. Two isoforms of DDAH have been identified in humans, DDAH-1 and DDAH-2. DDAH-1 inhibition represents a promising strategy to limit the overproduction of NO in pathological states without affecting the homeostatic role of this important messenger molecule. Here we describe the design and synthesis of 12 novel DDAH-1 inhibitors and report their derived kinetic parameters, IC50 and Ki. Arginine analogue 10a, characterized by an acylsulfonamide isosteric replacement of the carboxylate, showed a 13-fold greater inhibitory potential relative to the known DDAH-1 inhibitor, L-257. Compound 10a was utilized to study the putative binding interactions of human DDAH-1 inhibition using molecular dynamics simulations. The latter suggests that several stabilizing interactions occur in the DDAH-1 active-site, providing structural insights for the enhanced inhibitory potential demonstrated by in vitro inhibition studies. PMID:26420019

  18. Probing novel 1-aza-9-oxafluorenes as selective GSK-3beta inhibitors.

    Science.gov (United States)

    Voigt, Burkhardt; Krug, Martin; Schächtele, Christoph; Totzke, Frank; Hilgeroth, Andreas

    2008-01-01

    Within the histopathology of Alzheimer's disease (AD) certain hallmarks are beeing observed. The occurance of protein deposits belong to such characteristic features. Such deposits can be found extracellular as beta-amyloid (Abeta) plaques and intracellular as neurofibrillary tangles (NFTs). In the search for novel AD therapeutics it became of great interest to investigate the formation of NFTs and their contribution to the AD symptomatic. NFTs consist of hyperphosphorylated tau protein. Within the phosphorylation process of tau protein two kinases are of great importance: cyclin dependent kinase 5 (cdk5) and its truncated regulatory subunit p25 and glycogen synthase kinase 3beta (GSK-3beta). The role of both kinases within the NFT formation process is still under debate. To better understand the pathophysiological process highly selective inhibitors of both kinases are of value. Known inhibitors lack the necessary selectivity. We developed novel 1-aza-9-oxafluo-renes as selective GSK-3beta inhibitors. Structure-activity relationships of a series of 4-phenyl substituted derivatives are discussed. Variation of the 3-side chain led to selective carbonyl amide derivatives with selectivity factors of more than 100 at the tested ATP competitor concentrations. Such selectivities permit specific investigation of the role of GSK-3beta within the NFT formation processes. PMID:18000938

  19. Proteome of monocyte priming by lipopolysaccharide, including changes in interleukin-1beta and leukocyte elastase inhibitor

    Directory of Open Access Journals (Sweden)

    Beranova-Giorgianni Sarka

    2008-05-01

    Full Text Available Abstract Background Monocytes can be primed in vitro by lipopolysaccharide (LPS for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics. Results Monocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl-benzenesulfonyl fluoride (AEBSF, a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry. Conclusion We identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1? appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.

  20. In Silico Analysis of Sequence-Structure-Function Relationship of the Escherichia coli Methionine Synthase.

    Science.gov (United States)

    Kumar, Shiv; Bhagabati, Puja; Sachan, Reena; Kaushik, Aman Chandra; Dwivedi, Vivek Dhar

    2015-12-01

    The molecular evolution of various metabolic pathways in the organisms can be employed for scrutinizing the molecular aspects behind origin of life. In the present study, we chiefly concerned about the sequence-structure-function relationship between the Escherichia coli methionine synthase and their respective animal homologs by in silico approach. Using homology prediction technique, it was observed that only 79 animal species showed similarity with the E. coli methionine synthase. Also, multiple sequence alignment depicted only 25 conserved patterns between the E. coli methionine synthase and their respective animal homologs. Based on that, Pfam analysis identified the protein families of 22 conserved patterns among the attained 25 conserved patterns. Furthermore, the 3D structure was generated by HHpred and evaluated by corresponding Ramachandran plot specifying 93 % of the ? and ? residues angles in the most ideal regions. Hence, the designed structure was established as a good quality model for the full length of E. coli methionine synthase. PMID:26223547