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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

    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. Influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation, regulation and organisation of photosynthesis in Solanum nigrum.

    Riethmuller-Haage, Ingrid; Bastiaans, Lammert; Harbinson, Jeremy; Kempenaar, Corné; Kropff, Martin J

    2006-06-01

    The influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation of the photosynthetic apparatus was examined on 4-weeks-old climate chamber-grown Solanum nigrum plant. To have an indication on the relative performance of the photosynthetic apparatus of ALS-treated plants, the level of carbon dioxide (CO(2)) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) or photosystem II (Phi(PSII)) electron transport and leaf chlorophyll content were assessed for both control and treated plants at 2, 4 and 7 days after application of the herbicide. Results indicated a progressive inhibition of the level of CO(2) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) and II (Phi(PSII)) electron transport and the leaf chlorophyll content already 2 days after application of the herbicide. The linear relationship between the photosystem I and II was unaltered by herbicidal treatment and was sustained under conditions where large changes in pigment composition of the leaves occurred. It appears that the stress-induced loss of leaf chlorophyll is not a catastrophic process but rather is the consequence of a well-organised breakdown of components. Under photorespiratory and non-photorespiratory conditions, the relationship between the index of electron transport flow through photosystem I and II and the rate of CO(2) fixation is altered so that electron transport becomes less efficient at driving CO(2) fixation. PMID:16691366

  3. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl

    Yadav, Narendra; McDevitt, Raymond E.; Benard, Susan; Falco, S. Carl

    1986-01-01

    Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from Escherichia coli and Saccharomyces cerevisiae. The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucle...

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

    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

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

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

    1993-09-01

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

  6. Activity of Acetolactate Synthase from Maize (Zea mays L. ) as Influenced by Chlorsulfuron and Tribenuron-methyl

    FAN Zhi-jin; CHEN Jun-peng; HU Ji-ye; QIAN Chuan-fan; LI Zheng-ming

    2003-01-01

    Study on relative sensitivity of maize (Zea mays L. ) Nongda108 and Nongda3138 to sulfonylurea herbicide chlorsulfuron and tribenuron-methyl using maize taproot length by sand bioassy indicated that, Nongda3138 had higher tolerance to chlorsulfuron and tribenuron-methyl than Nongda108 did. Chlorsulfuron had stronger growth inhibition to maize Nongda108 and Nongda3138 than tribenuron-methyl did. Study on target enzyme of sulfonylurea herbicide acetolactate synthase (ALS) showed that, chlorsulfuron and tribenuron-methyl inhibited ALS in vitro strongly, and non-competitively. In the same concentration of inhibitors,chlorsuifuron had stronger ALS activity inhibition than tribenuron-methyl did. Lower level of chlorsulfuron and tribenuron-methyl has no ALS activity inhibition in vivo, the ALS inhibition only occurred in the condition of high concentration of chlorsulfuron and tribenuron-methyl in vivo.

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

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

    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.

  9. Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death.

    Zhao, Lun; Jing, Xue; Chen, Li; Liu, Yingjun; Su, Yanan; Liu, Tingting; Gao, Changbin; Yi, Bin; Wen, Jing; Ma, Chaozhi; Tu, Jinxing; Zou, Jitao; Fu, Tingdong; Shen, Jinxiong

    2015-12-01

    Tribenuron-methyl (TM) is a powerful sulfonylurea herbicide that inhibits branched-chain amino acid (BCAA) biosynthesis by targeting the catalytic subunit (CSR1) of acetolactate synthase (ALS). Selective induction of male sterility by foliar spraying of TM at low doses has been widely used for hybrid seed production in rapeseed (Brassica napus); however, the underlying mechanism remains unknown. Here, we report greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems after TM application. Constitutive or anther-specific expression of csr1-1D (a CSR1 mutant) eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis. The results of TM daub-stem experiments, combined with the observations of little TM accumulation in anthers and reversion of TM-induced male sterility by targeted expression of the TM metabolism gene Bel in either the mesophyll or phloem, suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem. Microscopy and immunoblotting revealed that autophagy, a bulk degradation process induced during cell death, was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS. Moreover, TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA. These data suggested that TM was polar-transported to anthers, resulting in BCAA starvation via anther-specific ALS inhibition and, ultimately, autophagic cell death in anthers. PMID:26362932

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

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

    1987-05-01

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

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

    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

  12. Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.).

    Vermeulen, A; Vaucheret, H; Pautot, V; Chupeau, Y

    1992-06-01

    Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. PMID:24203132

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

    Mourad, G; Williams, D; King, J

    1995-01-01

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

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

    Delye, Christophe; Pernin, Fanny; Scarabel, Laura

    2011-01-01

    We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg197, His197, Leu197, Ser197, Thr197 and Leu574 were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitu...

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

    Délye, Christophe; Pernin, Fanny; Scarabel, Laura

    2011-02-01

    We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg₁₉₇, His₁₉₇, Leu₁₉₇, Ser₁₉₇, Thr₁₉₇ and Leu₅₇₄ were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms. PMID:21421378

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

    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

  17. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

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

    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

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

    Silverman, P.M.; Eoyang, L.

    1987-06-01

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

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

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

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

    Silverman, P M; Eoyang, L

    1987-01-01

    Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a ...

  2. A Cellular Model for Screening Neuronal Nitric Oxide Synthase Inhibitors

    Fang, Jianguo; Silverman, Richard B.

    2009-01-01

    Nitric oxide synthase (NOS) inhibitors are potential drug candidates because it has been well demonstrated that excessive production of NO critically contributes to a range of diseases. Most inhibitors have been screened in vitro using recombinant enzymes, leading to the discovery of a variety of potent compounds. To make inhibition studies more physiologically relevant and bridge the gap between the in vitro assay and in vivo studies, we report here a cellular model for screening NOS inhibit...

  3. Inhibitors of nitric oxide synthase in inflammatory arthritis.

    Boughton-Smith, N K; Tinker, A C

    1998-07-01

    There is considerable evidence that excessive nitric oxide (NO) synthesized from L-arginine by inducible nitric oxide synthase (iNOS) plays an important pathological role in inflammatory arthritis. Since NO synthesized by constitutive isoforms of NOS has a physiological role, a great deal of activity has been directed at identifying inhibitors of NOS that are selective for the induced isoform. The major chemical areas that have been described so far in the search for such selective iNOS inhibitors and the activity of some of these compounds in animal models of arthritis are reviewed. PMID:18465556

  4. Protein inhibitor of neuronal nitric oxide synthase interacts with protein kinase A inhibitors.

    Yu, Jianqiang; Yu, Long; Chen, Zheng; Zheng, Lihua; Chen, Xiaosong; Wang, Xiang; Ren, Daming; Zhao, Shouyuan

    2002-03-28

    Protein kinase A (PKA) and neuronal nitric oxide synthase (nNOS) are important signaling molecules. It is well known that PKA can specifically phosphorylate nNOS. But the underlying molecular mechanism is still obscure. Our data indicate that the protein inhibitor of nNOS (PIN) binds to protein kinase A inhibitors (PKIs), which suggests that PKIs, together with PIN, might mediate the phosphorylation of nNOS by PKA. PMID:11978406

  5. Fatty acid synthase inhibitors isolated from Punica granatum L

    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)

  6. Fatty acid synthase inhibitors isolated from Punica granatum L

    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 μmol L-1. (author)

  7. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon. PMID:27338660

  8. Use of nitric oxide synthase inhibitors for the treatment of inflammatory disease and pain.

    Cheshire, D R

    2001-07-01

    This article reviews the recent literature on selective inhibitors of nitric oxide synthase (NOS) between 1999 and the first quarter of 2001. The introduction highlights the major therapeutic objectives for NOS inhibitors, including rheumatoid arthritis (RA), osteoarthritis (OA) and pain. The review attempts to cover the structural diversity of small molecule NOS inhibitors currently being explored in the pharmaceutical and academic communities. PMID:15995936

  9. Biochemistry: Acetohydroxyacid Synthase

    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.

  10. Fatty Acid Synthase Inhibitor C75 Ameliorates Experimental Colitis

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

    2013-01-01

    Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic α-methylene-γ-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or...

  11. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors. PMID:26864638

  12. New carboxylate and hydroxamate inhibitors of prostaglandin-H-synthase and their metal complexes

    Al Agha, Ahmed

    2009-01-01

    The thesis describes new carboxylate and hydroxamate inhibitors of prostaglandin-H-synthase (PGHS) as well as the synthesis and structures of metal complexes of the hydroxamates which were synthesised. This enzyme, also called cyclooxygenase (COX), has two active sites a cyclooxygenase (COX) active site which is the target for aspirin and a peroxidase (POX) active site, upon which the COX site depends. The thesis is divided in to three chapters. The first chapter describes the attempted sy...

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

    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.

  14. Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor.

    Montgomery, Heather J; Perdicakis, Basil; Fishlock, Dan; Lajoie, Gilles A; Jervis, Eric; Guy Guillemette, J

    2002-06-01

    Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited. PMID:11937350

  15. Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases.

    Adada, Mohamad; Luberto, Chiara; Canals, Daniel

    2016-05-01

    Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann-Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics. PMID:26200918

  16. Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

    Skovpen, Yulia V; Conly, Cuylar J T; Sanders, David A R; Palmer, David R J

    2016-02-17

    Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development. PMID:26836694

  17. Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity.

    Bommineni, Gopal R; Kapilashrami, Kanishk; Cummings, Jason E; Lu, Yang; Knudson, Susan E; Gu, Chendi; Walker, Stephen G; Slayden, Richard A; Tonge, Peter J

    2016-06-01

    β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection. PMID:27187871

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

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

    2010-01-01

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

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

    Yi-Hsuan Wu

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

  20. N-Substituted acetamidines and 2-methylimidazole derivatives as selective inhibitors of neuronal nitric oxide synthase.

    Maccallini, Cristina; Patruno, Antonia; Lannutti, Fabio; Ammazzalorso, Alessandra; De Filippis, Barbara; Fantacuzzi, Marialuigia; Franceschelli, Sara; Giampietro, Letizia; Masella, Simona; Felaco, Mario; Re, Nazzareno; Amoroso, Rosa

    2010-11-15

    A series of N-substituted acetamidines and 2-methylimidazole derivatives structurally related to W1400 were synthesized and evaluated as Nitric Oxide Synthase (NOS) inhibitors. Analogs with sterically hindering isopropyl and phenyl substituents on the benzylic carbon connecting the aromatic core of W1400 to the acetamidine nitrogen, showed good inhibitory potency for nNOS (IC(50)=0.2 and 0.3 μM) and selectivity over eNOS (500 and 1166) and to a lesser extent over iNOS (50 and 100). A molecular modeling study allowed to shed light on the effects of the structural modifications on the selectivity of the designed inhibitors toward the different NOS isoforms. PMID:20933416

  1. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

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

    Kamauchi, Shinya; Urade, Yoshihiro

    2011-11-01

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

  3. Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

    Wagner, Florence F; Bishop, Joshua A; Gale, Jennifer P; Shi, Xi; Walk, Michelle; Ketterman, Joshua; Patnaik, Debasis; Barker, Doug; Walpita, Deepika; Campbell, Arthur J; Nguyen, Shannon; Lewis, Michael; Ross, Linda; Weïwer, Michel; An, W Frank; Germain, Andrew R; Nag, Partha P; Metkar, Shailesh; Kaya, Taner; Dandapani, Sivaraman; Olson, David E; Barbe, Anne-Laure; Lazzaro, Fanny; Sacher, Joshua R; Cheah, Jaime H; Fei, David; Perez, Jose; Munoz, Benito; Palmer, Michelle; Stegmaier, Kimberly; Schreiber, Stuart L; Scolnick, Edward; Zhang, Yan-Ling; Haggarty, Stephen J; Holson, Edward B; Pan, Jen Q

    2016-07-15

    The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders. PMID:27128528

  4. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

    Liu, Ziyi; Cao, Zongxian; Jourdan, Tony; Erdelyi, Katalin; Godlewski, Grzegorz; Szanda, Gergő; Liu, Jie; Park, Joshua K.; Mukhopadhyay, Bani; Rosenberg, Avi Z.; Liow, Jeih-San; Lorenz, Robin G.; Pacher, Pal; Innis, Robert B.; Kunos, George

    2016-01-01

    Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1−/− but not in nos2−/− mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.

  5. Influence of nitric oxide synthase inhibitor on gerbil behavior after hyperbaric oxygen-induced convulsion

    Jianguang Zhou; Changyun Liu; Yiqun Fang; Yingqi Zhou; Erli Xu; Jingchang Liu

    2008-01-01

    BACKGROUND: Studies have reported that nitric oxide synthase (NOS) inhibitor can prolong the latency of hyperbaric oxygen-induced convulsion (HBOC). However, there are very few reports addressing the influence of NOS inhibitor on mental behavior.OBJECTIVE: To investigate behavioral changes after HBOC in gerbils, as well as the influence of NOS inhibitor.DESIGN, TIME AND SETTING: Randomized experiments were performed in the Laboratory of Hyperbaric Pressure and Diving Physiology, Naval Medical Research Institute of Chinese PLA (Shanghai,China) from March 2005 to June 2007.MATERIALS: Forty male gerbils were randomly divided into five groups: HBOC, saline control, NOS inhibitor, pressure control, and normal control. Each group contained eight animals.METHODS: In the HBOC group, once depression induction ended, animals were removed from the chamber five minutes after the first appearance of generalized convulsion induced by 0.5 MPa hyperbaric oxygen. Ten minutes before entering the chamber, saline control and NOS inhibitor animals were intraperitoneally injected with 1 mL saline and 20 mg/kg NG-nitro-L-arginine, respectively. The pressure control group was only exposed to 0.5 MPa. The remaining procedures in these three groups were identical to the HBOC group. The normal control group received no intervention.MAIN OUTCOME MEASURES: Open field test scores in gerbils prior to HBOC, as well as immediately,24 hours, and 72 hours after decompression ended.RESULTS: HBOC was not detected in either the normal control or the pressure control group, and there were no significant differences in opcn field test scores prior to and after HBOC (P > 0.05). HBOC occurred in the HBOC, saline control, and NOS inhibitor groups, with significant differences in open field test scores after decompression ended compared to normal control and pressure control groups (P < 0.05-0.01).Compared to the HBOC and saline control groups, the NOS inhibitor group exhibited a significantly lower score in

  6. Effect of nitric oxide synthase inhibitor on proteoglycan metabolism in repaired articular cartilage in rabbits

    孙炜; 金大地; 王吉兴; 秦立赟; 刘晓霞

    2003-01-01

    Objective: To study the effect of nitric oxide synthase inhibitor, S-methyl thiocarbamate (SMT), on proteoglycan metabolism in repaired articular cartilage in rabbits. Methods: Twenty-four male New Zealand white rabbits, aged 8 months and weighing 2.5 kg±0.2 kg, were used in this study. Cartilage defects in full thickness were created on the intercondylar articular surface of bilateral femurs of all the rabbits. Then the rabbits were randomly divided into 3 groups (n=8 in each group). The defects in one group were filled with fibrin glue impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2, BMP group), in one group with fibrin glue impregnated with rhBMP-2 and hypodermic injection with SMT (SMT group) and in the other group with nothing (control group). All the animals were killed at one year postoperatively. The tissue sections were stained with safranine O-fast green and analyzed by Quantiment 500 system to determine the content of glycosaminoglycan through measuring the percentage of safranine O-stained area, the thickness of cartilages and the mean gray scale (average stain intensity). Radiolabelled sodium sulphate (Na235SO4) was used to assess the proteoglycan synthesis. Results: At one year postoperatively, the percentage of safranine O-stained area, the mean gray scale and the cartilage thickness of the repaired tissues in SMT group were significantly higher than those of BMP group (P<0.01) and the control group (P<0.05). Result of incorporation of Na235SO4 showed that the proteoglycan synthesis in SMT group was higher than those of BMP group and the control group (P<0.01). Conclusions: SMT, a nitric oxide synthase inhibitor, can significantly increase the content of glycosaminoglycan and proteoglycan synthesis, and computer-based image analysis is a reliable method for evaluating proteoglycan metabolism.

  7. Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

    Carla S. Jung

    2014-03-01

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

  8. Effects of the inducible nitric oxide synthase inhibitor aminoguanidine in two different rat models of schizophrenia.

    Lafioniatis, Anastasios; Orfanidou, Martha A; Papadopoulou, Evangelia S; Pitsikas, Nikolaos

    2016-08-01

    Several lines evidence indicate that the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine and the mixed dopamine (DA) D1/D2 receptor agonist apomorphine induce schizophrenia-like symptoms in rodents, including memory impairments and social withdrawal. Nitric oxide (NO) has been proposed to act as an intracellular messenger in the brain and its overproduction is associated with schizophrenia. The current study was designed to investigate the ability of the inducible NO synthase (iNOS) inhibitor aminoguanidine (AG) to counteract schizophrenia-like behavioural deficits produced by ketamine and apomorphine in rats. The efficacy of AG to antagonize extinction of recognition memory, ketamine and apomorphine-induced recognition memory impairments was tested utilizing the novel object recognition task (NORT). Further, the efficacy of AG to attenuate ketamine-induced social withdrawal was examined in the social interaction test. AG (25 and 50mg/kg) antagonized extinction of recognition memory and reversed ketamine (3mg/kg) and apomorphine (1mg/kg)-induced recognition memory deficits. In contrast, AG (50 and 100mg/kg) did not counteract the ketamine (8mg/kg)-induced social isolation. The present data show that the iNOS inhibitor AG counteracted extinction of recognition memory and reversed recognition memory deficits produced by dysfunction of the glutamatergic and the dopaminergic (DAergic) system in rats. Therefore, AG may be efficacious in attenuating memory impairments often observed in schizophrenia patients. PMID:27132765

  9. Study on Inhibitors of Methionine Synthase Ⅷ: Synthesis of 2,5-Diamino-4-oxo-6- (3-butenyl) pyrimidine

    ZHANG Zhi-Li; WANG Hong-Tao; WANG Xiao-Wei; MA Xiao-Yan; LIU Jun-Yi; R.J. Griff; B.T. Golding

    2003-01-01

    @@ Cobalamin-dependent methionine synthase plays a crucial role in folate metabolism and such would appear to be an excellent target for rational antifolate drug design. However, to date, no anticancer agents directed at this enzyme are available, but the enzyme is efficiently and specifically inhibited by N2O and this has proven invaluable for evaluating the biochemical consequence of enzyme inhibition and for mechanistic studies. [1,2] 2,5-Diamino-4-oxo-6-(3-butenyl) pyrimidine, a key intermediate in synthetic inhibitors of methionine synthase, was first synthesized using γbutenyl-β-ketoester and guanidine carbonate (Scheme 1). [3

  10. Dynamic ligand-based pharmacophore modeling and virtual screening to identify mycobacterial cyclopropane synthase inhibitors

    CHINMAYEE CHOUDHURY; U DEVA PRIYAKUMAR; G NARAHARI SASTRY

    2016-05-01

    Multidrug resistance in Mycobacterium tuberculosis (M. Tb) and its coexistence with HIV arethe biggest therapeutic challenges in anti-M. Tb drug discovery. The current study reports a Virtual Screening(VS) strategy to identify potential inhibitors of Mycobacterial cyclopropane synthase (CmaA1), an importantM. Tb target considering the above challenges. Five ligand-based pharmacophore models were generatedfrom 40 different conformations of the cofactors of CmaA1 taken from molecular dynamics (MD) simulationstrajectories of CmaA1. The screening abilities of these models were validated by screening 23 inhibitors and1398 non-inhibitors of CmaA1. A VS protocol was designed with four levels of screening i.e., ligand-basedpharmacophore screening, structure-based pharmacophore screening, docking and absorption, distribution,metabolism, excretion and the toxicity (ADMET) filters. In an attempt towards repurposing the existing drugsto inhibit CmaA1, 6,429 drugs reported in DrugBank were considered for screening. To find compounds thatinhibit multiple targets of M. Tb as well as HIV, we also chose 701 and 11,109 compounds showing activitybelow 1 μM range on M. Tb and HIV cell lines, respectively, collected from ChEMBL database. Thus, a totalof 18,239 compounds were screened against CmaA1, and 12 compounds were identified as potential hits forCmaA1 at the end of the fourth step. Detailed analysis of the structures revealed these compounds to interactwith key active site residues of CmaA1.

  11. Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive rats.

    Han, Jie; Jiang, Dong-Mei; Ye, Yang; Du, Chang-Qing; Yang, Jian; Hu, Shen-Jiang

    2016-05-01

    Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium‑dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar‑Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration‑dependent manner. The Ang II‑induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium‑dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired

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

    Irene Mavelli

    2012-02-01

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

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

    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.

  14. Quinazoline thymidylate synthase inhibitors: methods for assessing the contribution of polyglutamation to their in vitro activity.

    Jackman, A L; Kimbell, R; Brown, M; Brunton, L; Boyle, F T

    1995-10-01

    Many quinazoline thymidylate synthase (TS) inhibitors undergo intracellular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibitory activity towards TS (IC50 0.001-2 microM) and the growth of L1210 cells (IC50 0.005-10 microM), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L1210: RD1694) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were validated using a series of quinazoline-based TS inhibitors with well-defined activity for TS, folypolyglutamate synthetase (FPGS) and the reduced-folate cell membrane carrier (RFC). Short-exposure growth-inhibition assays or the measurement of TS activity in situ after various incubation times, followed by different lengths of time in drug-free medium, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since only the growth-inhibitory potency of polyglutamated analogues is significantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyglutamation of a particular compound than isolated FPGS assays alone. PMID:7495479

  15. A New Route for the Synthesis of Thymidylate Synthase Inhibitor Raltitrexed

    CAO Sheng-Li; WAN Rong; FENG Yu-Ping

    2003-01-01

    @@ Raltitrexed (8), a new quinazoline-based inhibitor of thymidylate synthase (TS), has been registered widely for the first-line treatment of advanced colorectal cancer. [1,2] As reported in the literature, [3,4] it can be prepared from 2-thiophenecarboxylic acid via 7 steps in 3% overall yield, but n-BuLi and the low temperature at - 78 ℃ was needed for the introduction of 5-carboxyl group into thiophene ring through lithiation of 2-(N-Boc-N-methylamino) thiophene followed by the addition of CO2. Here we wish to report a new route for the synthesis of Raltitrexed which was obtained from 2,5-thiophenedicarboxylic acid via 6 steps in 18.2% overall yield (Scheme 1). The mild conditions utilized in the synthetic route avoid the use of n-BuLi, NaH and the experimental conditions of low temperature at - 78 ℃ and strictly free of water, and are suitable for the large-scale preparation.

  16. Molecular docking analysis of selected Clinacanthus nutans constituents as xanthine oxidase, nitric oxide synthase, human neutrophil elastase, matrix metalloproteinase 2, matrix metalloproteinase 9 and squalene synthase inhibitors

    Radhakrishnan Narayanaswamy

    2016-01-01

    Full Text Available Background: Clinacanthus nutans (Burm. f. Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO, nitric oxide synthase (NOS, human neutrophil elastase (HNE, matrix metalloproteinase (MMP 2 and 9, and squalene synthase (SQS using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET, and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0 toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS.

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

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

    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 c...

  18. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    Sebastián, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. PMID:27131621

  19. Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors.

    Meredith, Erik L; Ksander, Gary; Monovich, Lauren G; Papillon, Julien P N; Liu, Qian; Miranda, Karl; Morris, Patrick; Rao, Chang; Burgis, Robin; Capparelli, Michael; Hu, Qi-Ying; Singh, Alok; Rigel, Dean F; Jeng, Arco Y; Beil, Michael; Fu, Fumin; Hu, Chii-Whei; LaSala, Daniel

    2013-12-12

    Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure-activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11β-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing's syndrome. PMID:24900631

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

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

    2013-01-01

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

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

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

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

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

    1986-05-01

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

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

    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

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

    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

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

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

    2001-01-01

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

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

    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.

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

    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 β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-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

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

    Fernando Eduardo Padovan-Neto

    2011-06-01

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

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

    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.

  10. Nitric oxide synthase inhibitors can antagonize neurogenic and calcitonin gene-related peptide induced dilation of dural meningeal vessels

    Akerman, S; Williamson, D J; Kaube, H; Goadsby, P J

    2002-01-01

    The detailed pathophysiology of migraine is beginning to be understood and is likely to involve activation of trigeminovascular afferents. Clinically effective anti-migraine compounds are believed to have actions that include peripheral inhibition of calcitonin gene-related peptide (CGRP) release from trigeminal neurones, or preventing dural vessel dilation, or both. CGRP antagonists can block both neurogenic and CGRP-induced dural vessel dilation. Nitric oxide (NO) can induce headache in migraine patients and often triggers a delayed migraine. The initial headache is thought to be caused via a direct action of the NO–cGMP pathway that causes vasodilation by vascular smooth muscle relaxation, while the delayed headache is likely to be a result of triggering trigeminovascular activation. Nitric oxide synthase (NOS) inhibitors are effective in the treatment of acute migraine. The present studies used intravital microscopy to examine the effects of specific NOS inhibitors on neurogenic dural vasodilation (NDV) and CGRP-induced dilation. The non-specific and neuronal NOS (nNOS) inhibitors were able to partially inhibit NDV, while the non-specific and endothelial NOS (eNOS) inhibitors were able to partially inhibit the CGRP induced dilation. There was no effect of the inducible NOS (iNOS) inhibitor. The data suggest that the delayed headache response triggered by NO donors in humans may be due, in part, to increased nNOS activity in the trigeminal system that causes CGRP release and dural vessel dilation. Further, eNOS activity in the endothelium causes NO production and smooth muscle relaxation by direct activation of the NO–cGMP pathway, and may be involved in the initial headache response. PMID:12183331

  11. Concentrations of Nitric Oxide in Rat Brain Tissues after Diffuse Brain Injury and Neuroprotection by the Selective Inducible Nitric Oxide Synthase Inhibitor Aminoguanidine

    Yi-bao Wang; Shao-wu Ou; Guang-yu Li; Yun-hui Liu

    2005-01-01

    @@ To investigate the effects of nitric oxide (NO) and the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG) on trauma, we explored the concentrations of nitric oxide in rat brain tissues at different time stamps after diffuse brain injury (DBI) with or without AG treatment.

  12. Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as glycogen synthase kinase-3β inhibitors with anti-depressant activity.

    Khan, Imran; Tantray, Mushtaq A; Hamid, Hinna; Alam, Mohammad Sarwar; Kalam, Abul; Dhulap, Abhijeet

    2016-08-15

    A series of benzimidazole based thiadiazole and carbohydrazide conjugates have been synthesized and evaluated for inhibition of glycogen synthase kinase-3β and anti-depressant effect. Compounds 4f, 4j, 5b, 5g and 5i were found to be the most potent inhibitors of GSK-3β in vitro amongst the twenty-five benzimidazole based thiadiazole and carbohydrazide conjugates synthesized. Compound 5i was also found to exhibit significant antidepressant activity in vivo at 50mg/kg, when compared to fluoxetine, a known antidepressant drug. The molecular docking studies revealed multiple hydrogen bond interactions by the synthesized compounds with various amino acid residues, viz, ASP-133, LYS-183, PRO-136, VAL-135, TYR-134, or LYS-60 at the GSK-3β receptor site. PMID:27406796

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

    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 γ-rays and β-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

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

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

    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...

  15. Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase-a potential target to reduce Fusarium head blight disease.

    Bresso, E; Leroux, V; Urban, M; Hammond-Kosack, K E; Maigret, B; Martins, N F

    2016-07-01

    Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain. PMID:27324634

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

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

    2003-01-01

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

  17. Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment

    Ana Martinez

    2011-01-01

    Full Text Available Glycogen synthase kinase 3 (GSK-3, a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper.

  18. Inhibitor of fatty acid synthase induced apoptosis in human colonic cancer cells

    Pei Lin Huang; Zhen Sheng Dai; Yue Lin Jin; Shi Neng Zhu; Shi Lun Lu

    2000-01-01

    @@INTRODUCTION The treatment of human epithelial malignancies is limited by drug resistance and toxic and side effects,which results in the failure in the treatment of majority of advanced cancer victims. To seek for a new, and specific antineoplastic therapy will provide hope for tumor treatment. Although disordered intermediary metabolism in cancer cells has been known for many years, much of the work focused on abnormal glucose catabolism. At the same time, little attention has been paid to fatty acid synthasis in tumor tissues, dispite of the significance of fatty acid synthase (FAS) in some clinical human ovarian[1], breast[2], colorectal[3],and prostatic cancers[4,5]. Tumor cells which express high levels of fatty acid synthesizing enzymes use endogeneously synthesized fatty acids for membrance biosynthesis and appear to export large amounts of lipid. In contrast, normal cells preferentially utilize diary lipid.

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

    Tomishima Yoshiro

    2013-01-01

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

  20. Fatty acid synthase inhibitors from plants: isolation, structure elucidation, and SAR studies.

    Li, Xing-Cong; Joshi, Alpana S; ElSohly, Hala N; Khan, Shabana I; Jacob, Melissa R; Zhang, Zhizheng; Khan, Ikhlas A; Ferreira, Daneel; Walker, Larry A; Broedel, Sheldon E; Raulli, Robert E; Cihlar, Ronald L

    2002-12-01

    Fatty acid synthase (FAS) has been identified as a potential antifungal target. FAS prepared from Saccharomyces cerevisiae was employed for bioactivity-guided fractionation of Chlorophora tinctoria,Paspalum conjugatum, Symphonia globulifera, Buchenavia parviflora, and Miconia pilgeriana. Thirteen compounds (1-13), including three new natural products (1, 4, 12), were isolated and their structures identified by spectroscopic interpretation. They represented five chemotypes, namely, isoflavones, flavones, biflavonoids, hydrolyzable tannin-related derivatives, and triterpenoids. 3'-Formylgenistein (1) and ellagic acid 4-O-alpha-l-rhamnopyranoside (9) were the most potent compounds against FAS, with IC(50) values of 2.3 and 7.5 microg/mL, respectively. Furthermore, 43 (14-56) analogues of the five chemotypes from our natural product repository and commercial sources were tested for their FAS inhibitory activity. Structure-activity relationships for some chemotypes were investigated. All these compounds were further evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. Although there were several antifungal compounds in the set, correlation between the FAS inhibitory activity and antifungal activity could not be defined. PMID:12502337

  1. Impaired learning in rats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine.

    Meyer, R C; Spangler, E L; Patel, N; London, E D; Ingram, D K

    1998-01-01

    In previous experiments, it was demonstrated that systemic or central administration of the nitric oxide synthase (NO synthase) inhibitor, NG-nitro-L-arginine (N-Arg), produced dose-dependent learning impairments in rats in a 14-unit T-maze; and that sodium nitroprusside, a NO donor, could attenuate the impairment. Since N-Arg is not specific for neuronal NO synthase and produces hypertension, it is possible that effects on the cardiovasculature may have contributed to the impaired maze performance. In the present experiment, we have investigated the maze performance of 3-4 months old male Fischer-344 rats following treatment with 7-nitroindazole, a NO synthase inhibitor that is selective for neuronal NO synthase and does not produce hypertension. In addition, we examined the effects of the NO donor, molsidomine, which is much longer acting than sodium nitroprusside. Rats were pretrained to avoid footshock in a straight runway and received training in a 14-unit T-maze 24 h later. In an initial dose-response study, rats received intraperitoneal (i.p.) injections of either 7-nitroindazole (25, 50, or 65 mg/kg) or peanut oil 30 min prior to maze training. 7-nitroindazole produced significant, dose-dependent maze acquisition deficits, with 65 mg/kg producing the greatest learning impairment. This dose of 7-nitroindazole had no significant effect on systolic blood pressure. Following the dose-response study, rats were given i.p. injections of either 7-nitroindazole (70 mg/kg) plus saline, 7-nitroindazole (70 mg/kg) plus the NO donor, molsidomine (2 or 4 mg/kg), or peanut oil plus saline as controls. Both doses of molsidomine significantly attenuated the learning deficit induced by 7-nitroindazole relative to controls. These findings represent the first evidence that impaired learning produced by inhibition of neuronal NO synthase can be overcome by systemic administration of a NO donor. PMID:9489851

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

    Monnet, Christophe; Aymes, Frédéric; Corrieu, Georges

    2000-01-01

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

  3. Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs.

    Garima Khare

    Full Text Available Tuberculosis (TB continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50 values ranging from 20-100 µg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis.

  4. Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs.

    Khare, Garima; Kar, Ritika; Tyagi, Anil K

    2011-01-01

    Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50) values ranging from 20-100 µg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99) values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99) value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis. PMID:21818324

  5. Counteraction by nitric oxide synthase inhibitor of neurochemical alterations of dopaminergic system in 6-OHDA-lesioned rats under L-DOPA treatment.

    Del-Bel, Elaine; Padovan-Neto, Fernando Eduardo; Szawka, Raphael Escorsim; da-Silva, Célia Aparecida; Raisman-Vozari, Rita; Anselmo-Franci, Janete; Romano-Dutra, Angélica Caroline; Guimaraes, Francisco Silveira

    2014-01-01

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

  6. The use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide on periapical healing

    Ali Reza Farhad; Seyed Mohammad Razavi; Parnian Alavi Nejad

    2011-01-01

    Background: Nitric oxide (NO) is one of the many chemical mediators involved in inflammatory processes. In addition to periapical inflammation, NO can have a role in periapical healing. The purpose of this study was to evaluate the effect of aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS) on the degree of healing response of periapical lesions of the canine teeth of cats. Methods: In this interventional experimental study, the root canals of 48 cat c...

  7. Aspirin protected against endothelial damage induced by LDL:role of endogenous NO synthase inhibitors in rats

    Sheng DENG; Pan-yue DENG; Jun-lin JIANG; Feng YE; Jing YU; Tian-lun YANG; Han-wu DENG; Yuan-jian LI

    2004-01-01

    AIM: To study the protective effect of aspirin on damages of the endothelium induced by low-density lipoprotein (LDL), and whether the protective effect of aspirin is related to reduction of nitric oxide synthase inhibitor level.METHODS: Vascular endothelial injury was induced by a single injection of native LDL (4 mg/kg) in rats. Vasodilator responses to acetylcholine (Ach) in the isolated aortic rings were determined, and serum concentrations of asymmetric dimethylarginine (ADMA), malondialdehyde (MDA), tumour necrosis factor-α(TNF-α), and the activity of dimethylaminohydrolase (DDAH) were measured. RESULTS: A single injection of LDL (4 mg/kg)significantly decreased vasodilator responses to Ach, increased the serum level of ADMA, MDA, and TNF-α, and decreased DDAH activity. Aspirin (30 or 100 mg/kg) markedly reduced the inhibition of vasodilator responses to Ach by LDL, and the protective effect of aspirin at the lower dose was greater compared with high-dose aspirin group. Aspirin inhibited the increased level of MDA and TNF-α induced by LDL. Aspirin at the dose of 30 mg/kg,but not at higher dose (100 mg/kg), significantly reduced the concentration of ADMA and increased the activity of DDAH. CONCLUSION: Aspirin at the lower dose (30 mg/kg) protects the endothelium against damages elicited by LDL in vivo, and the protective effect of aspirin on endothelium is related to reduction of ADMA concentration by increasing DDAH activity.

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

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

    1996-02-01

    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 effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2. AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 microM (EC50 values for NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine were 159 and > 1000 microM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may complete with L-arginine for its binding site on NOS. 3. The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4. Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5. These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of i

  9. Effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthase inhibitor

    Jun-lin JIANG; De-jian JIANG; Yu-hai TANG; Nian-sheng LI; Han-wu DENG; Yuan-jian LI

    2004-01-01

    AIM: To investigate the effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthesis inhibitor asymmetric dimethylarginine (ADMA) in rats and cultured ECV304 cells. METHODS: Endothelial injury was induced by a single injection of low density lipoprotein (LDL) (4 mg/kg, 48 h) in rats or incubation with LDL (300 mg/L) or oxidative-modified LDL (100 mg/L) in cultured ECV304 cells, and vasodilator responses to acetylcholine (ACh) in the aortic rings and the level of ADMA, nitrite/nitrate (NO) and tumor necrosis factoralpha (TNF-α) in the serum or cultured medium were determined. And the adhesion of the monocytes to endothelial cells and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the cultured ECV304 cells were measured. RESULTS: A single injection of LDL decreased endothelium-dependent relaxation to ACh, markedly increased the serum level of endogenous ADMA and TNF-α, and reduced serum level of NO. Pretreatment with simvastatin (30 or 60 mg/kg) markedly attenuated inhibition of vasodilator responses to ACh, the increased level of TNF-α and the decreased level of NO by LDL, but no effect on serum concentration of endogenous ADMA. In cultured ECV304 cells, LDL or ox-LDL markedly increased the level of ADMA and TNF-α and potentiated the adhesion of monocytes to endothelial cells, concomitantly with a significantly decrease in the activity of DDAH and serum level of NO. Pretreatment with simvastatin (0.1, 0.5, or 2.5 μmol/L) markedly decreased the level of TNFo and the adhesion of monocytes to endothelial cells, but did not affect the concentration of endogenous ADMA and the activity of DDAH. CONCLUSION: Simvastatin protect the vascular endothelium against the damages induced by LDL or ox-LDL in rats or cultured ECV304 cells, and the beneficial effects of simvastatin may be related to the reduction of inflammatory cytokine TNF-o level.

  10. OP33GLYCOGEN SYNTHASE KINASE INHIBITORS REDUCE 3D MIGRATION OF PATIENT DERIVED GLIOBLASTOMA MULTIFORME STEM CELLS

    Tams, Daniel M.; Murray, Clare; Barry, Simon T.; Lawler, Sean; Bruning-Richardson, Anke; Short, Susan

    2014-01-01

    INTRODUCTION: Glioblastoma multiforme (GBM) is a fast growing, highly invasive malignant brain tumour. Inhibition of tumour cell migration into normal brain tissue represents a major target for treatment. Glycogen synthase kinase (GSK-3) inhibition has been associated with reduced GBM invasion in in vitro and in vivo models. Targeting this pathway with established and/or novel drugs may elucidate more effective treatment combinations. METHOD: The effect of GSK-3 inhibitors BIO, AZD2858, AZ1293 and AZ1080 on GBM migration was assessed in patient derived GBM stem cells (GBM-1) and two established cell lines (U251 and U87) using a 3D collagen based assay. Multiple drug concentrations were investigated with up to 72 hours exposure. A migration index was determined using aggregate core size and cell migration area. Immunohistochemistry and immunocytochemistry were used to assess cell morphology and cytoskeletal changes. RESULTS: All compounds inhibit migration in this model. AZD2858 was the most potent, causing significant effects at 1 micro molar. All compounds were cytotoxic at between 10 and 20 micro molar. Cytoskeletal and nuclear abnormalities were noted following drug exposure in all cell lines. These data suggest that possible mechanisms for the anti-migratory effect of these compounds include effects on F-actin localization and microtubule polarity. Inhibition of migration and cell architecture changes occurred at non-toxic doses. CONCLUSION: Inhibition of GSK3 significantly reduced migration of this highly invasive tumour. It is evident from these data that inhibiting the complex biological mechanisms driven by GSK3 may aid treatment of GBM through a number of different mechanisms.

  11. Mutations in the small subunit of acetolactate synthase from Streptomyces cinnamonensis

    Kopecký, Jan; Pospíšil, Stanislav; Janata, Jiří

    SissiHeraklion: Hellenic Society of Biological Sciences, 1999. s. 30. [International Symposium on the Biology of Actinomycetes /11./. 24.10.1999-28.10.1999, Sissi-Heraklion] Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology

  12. S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid: a model for potential bioreductively activated prodrugs for inhibitors of nitric oxide synthase (NOS) activity.

    Ulhaq, S; Naylor, M A; Chinje, E C; Threadgill, M D; Stratford, I J

    1997-01-01

    Treatment of 1,1-dimethylethyl S-(2-1,1-dimethylethoxycarbonylamino)-5-bromopentanoate with 1-potassio-2-nitroimidazole, followed by deprotection, afforded S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid, which was reduced to S-2-amino-5-(2-aminoimidazol-1-yl)pentanoic acid. This aminoimadazole inhibited rat brain nitric oxide synthase (NOS) activity 3.2 times more potently than did the nitro analogue. Thus S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid is a potent prodrug which may be bioreductively activated to a NOS inhibitor in hypoxic solid tumours. PMID:9051114

  13. Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase.

    Fishlock, Dan; Perdicakis, Basil; Montgomery, Heather J; Guillemette, J Guy; Jervis, Eric; Lajoie, Gilles A

    2003-03-20

    Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity. PMID:12614872

  14. Studies on the Compounds of d4T Combined with Nitric Oxide Donors and Nitric Oxide Synthase Inhibitors and their Anti-HIV and AIDS Activity

    KWALE MOLIME GUITREMBI Blaise(Central African); YAO Qi-zheng

    2004-01-01

    Stavudine, a potent anti-HIV and AiDS-related complex, is one of the Nucleoside Analogue Reverse Transcriptase Inhibitors (NARTIs). It is phosphorylated intracellularly and then inhibits the viral reverse transcriptase by acting as a false substrate. Modifications made on the hydrogen labile at the 5'-position on the sugar is an interesting template for the elaboration of new potent anti-HIV and AIDS drugs. The expected advantages of the modified stavudine prodrugs can be multiple: synergistic drug activities, enhancement of stavudine intracellular uptake, increase of stavudine brain delivery, and bypass of the first stavudine phosphorylation step into the cells. Nitric oxide synthase inhibitors of stavudine and nitric oxide donors of stavudine may hold significant promise for the treatment of HIV and AIDS.

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

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

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

    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

    Roč. 124, č. 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

  17. Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

    Bortolanza, Mariza; Cavalcanti-Kiwiatkoski, Roberta; Padovan-Neto, Fernando E; da-Silva, Célia Aparecida; Mitkovski, Miso; Raisman-Vozari, Rita; Del-Bel, Elaine

    2015-01-01

    l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinson's disease. PMID:25447229

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

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

    2015-01-01

    A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the ac...

  19. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β. PMID:26610618

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

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

    2013-08-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

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

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

    1999-05-01

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

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

    Highlights: •EV-077 reduced TNF-α 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 TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα 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 TNFα-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

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

    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-α 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 TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα 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 TNFα-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.

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

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

  5. The effect of nitric oxide synthase inhibitors nitro-L-arginine and 7-nitroindazole on spatial learning and motor functions in Lurcher mutant and wild type mice.

    Markvartová, V; Vozeh, F

    2008-01-01

    Nitric oxide (NO) is an intercellular messenger that, among other things, plays an important role in the nervous system as a gaseous neurotransmitter, modulating long-term potentiation (LTP) induction of synaptic transmission. LTP has been suggested to be the basis of memory formation. On the other hand NO also participates in excitotoxic processes which play an important role in many neuropathological states. The aim of this work was to observe the effect of two NO synthase (NOS) inhibitors (N omega-Nitro-L-arginine, NA; 7-nitroindazole, NI) on spontaneous behaviour, spatial learning and motor functions in Lurcher (+/Lc) and wild type (+/+) mice, derived from the B6CBA strain. Heterozygous Lurcher mutant mice represent a natural model of the olivocerebellar degeneration. They suffer from postnatal, practically total, extinction of cerebellar Purkinje cells (due to the excitotoxic apoptosis) and a partial decrease of granule cells and inferior olive neurons (ION) because of the lost target of their axons. +/+ animals are healthy littermates of +/Lc. NA is a nonselective NOS inhibitor which influences, except neuronal (n), also endothelial (e) NOS with an impact on blood pressure, NI is a selective nNOS inhibitor without any circulatory effect. The adult animals of both types (+/Lc; +/+) were influenced by acute administration of both inhibitors (25 mg/kg i.p. 30 min. before experiments) and newborns only by both acute and long-term administration of NI (1 month, starting from postnatal day 2, P2). Control solutions - saline or solvents of both NA and NI inhibitors--diluted 1M HCl and dimethyl sulfoxide (DMSO) respectively, were given at a relevant volume in the same way. The effect of both inhibitors and control solutions on motor functions was tested using four standard procedures (horizontal wire, slanting ladder, rotating cylinder, foot-bridge); in newborns at the age of 14 days. Spatial learning ability was examined in five-day long procedure in the Morris

  6. Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.

    Choi, Kyoung-Jae; Yu, Yeon Gyu; Hahn, Hoh Gyu; Choi, Jung-Do; Yoon, Moon-Young

    2005-08-29

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics. PMID:16111681

  7. The use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide on periapical healing

    Farhad, Ali Reza; Razavi, Seyed Mohammad; Nejad, Parnian Alavi

    2011-01-01

    Background: Nitric oxide (NO) is one of the many chemical mediators involved in inflammatory processes. In addition to periapical inflammation, NO can have a role in periapical healing. The purpose of this study was to evaluate the effect of aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS) on the degree of healing response of periapical lesions of the canine teeth of cats. Methods: In this interventional experimental study, the root canals of 48 cat canine teeth were infected with cat dental plaque and sealed. After induction of periapical lesions, root canal therapy (RCT) was performed. On the day of RCT phase, the cats were administered either AG (experimental group) or normal saline (control group), which was continued on a daily basis until the day of sacrifice. Four canine teeth in one cat served as negative and positive controls. The animals were sacrificed 6 weeks after RCT. The healing response of the periapical zones was analyzed histologically. The mean scores of healing for the two groups were compared using Mann–Whitney U test. Results: The mean scores of healing for the AG group (2.45±0.508) were significantly higher than those of the control group (2±0.510) (P<0.05). Conclusion: The use of an iNOS selective inhibitor such as AG can accelerate the healing process in periapical lesions. PMID:22135691

  8. Identification of novel membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitors with anti-influenza activities in vitro.

    Park, Ji Hoon; Park, Eun Beul; Lee, Jae Yeol; Min, Ji-Young

    2016-01-22

    Influenza A virus (IAV) is a major public health concern that leads to high morbidity and mortality worldwide. Despite various vaccination programs and development of drugs targeting essential viral proteins, the emergence of drug-resistant variants has been frequently reported and the therapeutic options are limited. Because exaggerated inflammation is considered as an important factor in disease pathogenesis, immunomodulatory agents that effectively suppress cytokine responses are needed for the treatment of IAV infection. Membrane-associated prostaglandin E synthase-1 (mPGES-1) is an enzyme responsible for the production of prostaglandin E2 (PGE2) that is the best-characterized immune modulatory lipid in vitro and in vivo models of inflammation. In the present study, we tested the anti-influenza activities of mPGES-1 inhibitors, using a phenotype-based assay involving image analyses. Seven primary hits among 49 compounds targeting mPGES-1 exhibited anti-influenza activities against A/Puerto Rico/8/1934 (H1N1) in a dose-dependent manner. The most effective hit, MPO-0047, suppressed influenza-induced p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) activation. We also showed that mRNA levels of TNF-α, IL-8, CCL5/RANTES, and CXCL10/IP-10 were significantly reduced by the treatment of influenza-infected cells with MPO-0047. Exogenous PGE2 reversed the inhibitory effects of MPO-0047. Our results showed that this selective mPGES-1 inhibitor has anti-influenza effects by inhibiting PGE2 production, which suppresses the induction of pro-inflammatory genes. Taken together our data revealed that mPGES-1 inhibitor has the potential for further development as an influenza therapeutic agent. PMID:26673392

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

    Xian-Cang Ma

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

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

    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 Å 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-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

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

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

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

    Darmani, Homa; Crossan, James C; Curtis, Adam

    2004-06-01

    The aim of the current study was to investigate the effect of inhibition of nitric oxide (NO) production after injury on inflammatory cell accumulation and fibrosis around digital flexor tendon and synovium. A standard crush injury was applied to the flexor tendons of the middle digit of the hindpaw and the overlying muscle and synovium of female Wistar rats. Thirty animals received an intraperitoneal injection of either isotonic saline or N(G)-nitro-l-arginine methyl ester (L-NAME; 5 mg/kg) immediately following the crush injury, and five animals were then sacrificed at various intervals and the paws processed for histology. Another group of five animals was sacrificed after 3 days for nitrite determinations. The results showed that nitrite production and hence NO synthase activity is doubled at the acute phase of tendon wound healing, and we can prevent this by administering a single dose of L-NAME immediately after injury. The incidence and severity of fibrocellular adhesions between tendon and synovium was much more marked in animals treated with L-NAME. Treatment with L-NAME elicited a chronic inflammatory response characterised by a persistent and extraordinarily severe accumulation of large numbers of inflammatory cells in the subcutaneous tissues, in muscle and in tendon. These findings indicate that in the case of injured tendon and synovium, NO could act to protect the healing tissue from an uncontrolled inflammatory response. PMID:15223606

  13. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

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

    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/μCT imaging. GSK-3 inhibitors caused β-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/μCT. 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

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

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

    2013-10-15

    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/μCT imaging. GSK-3 inhibitors caused β-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/μCT. 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

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

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

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

    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

  18. Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production.

    Geng, Feng; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-03-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of L-lysine biosynthesis in bacteria and plants and is allosterically regulated by L-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to L-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the L-lysine-sensitive DHDPS from Escherichia coli and L-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of L-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of L-lysine production yield by 46 %. PMID:22644522

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

    Zhao Xuan

    2013-01-01

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

  20. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

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

    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

  2. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    2010-04-01

    ... requirements for enzyme preparations in the Food Chemicals Codex, 4th ed., 1996, pp. 133-134, which is... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alpha-acetolactate decarboxylase (α-ALDC)...

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

    Dailey, F E; Cronan, J E

    1986-01-01

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

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

    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.

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

    L. A. RODRIGUES

    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, Nω-Nitro-L-Arginine (NωNLA. 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; NωNLA Escherichia coli endotoxin.

  6. Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

    Li-Bo Yu; Xin-Shu Dong; Wen-Zhou Sun; Dong-Lu Zhao; Yue Yang

    2005-01-01

    AIM: To investigate the effect and mechanism of action of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on invasion and metastasis of human colorectal cancer cell line SL-174T.METHODS: Human colorectal cancer cell line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h. Nitric oxide (NO) production was measured with Griess reagent. The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel).RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor metalloproteinase-2 (TIMP-2).RESULTS: L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L LNAME, respectively, the ability of the L-NAME treated SL174T cells to invade the reconstituted basement membrane decreased significantly (t = 8.056, P<0.05;t= 14.467, P<0.01;t= 27.785, P<0.01;and t= 29.405,P<0.01, respectively) and the inhibition rates were 10.29%,19.62%, 34.08%, and 42.23%, respectively. Moreover,L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46.85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L,respectively (t = 15.116, P<0.01). In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased (t = 71.238, P<0.01) and that of TIMP-2 mRNA was markedly increased (t = -13.020,P<O.01).CONCLUSION: L-NAME exerts anti-invasive and antimetastatic effects on SL-174T cell line via downregulating MMP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

  7. Inhibitors

    ... wrong place in the body. Immune Tolerance Induction (ITI) Therapy: The goal of ITI therapy is to stop the inhibitor reaction from ... body to accept clotting factor concentrate treatments. With ITI therapy, people receive large amounts of clotting factor ...

  8. "Zipped Synthesis" by Cross-Metathesis Provides a Cystathionine β-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model.

    McCune, Christopher D; Chan, Su Jing; Beio, Matthew L; Shen, Weijun; Chung, Woo Jin; Szczesniak, Laura M; Chai, Chou; Koh, Shu Qing; Wong, Peter T-H; Berkowitz, David B

    2016-04-27

    The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a "zipped" approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C 2-symmetric CBS product (l,l)-cystathionine. The "zipped" concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine-imine interchange. It is demonstrated that the most potent "zipped" inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia. PMID:27163055

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

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

  10. 1400W, a highly selective inducible nitric oxide synthase inhibitor is a potential disease modifier in the rat kainate model of temporal lobe epilepsy.

    Puttachary, Sreekanth; Sharma, Shaunik; Verma, Saurabh; Yang, Yang; Putra, Marson; Thippeswamy, Achala; Luo, Diou; Thippeswamy, Thimmasettappa

    2016-09-01

    Status epilepticus (SE) initiates epileptogenesis to transform normal brain to epileptic state which is characterized by spontaneous recurrent seizures (SRS). Prior to SRS, progressive changes occur in the brain soon after SE, for example, loss of blood-brain barrier (BBB) integrity, neuronal hyper-excitability (epileptiform spiking), neuroinflammation [reactive gliosis, high levels of reactive oxygen/nitrogen species (ROS/RNS)], neurodegeneration and synaptic re-organization. Our hypothesis was that modification of early epileptogenic events will alter the course of disease development and its progression. We tested the hypothesis in the rat kainate model of chronic epilepsy using a novel disease modifying drug, 1400W, a highly selective inhibitor of inducible nitric oxide synthase (iNOS/NOS-II). In an in vitro mouse brain slice model, using a multi-electrode array system, co-application of 1400W with kainate significantly suppressed kainate-induced epileptiform spiking. In the rats, in vivo, 4h after the induction of SE with kainate, 1400W (20mg/kg, i.p.) was administered twice daily for three days to target early events of epileptogenesis. The rats were subjected to continuous (24/7) video-EEG monitoring, remotely, for six months from epidurally implanted cortical electrodes. The 1400W treatment significantly reduced the epileptiform spike rate during the first 12-74h post-SE, which resulted in >90% reduction in SRS in long-term during the six month period when compared to the vehicle-treated control group (257±113 versus 19±10 episodes). Immunohistochemistry (IHC) of brain sections at seven days and six months revealed a significant reduction in; reactive astrogliosis and microgliosis (M1 type), extravascular serum albumin (a marker for BBB leakage) and neurodegeneration in the hippocampus, amygdala and entorhinal cortex in the 1400W-treated rats when compared to the vehicle control. In the seven day group, hippocampal Western blots revealed downregulation of

  11. Pulmonary hypertension triggered by lipopolysaccharide in ascites-susceptible and -resistant broilers is not amplified by aminoguanidine, a specific inhibitor of inducible nitric oxide synthase.

    Bowen, O T; Erf, G F; Anthony, N B; Wideman, R F

    2006-03-01

    Nitric oxide (NO) is a potent pulmonary vasodilator that modulates the pulmonary vasoconstriction and pulmonary hypertension (PH) triggered by bacterial lipopolysaccharide (LPS) in broilers. The amplitude and duration of the LPS-induced PH are markedly enhanced following pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME), which inhibits NO synthesis by both the constitutive (endothelial) and inducible (inflammatory) forms of nitric oxide synthase (eNOS and iNOS, respectively). In the present study L-NAME and the selective iNOS inhibitor aminoguanidine (AG) were administered to differentiate between iNOS and eNOS as the primary source of NO that attenuates the pulmonary vascular response to LPS. Clinically healthy male progeny from ascites-susceptible and ascites-resistant lines were anesthetized, and their pulmonary artery was cannulated. The initial pulmonary arterial pressure (PAP) was recorded, then the broilers either remained untreated (control group) or were injected i.v. with AG. Ten minutes later all birds received an i.v. injection of LPS, followed 40 min later by an i.v. injection of L-NAME. When compared with untreated controls, AG neither increased the baseline PAP nor did it increase or prolong the PH response to LPS. The ascites-susceptible broilers maintained a higher PAP than the ascites-resistant broilers throughout the experiment, and the ascites-resistant broilers exhibited greater relative increases in PAP in response to LPS than did the ascites-susceptible broilers. Within 40 min after the LPS injection, PAP subsided to a level that did not differ from the respective preinjection value for each line. Injecting L-NAME reversed the decline in PAP, and within 5 min PAP returned to hypertensive levels approaching the maximum peak PH response to LPS. The absence of any impact of AG coupled with the profound response to L-NAME indicates that NO synthesized by eNOS rather than iNOS likely modulated the acute (within 1 h) PH elicited by

  12. IL-1beta-Induced iNOS Expression, NO Release and Loss in Metabolic Cell Viability Are Resistant to Inhibitors of Ceramide Synthase and Sphingomyelinase in INS 832/13 Cells

    Rajakrishnan Veluthakal

    2006-11-01

    Full Text Available Context Emerging evidence indicates regulatory roles for ceramide in the metabolic dysfunction of the islet beta cell. Recently, potential similarities between IL-1beta and ceramide on their effects on islet beta cell have been reported, including reduction in mitochondrial membrane potential and loss in metabolic cell viability.Objective Herein, we investigated whether IL-1beta-induced nitric oxide synthetase (iNOS expression, nitric oxide (NO release and loss in metabolic cell viability require ceramide biosynthesis either via the activation of sphingomyelinase or ceramide synthase.Setting Insulin-secreting INS 832/13 cells.Results We found that two structurally-distinct inhibitors of sphingomyelinase activation (e.g., 3-O-methylsphingomyelin or desipramine or ceramide biosynthesis inhib-itor (e.g., fumonisin failed to exert clear effects on IL-1beta-induced iNOS expression, NO release and loss in cell viability.Conclusions Taken together, our findings indicate that neither the sphingomyelinase nor the ceramide synthase activation is required for IL-1beta-induced metabolic abnormalities in insulin-secreting INS 832/13 cells.

  13. Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

    Shen, Xiaoning; Liu, Dong; Liu, Jun; Wang, Yanyan; Xu, Jiahui; Yang, Zhengjiao; Guo, Ting; Niu, Huanqing; Ying, Hanjie

    2016-09-01

    Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production. PMID:27285575

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

    Graphical abstract: - Abstract: Cisplatin is used in the treatment of a wide variety of solid tumors, but its use is limited by its serious adverse effects, including ototoxicity. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions by phosphorylating its substrates. However, the otoprotective effect of GSK-3 inhibitors is poorly understood. Here, we investigated whether GSK-3 is involved in cisplatin-induced ototoxicity in HEI-OC1 cells and organs of Corti (OCs). GSK-3 inhibitors suppressed cisplatin-induced apoptosis determined by decreased p53 activity, and also decreased expression of PARP and p53 target genes such as p21 and PUMA. The effect of GSK-3 inhibitors was mediated by markedly increased nuclear β-catenin that in turn blocked nuclear translocation of NF-κB. siRNA-mediated β-catenin knockdown markedly increased the expression of NF-κB target genes, such as TNF-α and IL-6. Our data suggest that the GSK-3/β-catenin pathway may play a central role in cisplatin-mediated cytotoxicity in HEI-OC1 cells and hair cells of OCs in vitro

  15. Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

    Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

    2015-11-25

    Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders. PMID:26486317

  16. Benzalacetone Synthase

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

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

    Brožíčková, Carole; Otáhal, Jakub

    2013-01-01

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

  18. Inducible nitric oxide synthase and inflammation.

    Salvemini, D; Marino, M H

    1998-01-01

    Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications. PMID:15991919

  19. Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

    Jennifer M Atkinson

    Full Text Available It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment in contrast to other solid tumor cell lines (IC50 >10 uM as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

  20. Highly Efficient Synthesis of Two Hyaluronan Trisaccharide Analogues for Potential Hyaluronic Acid Synthases Inhibitors%透明质酸三糖模拟物的高效合成

    魏国华; 杜宇国; Khushi L. Matta

    2009-01-01

    The syntheses of two hyaluronan trisaccharide analogues, naphthyl 0-(3-methoxy-B-D-glucopy-ranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-B-D-glucopyranosyl)-(1,4)-0-B-D-glucopyranosyluronic acid and naphthyl O-(3-methoxy-2-acetamido-2-deoxy-B-D-glucopyranosyl)-(1,4)-O-(B-D-glucopyranosylu-ronic acid)-(1,3 )-O-2-acetamido-2-deoxy-B-D-glucopyranoside, were described. Construction of the target molecules was achieved through a combination of BF_3·Et_2O/toluene system and trichloroacetimidate glycosyia-tion methodology. This is the first report on the synthesis of the 3-methoxyl derivatives, which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan and can be used for potential hyaluronic acid synthases inhibitors.%设计合成了2个透明质酸(HA)模拟物1和2, 通过最小基团MeO的引入修饰, 模拟天然HA片段的特性, 用于透明质酸合成酶(HAS)催化机理与抑制剂的研究.

  1. [Heme oxygenase activity in the tissues of the vessels and heart of rats under co-administration of NO-synthase inhibitor and hemin chloride].

    Kaliman, P A; Filimonenko, V P; Nikitchenko, I V

    2008-01-01

    The administration of hemin chloride in a dose of 1.5 mg/100 g of the body weight was found to cause accumulation of the total heme and TBA-reactive products in the rat blood serum and vessels. Pretreatment by N(omega)-nitro-L-arginine (0.5 h before hemin chloride administration) did not affect the dynamics of the total heme and TBA-reacting products accumulation. The increase of heme oxygenase activity was observed in the vessels after hemin chloride administration. This effect was strengthened by N(omega)-nitro-L-arginine pretreatment. The changes of heme oxygenase activity and the total heme level in heart were not observed at any periods studied. The increase of the TBA-reactive products level in the heart after exogenous hemin injection was accompanied by an increase of nitrites content and blocked by pretreatment of NOS inhibitor. The N(omega)-nitro-L-arginine alone caused the accumulation of the total heme, TBA-reacting products and the increase of heme oxygenase activity in the vessels. The role of heme and NO in regulation of the heme oxygenase activity is discussed. PMID:18819384

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

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

    1999-01-01

    This study examined whether a prior electrolytic lesion of the tissue surrounding the anteroventral third ventricle (AV3V) would affect the increase in mean arterial blood pressure (MAP) and the fall in heart rate (HR) produced by systemic injection of the nitric oxide synthesis (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 25 micromol/kg, i.v.) in conscious rats. L-NAME produced a smaller increase in MAP in AV3V-lesion than in sham-lesion rats (+19+/-3 vs. +40+/-3 mmHg, respectively; P<0.05). In contrast, L-NAME produced similar falls in HR in the AV3V-lesion and sham-lesion rats (-103+/-15 vs. -97+/-8 bpm, respectively; P<0.05). These findings demonstrate that the L-NAME-induced pressor response is dependent upon the integrity of the AV3V region, whereas the L-NAME-induced bradycardia is not. Copyright 1999 Elsevier Science B. V.

  3. Nitric Oxide Synthase Inhibitors as Antidepressants

    Wegener, Gregers; Volke, Vallo

    2010-01-01

    , 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....

  4. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  5. An Arabidopsis callose synthase

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

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......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...

  6. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    Tanaka, Hiroaki, E-mail: tanakah@confsci.co.jp [Confocal Science Inc. (Japan); Tsurumura, Toshiharu; Aritake, Kosuke [Osaka Bioscience Institute (Japan); Furubayashi, Naoki [Maruwa Foods and Biosciences Inc. (Japan); Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika [Confocal Science Inc. (Japan); Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo [Japan Aerospace Exploration Agency (Japan); Inaka, Koji [Maruwa Foods and Biosciences Inc. (Japan); Urade, Yoshihiro [Osaka Bioscience Institute (Japan)

    2011-01-01

    Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality. Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

  7. In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of This Activity: Unexpected Influence of the Tetrachloroplatinate(II) Counteranion. Crystal Structures of Bis(S-Methyl-Isothiouronium)-N,N'-Bis(3-Guanidinopropyl)Piperazinium and Hexamidinium Tetrachloroplatinates(II) Salts.

    Morgant, G; Viossat, B; Roch-Arveiller, M; Prognon, P; Giroud, J P; Lancelot, J C; Robba, M; Huy, D N

    1998-01-01

    The synthesis and crystal structures of bis(S-methylisothiouronium) (MSTUH)(+), N,N'-bis((3- guanidinopropyl)piperazinium (PipeC3GuaH4)(4+) and hexamidinium (HexaH2)(2+) tetrachloro platinate(ll) salts ( called hereafter PtMSTU, PtPipeC3Gua and PtHexa respectively ) were investigated. These compounds contain the "amidine" function ( - C(=NH)NH(2) ) in which the H atoms supplied by the acid have become attached to the imino group of each terminal amidino function. Moreover, in PtPipeC3Gua, the nitrogen atoms of the chair-piperazine moiety are also protonated. The influence of tetrachloroplatinate(ll) counteranion ( versus sulfate, nitrate and diisethionate ) in the in vivo nitrite inhibition by the (MSTUH)(+), (PipeC3GuaH4)(4+) and (HexaH2)(2+) cations was investigated. The three tetrachloroplatinate(ll) salts, unexpectedly, do not inhibit significantly the in vivo nitrite production in comparison with the other salts (sulfate, nitrate and diisethionate and their corresponding previous countercations) which exhibit NO synthase inhibition, especially bis(S-methylisothiouronium) sulfate, a selective and potent inducible NO synthase (iNOS) inhibitor commonly used as standard. PMID:18475834

  8. Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase; Sintese e modificacoes de derivados heterociclicos de d-arabinose: potenciais inibidores de glicose-6-fosfato isomerase e de glicosamina-6-fosfato sintase

    Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Fac. de Farmacia. Dept. de Produtos Farmaceuticos]. E-mail: ricardodylan@farmacia.ufmg.br

    2008-07-01

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

  9. Geranyl diphosphate synthase from mint

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    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.

  10. Geranyl diphosphate synthase from mint

    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.

  11. Tapentadol and nitric oxide synthase systems.

    Bujalska-Zadrożny, Magdalena; Wolińska, Renata; Gąsińska, Emilia; Nagraba, Łukasz

    2015-04-01

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

  12. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    Skov, Søren; Pedersen, Marianne Terndrup; Andresen, Lars; Straten, Per Thor; Woetmann, Anders; Odum, Niels

    2005-01-01

    We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...

  13. Hybrid polyketide synthases

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  14. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  15. Monoterpene synthases from common sage (Salvia officinalis)

    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.

  16. Triazolopyrimidines as a New Herbicidal Lead for Combating Weed Resistance Associated with Acetohydroxyacid Synthase Mutation.

    Liu, Yu-Chao; Qu, Ren-Yu; Chen, Qiong; Yang, Jing-Fang; Cong-Wei, Niu; Zhen, Xi; Yang, Guang-Fu

    2016-06-22

    Acetohydroxyacid synthase (AHAS; also known as acetolactate synthase; EC 2.2.1.6, formerly EC 4.1.3.18) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. Weed resistance to AHAS-inhibiting herbicides, increasing at an exponential rate, is becoming a global problem and leading to an urgent demand of developing novel compounds against both resistant and wild AHAS. In the present work, a series of novel 2-aroxyl-1,2,4-triazolopyrimidine derivatives (a total of 55) were designed and synthesized with the aim to discover an antiresistant lead compound. Fortunately, the screening results indicated that many of the newly synthesized compounds showed a better, even excellent, inhibition effect against both the wild-type Arabidopsis thaliana AHAS and P197L mutants. Among them, compounds 5-3 to 5-17, compounds 5-19 to 5-26, compounds 5-28 to 5-45, and compound 5-48 have the lower values of resistance factor (RF) and display a potential power to overcome resistance associated with the P197L mutation in the enzyme levels. Further greenhouse in vivo assay showed that compounds 5-15 and 5-20 displayed "moderate" to "good" herbicidal activity against both the wild type-and the resistant (P197L mutation) Descurainia sophia, even at a rate as low as 0.9375 (g of ai/ha). The above results indicated that these two compounds could be used as new leads for the future development of antiresistance herbicides. PMID:27265721

  17. Ectopic ATP synthase in endothelial cells: a novel cardiovascular therapeutic target.

    Fu, Yi; Zhu, Yi

    2010-01-01

    Adenosine triphosphate (ATP) synthase produces ATP in cells and is found on the inner membrane of mitochondria or the cell plasma membrane (ectopic ATP synthase). Here, we summarize the functions of ectopic ATP synthase in vascular endothelial cells (ECs). Ectopic ATP synthase is involved in adenosine metabolism on the cell surface through its ATP generation or hydrolysis activity. The ATP/ADP generated by the enzyme on the plasma membrane can bind to P2X/P2Y receptors and activate the related signalling pathways to regulate endothelial function. The β-chain of ectopic ATP synthase on the EC surface can recruit inflammatory cells and activate cytotoxic activity to damage ECs and induce vascular inflammation. Angiostatin and other angiogenesis inhibitors can have anti-angiogenic functions by inhibiting ectopic ATP synthase on ECs. Moreover, ectopic ATP synthase on ECs is a receptor for apoA-I, the acceptor of cholesterol efflux, which implies that endothelial ectopic ATP synthase is involved in cholesterol metabolism. Coupling factor 6 (CF6), a part of ectopic ATP synthase, is released from ECs and can inhibit prostacyclin synthesis and promote nitric oxide (NO) degradation to enhance NO bioactivity. Because ATP/ADP generated by ectopic ATP synthase can induce NO production, substances such as CF6 can inhibit NO generation by inhibiting surface ATP/ADP production. Thus, the components of ectopic ATP synthase are associated with regulation of vascular tone. Through these functions, ectopic ATP synthase on ECs is considered a potential and novel therapeutic target for atherosclerosis, hypertension and lipid disorders. PMID:21247400

  18. Prenyldiphosphate synthases and gibberellin biosynthesis

    C.C.N. van Schie; M.A. Haring; R.C. Schuurink

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  19. Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin*

    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 β-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...

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

    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...

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

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

    2002-01-01

    Background Previous work has suggested that in the liver, adenosine preconditioning is mediated by nitric oxide. Whether the endothelial isoform of nitric oxide synthase plays a part in this mechanism has however not yet been investigated. Methods Wistar rats were used (6 in each group) – Groups: (1) sham, (2) ischemia-reperfusion, (3) adenosine + ischemia-reperfusion, (4) endothelial isoform inhibitor + adenosine + ischemia-reperfusion. Results Using immunohistochemistry, this study has revealed a decrease in the expression of endothelial nitric oxide synthase following hepatic ischemia-reperfusion. This was prevented by adenosine pre-treatment. When an inhibitor of endothelial nitric oxide synthase was administered prior to adenosine pre-treatment, pre-conditioning did not occur despite normal expression of endothelial nitric oxide synthase. Conclusions These findings suggest that adenosine attenuates hepatic injury by preventing the downregulation of endothelial nitric oxide synthase that occurs during ischemia-reperfusion. PMID:12241560

  2. Synthesis of Novel Oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles as Glycogen Synthase Kinase-3β Inhibitors with Anti-inflammatory Potential.

    Tantray, Mushtaq A; Khan, Imran; Hamid, Hinna; Alam, Mohammad Sarwar; Umar, Sadiq; Ali, Yakub; Sharma, Kalicharan; Hussain, Firasat

    2016-06-01

    A novel series of oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles has been synthesized by click chemistry approach and evaluated for in vitro GSK-3β inhibitory activity. Compound 4g showed maximum inhibition with IC50 value of 0.19 μm. Keeping in view the effect of GSK-3β inhibition on inflammation, compounds 4g, 4d, 4f, 4i, 4n and 4q exhibiting significant GSK-3β inhibition were examined for in vivo anti-inflammatory activity in rat paw edema model. The compounds 4g, 4d, 4f and 4i showed pronounced in vivo anti-inflammatory activity (76.36, 74.54, 72.72 and 70.90%, respectively, after 5h post-carrageenan administration) and were further found to inhibit the pro-inflammatory mediators, viz. NO, TNF-α, IL-1β, and IL-6 substantially in comparison with indomethacin, an anti-inflammatory drug as well as SB216763, a GSK-3β inhibitor, reported to exert a similar effect. Histopathology studies confirmed the tolerance of gastric mucosa to these compounds. PMID:26804375

  3. Cellulose synthase complexes: structure and regulation

    Lei eLei

    2012-04-01

    Full Text Available This review is to update the most recent progress on characterization of the composition, regulation, and trafficking of cellulose synthase complexes. We will highlight proteins that interact with cellulose synthases, e.g. cellulose synthase-interactive protein 1 (CSI1. The potential regulation mechanisms by which cellulose synthase interact with cortical microtubules in primary cell walls will be discussed.

  4. Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region.

    Kulmacz, R J

    1989-08-25

    Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these

  5. 新型苯并硫氮杂(卓)酮类非ATP竞争GSK-3β抑制剂的设计、合成和活性评价%Design, Synthesis and in Vitro Test of Novel Non-ATP Competitive Glycogen Synthase Kinase-3β(GSK-3β)Inhibitors

    黄朝辉; 胡海荣; 雷贾毅; 楚勇; 叶德泳

    2012-01-01

    OBJECTIVE To discover novel non-ATP competitive glycogen synthase kinase-3P(GSK-3P) inhibitors. METHODS A virtual screening was conducted by Autodock program, which docked the small drug-like molecules of Maybridge library at the non-ATP binding site of GSK-3β The target compounds had been designed based on the virtual screening result and successfully synthesized through Knoevenagel reaction, cyclization and Af-alkylation. The inhibition to GSK-3P was tested by in vitro enzamic test. RESULTS 5-benzyl-2-(furan-2-yl)-2,3-dihydrobenzo[b][l,4] thiazepin-4(5H)-one showed moderate inhibition to GSK-3P in vitro (IC50 47.69±2.38 μmol·L-1). CONCLUSION The discovered new active compound is structurally different to other inhibitors of GSK-3P and worthy of further study as a novel lead compound.%目的 寻找新型的非ATP竞争糖原合成酶激酶-3β(GSK-3β)抑制剂.方法 针对GSK-3β的非ATP结合的底物作用位点为靶点,采用Autodock程序对类药性小分子库Maybridge进行虚拟筛选寻找新型GSK-3β抑制剂.采用克脑文格尔反应,环合及N-烷基化反应制备目标化合物.采用体外酶抑制活性测试目标化合物的活性.结果 化合物2-(2-呋喃基)-5-苄基-2,3-二氢苯并[b][1,4]硫氮杂(卓)-4(5H)-酮对GSK-3β具有中等抑制活性(IC50 47.69±2.38 μmol·L-1).结论 活性化合物的结构与目前报道的其他GSK-3β抑制剂不同,可望作为新的先导化合物,值得进一步研究.

  6. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  7. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    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.

  8. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein. PMID:21169700

  9. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2010-01-01

    Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

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

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

    2011-11-17

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

  11. HYPOTHALAMIC BLOOD-FLOW REMAINS UNALTERED FOLLOWING CHRONIC NITRIC-OXIDE SYNTHASE BLOCKADE IN RATS

    BENYO, Z; SZABO, C; STUIVER, BT; BOHUS, B; SANDOR, P

    1995-01-01

    The effect of the chronic oral application of N-G-nitro-L-arginine methyl eater (L-NAME), a potent inhibitor of nitric oxide (NO) production, was studied on hypothalamic blood flow (HBF) and hypothalamic nitric oxide synthase (NOS) activity in rats. L-NAME was dissolved in the drinking water, in a c

  12. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs. PMID:26976449

  13. Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

    Hui LU; Bing Zhu; Xin-Dong Xue

    2006-01-01

    AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue.METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (LPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined.RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury.CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

  14. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

    Grundy, Daniel J; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J; Christianson, David W; Allemann, Rudolf K

    2016-04-12

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues

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

    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

  16. Producing biofuels using polyketide synthases

    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.

  17. Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

    Cai, G.; Faleri, C.; Casino, C.; Emons, A.M.C.; Cresti, M.

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tub

  18. Properties of phosphorylated thymidylate synthase

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Pawel; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    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, Trichin......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 m......RNAs (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 Fd...

  19. Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

    Roberta d'Emmanuele di Villa Bianca

    Full Text Available Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

  20. Nitric Oxide synthases and atrial fibrillation

    CynthiaAnnCarnes

    2012-04-01

    Full Text Available Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases, which normally produce nitric oxide in the heart. Two nitric oxide synthase isoforms (1 and 3 are normally expressed in the heart. During pathologies such as heart failure, there is induction of nitric oxide synthase 2 in multiple cell types in the myocardium. In certain conditions, the NOS enzymes may become uncoupled, shifting from production of nitric oxide to superoxide anion, a potent free radical and oxidant. Multiple lines of evidence suggest a role for nitric oxide synthases in the pathogenesis of atrial fibrillation. Therapeutic approaches to reduce atrial fibrillation by modulation of nitric oxide synthase activity may be beneficial, although further investigation of this strategy is needed.

  1. Threonine phosphorylation of rat liver glycogen synthase

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

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

    Beurel, Eléonore; Kaidanovich-Beilin, Oksana; Yeh, Wen-I; Song, Ling; Palomo, Valle; Michalek, Suzanne M.; Woodgett, James R.; Harrington, Laurie E.; Eldar-Finkelman, Hagit; Martinez, Ana; Jope, Richard S.

    2013-01-01

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

  3. Selective inhibition of inducible nitric oxide synthase by derivatives of acetamidine.

    Maccallini, Cristina; Patruno, Antonia; Ammazzalorso, Alessandra; De Filippis, Barbara; Fantacuzzi, Marialuigia; Franceschelli, Sara; Giampietro, Letizia; Masella, Simona; Tricca, Maria Luisa; Amoroso, Rosa

    2012-11-01

    A new series of phenyl- and heteryl acetamidines were synthesized and evaluated as inhibitors of nitric oxide synthases (NOS). While the N-substitution of the acetamidine moiety with different heterocycles appears to completely destroy the activity, linking the phenyl core preserves it. Moreover, it was observed a strong dependence of the phenylacetamidines potency of action from the length of the alkyl chain that connects the aromatic ring to the acetamidine moiety. PMID:22741778

  4. Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

    Patel, S.; Gaspers, L. D.; Boucherie, S.; Memin, E.; Stellato, K. A.; Guillon, G; Combettes, L; Thomas, A P

    2002-01-01

    Increases in both Ca2+ and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca2+ signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mim...

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

    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

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

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger; Harholt, Jesper;

    2007-01-01

    Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are...... unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module......, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed....

  7. Crystal structure of riboflavin synthase

    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.

  8. Effects of nitric oxide synthase inhibitor in two-week oral treatment on hyperdynamic circulatory state in cirrhotic rats%一氧化氮合酶抑制剂口服两周治疗对肝硬化大鼠高动力循环状态的影响

    黄颖秋; 萧树东; 莫剑忠; 张德中

    2000-01-01

    To investigate the effects of low dosage of nitric oxide synthase (NOS) inhibitor Nc-nitro -L-arginine methyl ester ( L-NAME) in two-week treatment on the hyperdynamic circulatory state in rats with cirrhosis. METHODS: Cirrhosis model was induced in male SD rats by injection of 60 % CCL4 oily solution subcutaneously. Cirrhotic rats were treated with L-NAME ( 0.5 mg·kg-1·d-1) by gavage for two weeks. Mean arterial pressure ( AP ), portal pressure(PP), cardiac output ( CO ), cardiac index ( CI ), splanchnic vascular resistance ( SVR ), splanchnic blood flow(SBF) and serum nitrite levels were determined in L-NAME-treated, L-NAME-untreated cirrhotic rats and controls by using 57Co-labled microsphere technique and a fluorometric assay, respectively. RESULTS: Untreated cirrhotic rats had significantly lower MAP, SVR and higher PP, CO, CI, SBF and nitrite concentration than those of the controls (all,P< 0.01 ). In treated cirrhotic rats, L-NAME significantly attenuated the increase of CO, CI, SBF, nitrite concentration and the decrease of MAP and SVR. In treated cirrhotic rats, L-NAME induced a marked decrease of nitrite concentration than untreated cirrhotic rats[(1.471±0.907)μmol/L vs (4.204±1.253) μmol/L, P<0.01]. CONCLUSION: The endogenous NO may play an important role in the changes of hemodynamics pattern in cirrhosis, and hyperdynamic circulatory state in rats with cirrhosis can be ameliorated by oral two-week administration of lower dose of L-NAME.%目的:观察小剂量一氧化氮合酶(N0S)抑制剂N-硝基-L-精氨酸甲酯(L-NAME)连续2周治疗对肝硬化大鼠高动力循环状态的影响.方法:用60%四氯化碳油性溶液皮下注射SD大鼠制造肝硬化大鼠模型.对肝硬化大鼠,用L-NAME(0.5mg·kg·d-1)胃管内注入连续治疗2周.用57Co同位素微球技术分别测定L-NAME治疗组、未治疗组及正常对照组的平均动脉压(MAP)、门静脉压(PP)、心输出量(C0)、心脏指数(CI)、内脏血管阻力(SVR)及内脏

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

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

    2015-05-01

    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. PMID:26601184

  10. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Edwin R Lampugnani; Persson, Staffan

    2016-01-01

    ABSTRACT Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated ...

  11. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-01-01

    Background Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Methods Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Results Each of the respective gene...

  12. Arabidopsis CDS blastp result: AK242817 [KOME

    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 ...

  13. Arabidopsis CDS blastp result: AK058963 [KOME

    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 ...

  14. Arabidopsis CDS blastp result: AK109628 [KOME

    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 ...

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

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

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

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

    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...

  17. Nitric Oxide Synthases and Atrial Fibrillation

    CynthiaAnnCarnes; ArunSridhar; SandorGyorke

    2012-01-01

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

  18. Corrosion inhibitors

    In this paper, we briefly describe the characteristics, cost and electrochemical nature of the corrosion phenomena as well as some of the technologies that are currently employed to minimize its effect. The main subject of the paper however, deals with the description, classification and mechanism of protection of the so-called corrosion inhibitors. Examples of the use of these substances in different aggressive environments are also presented as means to show that these compounds, or their combination, can in fact be used as excellent and relatively cheap technologies to control the corrosion of some metals. In the last part of the paper, the most commonly used techniques to evaluate the efficiency and performance of corrosion inhibitors are presented as well as some criteria to make a careful and proper selection of a corrosion inhibitor technology in a given situation. (Author) 151 refs

  19. Unique animal prenyltransferase with monoterpene synthase activity

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

    2009-06-01

    Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

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

    2010-01-01

    The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase. PMID:20704760

  1. Properties of phosphorylated thymidylate synthase.

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    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. PMID:26315778

  2. Molecular evolution of dihydrouridine synthases

    Kasprzak Joanna M

    2012-06-01

    Full Text Available Abstract Background Dihydrouridine (D is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS. DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as “predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family”. Results To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model. Conclusions We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS

  3. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa.

    Ninnemann, H; Maier, J

    1996-08-01

    Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitic oxide donor, sodium nitroprusside, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives NG -nitro-L-arginine (L-NA) and NG-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness nad in the light, whereas the stereoisomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photo-physiology. PMID:8760579

  4. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...... gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise...... combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no...

  5. A pentacyclic reaction intermediate of riboflavin synthase

    Illarionov, Boris; Eisenreich, Wolfgang; Bacher, Adelbert

    2001-01-01

    The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermedi...

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

    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. Green tea catechin inhibits fatty acid synthase without stimulating carnitine Palmitoyltransferase-1 or inducing weight loss in experimental animals

    Puig i Miquel, Teresa; Relat Pardo, Joana; Marrero González, Pedro F.; Haro Bautista, Diego; Brunet, Joan; Colomer Bosch, Ramón

    2008-01-01

    Background: The enzyme fatty acid synthase (FASN) is highly expressed in many human carcinomas and its inhibition is cytotoxic to human cancer cells. The use of FASN inhibitors has been limited until now by anorexia and weight loss, which is associated with the stimulation of fatty acid oxidation. Materials and Methods: The in vitro effect of (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism enzymes, on apoptosis and on cell signalling was evaluated. In vivo, the effect of EGCG o...

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

    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 ...

  9. Inducible nitric oxide synthase is involved in the modulation of depressive behaviors induced by unpredictable chronic mild stress

    Peng Yun-Li; Liu Yu-Ning; Liu Lei; Wang Xia; Jiang Chun-Lei; Wang Yun-Xia

    2012-01-01

    Abstract Background Experiences and inflammatory mediators are fundamental in the provocation of major depressive disorders (MDDs). We investigated the roles and mechanisms of inducible nitric oxide synthase (iNOS) in stress-induced depression. Methods We used a depressive-like state mouse model induced by unpredictable chronic mild stress (UCMS). Depressive-like behaviors were evaluated after 4 weeks of UCMS, in the presence and absence of the iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamid...

  10. Inhibition of Glycogen Synthase Kinase-3β Improves Tolerance to Ischemia in Hypertrophied Hearts

    Barillas, Rodrigo; Friehs, Ingeborg; Cao-Danh, Hung; Martinez, Joseph F.; del Nido, Pedro J.

    2012-01-01

    Background Hypertrophied myocardium is more susceptible to ischemia/reperfusion injury, in part owing to impaired insulin-mediated glucose uptake. Glycogen synthase kinase-3β (GSK-3β) is a key regulatory enzyme in glucose metabolism that, when activated, phosphorylates/inactivates target enzymes of the insulin signaling pathway. Glycogen synthase kinase-3β is regulated upstream by Akt-1. We sought to determine whether GSK-3β is activated in ischemic hypertrophied myocardium owing to impaired Akt-1 function, and whether inhibition with lithium (Li) or indirubin-3′-monoxime,5-iodo- (IMI), a specific inhibitor, improves post-ischemic myocardial recovery by improving glucose metabolism. Methods Pressure-overload hypertrophy was achieved by aortic banding in neonatal rabbits. At 6 weeks, isolated hypertrophied hearts underwent 30 minutes of normothermic ischemia and reperfusion with or without GSK-3β inhibitor (0.1 mM Li; 1 µM IMI) as cardioplegic additives. Cardiac function was measured before and after ischemia. Expression, activity of Akt-1 and GSK-3β, and lactate were determined at end-ischemia. Results Contractile function after ischemia was better preserved in hypertrophied hearts treated with GSK-3β inhibitors. Activity of Akt-1 was significantly impaired in hypertrophied myocardium at end-ischemia. Glycogen synthase kinase-3β enzymatic activity at end-ischemia was increased in hypertrophied hearts and was blocked by Li or IMI concomitant with significantly increased lactate production, indicating increased glycolysis. Conclusions Regulatory inhibition of GSK-3β by Akt-1 in hypertrophied hearts is impaired, leading to activation during ischemia. Inhibition of GSK-3β by Li or IMI improves tolerance to ischemia/reperfusion injury in hypertrophied myocardium. The likely protective mechanism is an increase in insulin-mediated glucose uptake, resulting in greater substrate availability for glycolysis during ischemia and early reperfusion. PMID:17588398

  11. Nuclear genetic defects of mitochondrial ATP synthase

    Houštěk, Josef; Kmoch, S.; Mayr, J. A.; Sperl, W.; Zeman, J.

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

  12. Inducible nitric oxide synthase in renal transplantation

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  13. Hyaluronan synthase in trabecular meshwork cells

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

    2003-01-01

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

  14. Activities and regulation of peptidoglycan synthases

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

    2015-01-01

    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 b

  15. The tomato terpene synthase gene family

    V. Falara; T.A. Akhtar; T.T.H. Nguyen; E.A. Spyropoulou; P.M. Bleeker; I. Schauvinhold; Y. Matsuba; M.E. Bonini; A.L. Schilmiller; R.L. Last; R.C. Schuurink; E. Pichersky

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  16. Loop residues and catalysis in OMP synthase

    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...

  17. Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

    Burnett, A. L.; Nelson, R.J.; Calvin, D. C.; Liu, J. X.; Demas, G E; Klein, S. L.; Kriegsfeld, L. J.; Dawson, V L; Dawson, T. M.; Snyder, S H

    1996-01-01

    BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mat...

  18. 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

    L. Vargas

    2013-06-01

    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.

  19. Cellulose Synthases and Synthesis in Arabidopsis

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  20. Effects of NOS inhibitor on dentate gyrus neurogenesis after diffuse brain injury in the adult rats

    SunLi-Sha; XuJiang-ping

    2004-01-01

    Objective To investigate the effects of selective nitric oxide synthase (NOS) inhibitors on dentate gyrus neurogenesis after diffuse brain injury (DBI) in the adult rat brain. Methods Adult male SD rats were subjected to diffuse brain injury (DBI) model. By using systemic bromodeoxyuridine (BrdU) to label dividing cells, we compared the proliferation rate of

  1. Evolution of polyketide synthases in bacteria

    Ridley, Christian P.; Lee, Ho Young; Khosla, Chaitan

    2008-01-01

    The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of polyketides as antimicrobial agents, provides strong motivation for understanding how polyketide antibiotics have evolved and diversified in nature. Insights into how bacterial polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory efforts in generating the next generation of polyketide antibiotics. Here, we examine phylogenetic and structura...

  2. Nitric oxide synthase in the pineal gland

    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 ...

  3. Caffeine synthase and related methyltransferases in plants.

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region. PMID:14977590

  4. Nitric Oxide Synthases in Heart Failure

    Carnicer, Ricardo; Crabtree, Mark J; Sivakumaran, Vidhya; Casadei, Barbara; Kass, David A

    2013-01-01

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

  5. The tomato terpene synthase gene family

    Falara, V.; Akhtar, T.A.; NGUYEN, T. T. H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R. C.; Pichersky, E

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28 which are functional or potentially functional. Of these 28 TPS genes, 25 were expressed in at least some parts of the plant. The enzymatic functions of eight of the TPS proteins were previously r...

  6. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    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

  7. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    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.

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

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

    2013-04-01

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

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

    Gea Guerriero

    Full Text Available 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

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

    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.

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

    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...

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

    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...

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

    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. Evolutinoary Consideration on 5-Aminolevulinate Synthase in Nature

    Oh-Hama, Tamiko

    1997-08-01

    5-Aminolevulinic acid (ALA), a universal precursor of tetrapyrrole compounds can be synthesized by two pathways: the C5 (glutamate) pathway and ALA synthase. From the phylogenetic distribution it is shown that distribution of ALA synthase is restricted to the α subclass of purple bacteria in prokaryotes, and further distributed to mitochondria of eukaryotes. The monophyletic origin of bacterial and eukaryotic ALA synthase is shown by sequence analysis of the enzyme. Evolution of ALA synthase in the α subclass of purple bacteria is discussed in relation to the energy-generating and biosynthetic devices in subclasses of this bacteria.

  15. Sphingomyelin Synthases Regulate Protein Trafficking and Secretion

    Subathra, Marimuthu; Qureshi, Asfia; Luberto, Chiara

    2011-01-01

    Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 r...

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

    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-Nω-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)

  17. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G; Köllner, Tobias G

    2016-03-15

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  18. Loss of LRPPRC causes ATP synthase deficiency.

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Kühlbrandt, Werner; Larsson, Nils-Göran

    2014-05-15

    Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria. PMID:24399447

  19. Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae.

    Langdon, S D; Jones, M E

    1987-09-25

    In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form. PMID:3308878

  20. Localization of nitric oxide synthase in human skeletal muscle

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    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...

  1. The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

    Vain, T.; Crowell, E.F.; Timpano, H.; Biot, E.; Desprez, T.; Mansoori Zangir, N.; Trindade, L.M.; Pagant, S.; Robert, S.; Hofte, H.; Gonneau, M.; Vernhettes, S.

    2014-01-01

    Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis

  2. p63 promotes cell survival through fatty acid synthase.

    Venkata Sabbisetti

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

  3. STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE

    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.

  4. Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

    Meleza, Cesar; Thomasson, Bobbie; Ramachandran, Chidambaram; O'Neill, Jason W; Michelsen, Klaus; Lo, Mei-Chu

    2016-10-15

    Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay. PMID:27485270

  5. Proton pump inhibitors

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

  6. Proton pump inhibitors

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

  7. Structure and reaction mechanism of basil eugenol synthase.

    Gordon V Louie

    Full Text Available Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs, and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H and a mixed competitive inhibitor EMDF ((7S,8S-ethyl (7,8-methylene-dihydroferulate provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent

  8. Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum

    Bitencourt, Tamires Aparecida; Komoto, Tatiana Takahasi; Massaroto, Bruna Gabriele; Miranda, Carlos Eduardo Saraiva; Beleboni, Rene Oliveira; Marins, Mozart; Fachin, Ana Lúcia

    2013-01-01

    Background Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis. Methods The antifungal activity of th...

  9. Functional dissection of N-acetylglutamate synthase (ArgA) of Pseudomonas aeruginosa and restoration of its ancestral N-acetylglutamate kinase activity

    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...

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

    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)

  11. Biosynthetic potential of sesquiterpene synthases: Alternative products of tobacco 5-epi-aristolochene synthase

    O’Maille, Paul E.; Chappell, Joe; Noel, Joseph P.

    2006-01-01

    Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC–MS assay. The combined use of chiral and non...

  12. All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity[S

    Ding, Tingbo; Kabir, Inamul; Li, Yue; Lou, Caixia; Yazdanyar, Amirfarbod; Xu, Jiachen; Dong, Jibin; Zhou, Hongwen; Park, Taesik; Boutjdir, Mohamed; Li, Zhiqiang; Jiang, Xian-Cheng

    2015-01-01

    Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide a...

  13. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-01

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed. PMID:14685317

  14. Interleukin 1 beta induces diabetes and fever in normal rats by nitric oxide via induction of different nitric oxide synthases

    Reimers, J I; Bjerre, U; Mandrup-Poulsen, T;

    1994-01-01

    Substantial in vitro evidence suggests that nitric oxide may be a major mediator of interleukin 1 (IL-1) induced pancreatic beta-cell inhibition and destruction in the initial events leading to insulin-dependent diabetes mellitus. Using NG-nitro-L-arginine methyl ester, an inhibitor of both the......, glucagon, corticosterone and leukocyte- and differential-counts in normal rats injected once daily for 5 days with interleukin 1 beta (IL-1 beta) (0.8 microgram/rat = 4.0 micrograms/kg). Inhibition of both the constitutive and the inducible forms of nitric oxide synthase prevented IL-1 beta-induced fever...

  15. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly. PMID:26734760

  16. Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase.

    Iwata-Reuyl, Dirk; Math, Shivanand K; Desai, Shrivallabh B; Poulter, C Dale

    2003-03-25

    Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene. PMID:12641468

  17. Microsomal prostaglandin E synthase-1 protects against Fas-induced liver injury.

    Yao, Lu; Chen, Weina; Han, Chang; Wu, Tong

    2016-06-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme for the synthesis of prostaglandin E2 (PGE2), a proproliferative and antiapoptotic lipid molecule important for tissue regeneration and injury repair. In this study, we developed transgenic (Tg) mice with targeted expression of mPGES-1 in the liver to assess Fas-induced hepatocyte apoptosis and acute liver injury. Compared with wild-type (WT) mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, less hepatic necrosis/apoptosis, and lower level of caspase cascade activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analysis revealed increased expression and activation of the serine/threonine kinase Akt and associated antiapoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling and suggest that induction of mPGES-1 or treatment with PGE2 may represent important therapeutic strategy for the prevention and treatment of Fas-associated liver injuries. PMID:27102561

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

    Marjelo M. Mines

    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.

  19. Para-aminobenzoic acid (PABA synthase enhances thermotolerance of mushroom Agaricus bisporus.

    Zhonglei Lu

    Full Text Available Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom's innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins.

  20. Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

    The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7,500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to subunit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degree C. The monomer is formed upon heating with SDS to 121 degree C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m-chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter

  1. Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

    Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

    2015-08-01

    This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials. PMID:26135986

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

    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.

  3. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target. PMID:26621635

  4. Pseudoguaianolides isolated from Inula britannica var. chinenis as inhibitory constituents against inducible nitric oxide synthase.

    Lee, Hyun-Tai; Yang, Seung-Won; Kim, Kyeong Ho; Seo, Eun-Kyoung; Mar, Woongchon

    2002-04-01

    Three pseudoguaianolide type sesquiterpenes, bigelovin (1), 2,3-dihydroaromaticin (2), and ergolide (3) were isolated as inhibitory constituents against inducible nitric oxide synthase (iNOS) from the flowers of Inula britannica var. chinensis. Bigelovin (1) exhibited a highly potent inhibitory activity on lipopolysaccharide (LPS)-induced iNOS in murine macrophage RAW 264.7 cells with an IC50 value of 0.46 mM, which is about 8 times more potent than the known selective inhibitor of iNOS, L-N6-(1-iminoethyl)lysine (IC50 3.49 microM). 2,3-Dihydroaromaticin (2) and ergolide (3) also exhibited potent inhibitory activities on LPS-induced iNOS with IC50 values of 1.05 and 0.69 microM, respectively. PMID:12009027

  5. Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

    Patel, Sandip; Gaspers, Lawrence D; Boucherie, Sylviane; Memin, Elisabeth; Stellato, Kerri Anne; Guillon, Gilles; Combettes, Laurent; Thomas, Andrew P

    2002-09-13

    Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation. PMID:12097323

  6. Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-1-Carboxylate Synthase

    Jian-Feng LI; Liang-Hu QU; Ning LI

    2005-01-01

    1-aminocyclopropane-1-carboxylate (ACC) synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase (LeACS2) in tomato (Lycopersicon esculentum Mill.) fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ℃. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthaseprocessing protease from plant tissues.

  7. Effect of vardenafil on nitric oxide synthase expression in the paraventricular nucleus of rats without sexual stimulation.

    Shin, M-S; Ko, I-G; Kim, S-E; Kim, B-K; Kim, C-J; Kim, D-H; Yoon, S-J; Kim, K-H

    2012-05-01

    Vardenafil hydrochloride (HCl) is a potent and selective phosphodiesterase type-5 (PDE-5) inhibitor that enhances nitric oxide (NO)-mediated relaxation of human corpus cavernosum and NO-induced rabbit penile erection, and enhances erectile function in patients. In the present study, the effect of vardenafil on nitric oxide synthase (NOS) and neuronal NOS expressions in the paraventricular nucleus (PVN) of rats without sexual stimulation was investigated using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal NOS (nNOS) immunohistochemistry and western blot analysis. The present results showed that NOS and nNOS expression in the PVN was increased by vardenafil treatment as the dose- and duration-dependently without sexual stimulation. The phosphodiesterase type-5 inhibitor, vardenafil, augmented NOS expression in the brain without sexual stimulation. The present study suggests that sexual behaviour can be directly modulated by neurotransmitters such as nitric oxide. PMID:21950284

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

    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)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identificat...

  9. Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum

    Nakai, Tomonori; Tonouchi, Naoto; Konishi, Teruko; Kojima, Yukiko; Tsuchida, Takayasu; Yoshinaga, Fumihiro; Sakai, Fukumi; Hayashi, Takahisa

    1999-01-01

    Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-β-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the...

  10. Glycogen synthase kinase 3: more than a namesake

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

    2009-01-01

    Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, τ protein and β catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as...

  11. Subcellular Targeting Domains of Sphingomyelin Synthase 1 and 2

    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...

  12. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

    Khosla, Chaitan; Herschlag, Daniel; Cane, David E.; Walsh, Christopher T.

    2014-01-01

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase doma...

  13. Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

    Wong, H C; Fear, A L; Calhoon, R D; Eichinger, G H; Mayer, R; Amikam, D; Benziman, M; Gelfand, D H; Meade, J H; Emerick, A W

    1990-01-01

    An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the co...

  14. Computational design and selections for an engineered, thermostable terpene synthase

    Diaz, JE; Lin, CS; Kunishiro, K; Feld, BK; Avrantinis, SK; Bronson, J.; J. Greaves; Saven, JG; Weiss, GA

    2011-01-01

    Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact protein...

  15. Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica

    Hyo-Jin An; Agung Nugroho; Byong-Min Song; Hee-Juhn Park

    2015-01-01

    Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic ac...

  16. New evolution: Inhibitors of fatty acid synthase and fat-reducing study

    2002-01-01

    @@ Obesity sweeps all over the world presently at an astonishing speed. The rate of the disease ascends in both the developed and developing countries. According to a recent report from the World Health Organization, it can be estimated that about 250 million adults are obese all over the world, and that at least 500 million adults are overweight[1]. In the United States, more than 10% of children are obese and more than 15% of children are overweight[2]. About 2%-7% annual medical expenses of the developed countries are used for obesity by conservative estimation[3].

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

    Brožíčková, Carole; Mikulecká, Anna; Otáhal, Jakub

    2014-01-01

    Roč. 63, č. 5 (2014), s. 637-648. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/10/0999; GA ČR(CZ) GPP304/11/P386; GA ČR(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

  18. Bedaquiline – The first ATP synthase inhibitor against multi drug resistant tuberculosis

    Lakshmanan, Mageshwaran; Xavier, Alphienes Stanley

    2013-01-01

    Increasing incidence of MDR-TB, long duration of treatment and co-infection with HIV are the significant problems in achieving the eradication of tuberculosis. Bedaquiline is an anti-tuberculosis drug with unique mechanism of action. It selectively inhibits the mycobacterial energy metabolism i.e. ATP synthesis and found to be effective against all states of Mycobacterium tuberculosis like active, dormant, replicating, non-replicating, intracellular and extracellular. Preclinical studies have...

  19. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  20. Regulation by glycogen synthase kinase-3 of inflammation and T cells in CNS diseases

    Eleonore Beurel

    2011-08-01

    Full Text Available Elevated markers of neuroinflammation have been found to be associated with many psychiatric and neurodegenerative diseases, such as mood disorders, Alzheimer's disease, and multiple sclerosis. Since neuroinflammation is thought to contribute to the pathophysiology of these diseases and to impair responses to therapeutic interventions and recovery, it is important to identify mechanisms that regulate neuroinflammation and potential targets for controlling neuroinflammation. Recent findings have demonstrated that glycogen synthase kinase-3 (GSK3 is an important regulator of both the innate and adaptive immune systems' contributions to inflammation. Studies of the innate immune system have shown that inhibitors of GSK3 profoundly alter the repertoire of cytokines that are produced both by peripheral and central cells, reducing proinflammatory cytokines and increasing anti-inflammatory cytokines. Furthermore, inhibitors of GSK3 promote tolerance to inflammatory stimuli, reducing inflammatory cytokine production upon repeated exposure. Studies of the adaptive immune system have shown that GSK3 regulates the production of cytokines by T cells and the differentiation of T cells to subtypes, particularly Th17 cells. Regulation of transcription factors by GSK3 appears to play a prominent role in its regulation of immune responses, including of NF-κB, cyclic AMP response element binding protein (CREB, and signal transducer and activator of transcription-3 (STAT3. In vivo studies have shown that GSK3 inhibitors ameliorate clinical symptoms of both peripheral and central inflammatory diseases, particularly experimental autoimmune encephalomyelitis (EAE, the animal model of multiple sclerosis. Therefore, the development and application of GSK3 inhibitors may provide a new therapeutic strategy to reduce neuroinflammation associated with many CNS diseases.

  1. Homocystinuria due to cystathionine beta synthase deficiency

    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.

  2. Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase

    Danley, Dennis E; Baima, Eric T.; Mansour, Mahmoud; Fennell, Kimberly F.; Chrunyk, Boris A.; Mueller, John P.; Liu, Shenping; Qiu, Xiayang

    2014-01-01

    Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2....

  3. What Is the True Structure of D609, a Widely Used Lipid Related Enzyme Inhibitor?

    Kato, Mikako; Hammam, Mostafa A S; Taniguchi, Tohru; Suga, Yoshiko; Monde, Kenji

    2016-02-19

    D609 (1) has been used as a lipid-related enzyme inhibitor during the past three decades. Although it has eight possible stereoisomers, no systematic research considering its chirality has been performed. In this paper, eight possible chiral alcohols as direct precursors of D609 were synthesized, and their stereochemistries were elucidated by a vibrational circular dichroism (VCD) technique. Phosphatidylcholine-specific phospholipase C and sphingomyelin synthase inhibition assays of these isomers showed considerable differences in their activities. PMID:26854352

  4. Changes in the profile of NO synthases affect coronary blood flow autoregulation and myocardial contractile activity during restraint stress in rats.

    Solodkov, A P; Lazuko, S S; Knyazev, E N; Nechaev, I N; Krainova, N A

    2014-12-01

    The efficiency of autoregulation of the coronary blood flow and contractile activity of the myocardium in the presence of inhibitors of constitutive and inducible NO synthases was studied in rats exposed to 6-h restraint stress. Intracoronary administration of S-methylisothiourea (10 μmol/liter), but not L-NAME (60 μmol/liter) fully prevented post-stress increase in the volume coronary blood flow rate, intensity of heart perfusion, and reduction of ventricular developed pressure at all levels of perfusion pressure. Real-time PCR showed 6-fold increased expression of inducible NO-synthase mRNA in the heart tissue against the background of unchanged expression of neuronal and endothelial NO synthases and 2-3-fold elevated content of transcripts of stress-inducible genes Hspa1a and Hspbp1. It was shown that the hypotension of coronary vessels and reduced contractile function of the myocardium are related to NO production by inducible NO synthase in endotheliocytes of coronary vessels and cardiomyocytes. PMID:25430647

  5. Low toxicity corrosion inhibitors

    This paper discusses the design and testing of low toxicity corrosion inhibitors. New chemistries have been investigated with respect to corrosion protection and impact on the marine environment. The resulting chemicals, while they are effective corrosion inhibitors, present significant improvements in terms of environmental properties over current products. The discussion includes results of the corrosion inhibition, toxicity, biodegradability and partitioning studies

  6. 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.

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

    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 S-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. PMID:20689184

  7. 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

    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.

  8. Characterisation of the tryptophan synthase alpha subunit in maize

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  9. Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells

    2007-01-01

    AIM: To investigate the link between chronic biliary inflammation and carcinogenesis using hamster gallbladder epithelial cells.METHODS: Gallbladder epithelial cells were isolated from hamsters and cultured with a mixture of inflammatory cytokines including interleukin-1β, interferon-γ, and tumor necrosis factor-α. Inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) generation, and DNA damage were evaluated.RESULTS: NO generation was increased significantly following cytokine stimulation, and suppressed by an iNOS inhibitor. iNOS mRNA expression was demonstrated in the gallbladder epithelial cells during exposure to inflammatory cytokines. Furthermore, NO-dependent DNA damage, estimated by the comet assay, was significantly increased by cytokines, and decreased to control levels by an iNOS inhibitor.CONCLUSION: Cytokine stimulation induced iNOS expression and NO generation in normal hamster gallbladder epithelial cells, which was sufficient to cause DNA damage. These results indicate that NO-mediated genotoxicity induced by inflammatory cytokines through activation of iNOS may be involved in the process of biliary carcinogenesis in response to chronic inflammation of the biliary tree.

  10. Comparing Drug Images and Repurposing Drugs with BioGPS and FLAPdock: The Thymidylate Synthase Case.

    Siragusa, Lydia; Luciani, Rosaria; Borsari, Chiara; Ferrari, Stefania; Costi, Maria Paola; Cruciani, Gabriele; Spyrakis, Francesca

    2016-08-01

    Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent in vitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinase IIα as a control and tested a series of its inhibitors against the TS template. Ellagic acid and apigenin were identified as TS inhibitors, and various flavonoids were selected and synthesized in a second-round selection. The compounds were demonstrated to be active in the low-micromolar range. PMID:27404817

  11. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression.

    Kim, Sun-Hee; Hashimoto, Yuuri; Cho, Sung-Nam; Roszik, Jason; Milton, Denái R; Dal, Fulya; Kim, Sangwon F; Menter, David G; Yang, Peiying; Ekmekcioglu, Suhendan; Grimm, Elizabeth A

    2016-05-01

    COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1-specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy. PMID:26801201

  12. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose

    Huan Huang; McIntosh, Avery L.; Martin, Gregory G.; Petrescu, Anca D.; Landrock, Kerstin K.; Danilo Landrock; Kier, Ann B.; 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-α (PPARα) in the nucleus, was found to bind TOFA and its activated CoA th...

  13. Molecular mechanism underlying the synergistic interaction between trifluorothymidine and the epidermal growth factor receptor inhibitor erlotinib in human colorectal cancer cell lines

    Bijnsdorp, Irene V.; Kruyt, Frank A. E.; Fukushima, Masakazu; Smid, Kees; Gokoel, Shanti; Peters, Godefridus J.

    2010-01-01

    The pyrimidine trifluorothymidine (TFT) inhibits thymidylate synthase (TS) and can be incorporated into the DNA. TFT, as part of TAS-102, is clinically evaluated in phase II studies as an oral chemotherapeutic agent. Erlotinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (E

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

    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.

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

    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

  16. Divergence of multimodular polyketide synthases revealed by a didomain structure.

    Zheng, Jianting; Gay, Darren C; Demeler, Borries; White, Mark A; Keatinge-Clay, Adrian T

    2012-07-01

    The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 Å-resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ∼600-Å(2) interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues. PMID:22634636

  17. Neuronal nitric oxide synthase and N-methyl-D-aspartate neurons in experimental carbon monoxide poisoning

    We measured changes in nitric oxide (·NO) concentration in the cerebral cortex during experimental carbon monoxide (CO) poisoning and assessed the role for N-methyl-D-aspartate receptors (NMDARs), a glutamate receptor subtype, with progression of CO-mediated oxidative stress. Using microelectrodes, ·NO concentration was found to nearly double to 280 nM due to CO exposure, and elevations in cerebral blood flow, monitored as laser Doppler flow (LDF), were found to loosely correlate with ·NO concentration. Neuronal nitric oxide synthase (nNOS) activity was the cause of the ·NO elevation based on the effects of specific NOS inhibitors and observations in nNOS knockout mice. Activation of nNOS was inhibited by the NMDARs inhibitor, MK 801, and by the calcium channel blocker, nimodipine, thus demonstrating a link to excitatory amino acids. Cortical cyclic GMP concentration was increased due to CO poisoning and shown to be related to ·NO, versus CO, mediated guanylate cyclase activation. Elevations of ·NO were inhibited when rats were infused with superoxide dismutase and in rats depleted of platelets or neutrophils. When injected with MK 801 or 7-nitroindazole, a selective nNOS inhibitor, rats did not exhibit CO-mediated nitrotyrosine formation, myeloperoxidase (MPO) elevation (indicative of neutrophil sequestration), or impaired learning. Similarly, whereas CO-poisoned wild-type mice exhibited elevations in nitrotyrosine and myeloperoxidase, these changes did not occur in nNOS knockout mice. We conclude that CO exposure initiates perivascular processes including oxidative stress that triggers activation of NMDA neuronal nNOS, and these events are necessary for the progression of CO-mediated neuropathology

  18. A large blood pressure-raising effect of nitric oxide synthase inhibition in humans

    Sander, M.; Chavoshan, B.; Victor, R. G.; Blomqvist, C. G. (Principal Investigator)

    1999-01-01

    In experimental animals, systemic administration of nitric oxide synthase (NOS) inhibitors causes large increases in blood pressure that are in part sympathetically mediated. The aim of this study was to determine the extent to which these conclusions can be extrapolated to humans. In healthy normotensive humans, we measured blood pressure in response to two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME), the latter of which recently became available for use in humans. The major new findings are 3-fold. First, L-NAME produced robust increases in blood pressure that were more than 2 times larger than those previously reported in humans with L-NMMA and approximated those seen in experimental animals. L-NAME (4 mg/kg) raised mean arterial pressure by 24+/-2 mm Hg (n=27, P<0.001), whereas in subjects who received both inhibitors, a 12-fold higher dose of L-NMMA (50 mg/kg) raised mean arterial pressure by 15+/-2 mm Hg (n=4, P<0.05 vs L-NAME). Second, the L-NAME-induced increases in blood pressure were caused specifically by NOS inhibition because they were reversed by L-arginine (200 mg/kg, n=12) but not D-arginine (200 mg/kg, n=6) and because NG-nitro-D-arginine methyl ester (4 mg/kg, n=5) had no effect on blood pressure. Third, in humans, there is an important sympathetic component to the blood pressure-raising effect of NOS inhibition. alpha-Adrenergic blockade with phentolamine (0.2 mg/kg, n=9) attenuated the L-NAME-induced increase in blood pressure by 40% (P<0.05). From these data, we conclude that pharmacological inhibition of NOS causes large increases in blood pressure that are in part sympathetically mediated in humans as well as experimental animals.

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

    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

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

    Colpo Anna

    2010-10-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK-3 α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB 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-3α and GSK-3β function in multiple myeloma (MM. Methods GSK-3 α and β 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 α and β isoforms. Survival signaling pathways were studied with WB analysis. Results GSK-3α and GSK-3β 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-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α 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-3α and β i play distinct roles in cell survival and ii modulate the sensitivity to proteasome inhibitors.

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

    Glycogen Synthase Kinase-3 (GSK-3) α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB 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-3α and GSK-3β function in multiple myeloma (MM). GSK-3 α and β 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 α and β isoforms. Survival signaling pathways were studied with WB analysis. GSK-3α and GSK-3β 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-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α 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-3α and β i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

  2. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

    Lim, Yan Ping; Go, Maybelle K; Yew, Wen Shan

    2016-01-01

    Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development. PMID:27338328

  3. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

    2009-01-01

    8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the sub...

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

    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, ...

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

    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...

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

    Zeliha Selamoglu-Talas

    2015-10-01

    Full Text Available Background: 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 Nω-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  (P<0.001  in  the  malondialdehyde  levels  in  the  L-NAME treatment groups when compared to control rats, but the decrease (P<0.001 in the catalase activities in both brain and lung tissues. There were statistically changes (P<0.001 in these parameters of L-NAME+propolis treated rats as compared with L-NAME-treated group.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.

  7. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-01

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed. PMID:26546087

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

    Igamberdiev, Abir U.; 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+, ...

  9. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  10. Small-molecule caspase inhibitors

    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.

  11. Small-molecule caspase inhibitors

    Zhenodarova, S M [Institute for Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation)

    2010-02-28

    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.

  12. Small-molecule caspase inhibitors

    Zhenodarova, S. M.

    2010-02-01

    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.

  13. Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases*

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

    2011-01-01

    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 α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). ...

  14. Characterising the cellulose synthase complexes of cell walls

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  15. A particular phenotype in a girl with aldosterone synthase deficiency.

    Williams, Tracy A; Mulatero, Paolo; Bosio, Maurizio; Lewicka, Sabina; Palermo, Mario; Veglio, Franco; Armanini, Decio

    2004-07-01

    Aldosterone synthase deficiency (ASD) usually presents in infancy as a life-threatening electrolyte imbalance. A 4-wk-old child of unrelated parents was examined for failure to thrive and salt-wasting. Notable laboratory findings were hyperkalemia, high plasma renin, and low-normal aldosterone levels. Urinary metabolite ratios of corticosterone/18-hydroxycorticosterone and 18-hydroxycorticosterone/aldosterone were intermediate between ASD type I and type II. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (P450c11AS), revealed that the patient was a compound heterozygote carrying a previously described mutation located in exon 4 causing a premature stop codon (E255X) and a further, novel mutation in exon 5 that also causes a premature stop codon (Q272X). The patient's unaffected father was a heterozygous carrier of the E255X mutation, whereas the unaffected mother was a heterozygous carrier of the Q272X mutation. Therefore, the patient's CYP11B2 encodes two truncated forms of aldosterone synthase predicted to be inactive because they lack critical active site residues as well as the heme-binding site. This case of ASD is of particular interest because despite the apparent lack of aldosterone synthase activity, the patient displays low-normal aldosterone levels, thus raising the question of its source. PMID:15240589

  16. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    Napolitano, H. B.; Sculaccio, S. A.; Thiemann, O H; G Oliva

    2004-01-01

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa.

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

    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.

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

    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

  19. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

    Gibson, Marc W; Dewar, Simon; Ong, Han B; Sienkiewicz, Natasha; Fairlamb, Alan H

    2016-05-01

    Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed. PMID:27175479

  20. Inhibition of small G proteins of the Rho family by statins or Clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II

    Hausding, Michael; Witteck, Andrea; Rodriguez-Pascual, Fernando; von Eichel-Streiber, Christian; Förstermann, Ulrich; Kleinert, Hartmut

    2000-01-01

    In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly.Human A549/8- and DLD-1 cells a...

  1. Cholinesterase inhibitors from botanicals

    Faiyaz Ahmed

    2013-01-01

    Full Text Available Alzheimer′s disease (AD is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh, appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE and butyrylcholinesterase (BChE. Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com are also presented and the scope for future research is discussed.

  2. Phosphodiesterase-5 inhibitors.

    Cockrill, Barbara A; Waxman, Aaron B

    2013-01-01

    Nitric oxide (NO) signaling plays a key role in modulating vascular tone and remodeling in the pulmonary circulation. The guanylate cyclase/cyclic guanylate monophosphate-signaling pathway primarily mediates nitric oxide signaling. This pathway is critical in normal regulation of the pulmonary vasculature, and is an important target for therapy in patients with pulmonary hypertension. In the pulmonary vasculature, degradation of cGMP is primarily regulated by PDE-5, and inhibition of this enzyme has important effects on pulmonary vasculature smooth muscle tone. Large randomized placebo-controlled trials of PDE-5 inhibitors demonstrated improved exercise capacity, hemodynamics and quality of life in adult patients with PAH. This chapter will discuss the mechanisms of NO signaling in the vasculature, characteristics of the PDE5-inhibitors approved for treatment of PH, and review available data on the use of phosphodiesterase inhibitors in PH. PMID:24092343

  3. A new type of Na(+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

    Sarah Schulz

    Full Text Available The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

  4. Hematopoetic prostaglandin D synthase: an ESR1-dependent oviductal epithelial cell synthase.

    Bridges, Phillip J; Jeoung, Myoungkun; Shim, Sarah; Park, Ji Yeon; Lee, Jae Eun; Sapsford, Lindsay A; Trudgen, Kourtney; Ko, Chemyong; Gye, Myung Chan; Jo, Misung

    2012-04-01

    Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD₂: PGD₂ receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct. PMID:22374975

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

    Eeva Moilanen; Hannu Kankaanranta; Riina Nieminen; Outi Kärpänniemi; Riku Korhonen; Outi Sareila

    2006-01-01

    In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ (IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α (STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also...

  6. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    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.

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

    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

  8. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions. PMID:26589785

  9. Expression in Arabidopsis of a Strawberry Linalool Synthase Gene Under the Control of the Inducible Potato P12 Promoter

    YANG Li-mei; Per Mercke; Joop J A van Loon; FANG Zhi-yuan; Marcel Dicke; Maarten A Jongsma

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FANESl 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-PP12-LIS' was transformed to Arabidopsis thaliana ecotype Columbia O. Kanamycin resistant T0 seedlings were confirmed for the presence and transcription of the LIS' gene by PCR analysis on genomic DNA and by RT-PCR analysis on RNA. Genomic and RT-PCR products were sequenced to confirm correct splicing of the synthetic intron. The expression of active linalool synthase by the PP12-LIS' gene construct in the transgenic lines was assessed by measuring linalool emission using solid phase micro-extraction (SPME) GC-MS measurements after induction with methyl jasmonate. Among 30 tested independent T2 transgenic lines, 10 exhibited linalool production.Linalool expression could be induced by methyl jasmonate treatment, but not by diamondback moth larvae.

  10. Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides.

    Park, J M; Higuchi, T; Kikuchi, K; Urano, Y; Hori, H; Nishino, T; Aoki, J; Inoue, K; Nagano, T

    2001-04-01

    The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay. PMID:11309260

  11. Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

    Dias, Danielle F.; Alves, Ricardo J., E-mail: ricardodylan@farmacia.ufmg.b [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia; Roux, Celine; Durand, Philippe; Iorga, Bogdan; Badet-Denisot, Marie A.; Badet, Bernard [Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette (France). Inst. de Chimie des Substances Naturelles

    2010-07-01

    The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-phosphate, is therefore an interesting target in the fight against microorganisms. In this work is described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC{sub 50} in the mmol L{sup -1} range. (author)

  12. Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

    The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-phosphate, is therefore an interesting target in the fight against microorganisms. In this work is described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC50 in the mmol L-1 range. (author)

  13. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  14. Corrosion inhibitor compositions

    A corrosion inhibitor compositon for hydrocarbon fuels consisting essentially of, by weight, (A) about 75% to 95% of at least one polymerized unsaturated aliphatic monocarboxylic acid, said unsaturated acid having 16 to 18 carbons per molecule, and (B) about 5% to 25% of at least one monoalkenylsuccinic acid in which the alkenyl group as 8 to 18 carbons

  15. Thymidine Phosphorylase Inhibitors

    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 ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors * angiogenesis * cancer chemotherapy Subject RIV: CC - Organic Chemistry

  16. Sunflower trypsin inhibitor-1.

    Korsinczky, Michael L J; Schirra, Horst Joachim; Craik, David J

    2004-10-01

    SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors. PMID:15544530

  17. Inhibitors of histone demethylases

    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...

  18. Molecular Docking Analysis of Selected Clinacanthus nutans Constituents as Xanthine Oxidase, Nitric Oxide Synthase, Human Neutrophil Elastase, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9 and Squalene Synthase Inhibitors

    Radhakrishnan Narayanaswamy; Azizul Isha; Lam Kok Wai; Intan Safinar Ismail

    2016-01-01

    Background: Clinacanthus nutans (Burm. f.) Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (X...

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

    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.

  20. A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

    Dashuang Shi

    Full Text Available 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 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° 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.

  1. A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

    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 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° 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. PMID:22174908

  2. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

    Nanson, Jeffrey D; Himiari, Zainab; Swarbrick, Crystall M D; Forwood, Jade K

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  3. Decreased glycogen synthase kinase-3 levels and activity contribute to Huntington's disease.

    Fernández-Nogales, Marta; Hernández, Félix; Miguez, Andrés; Alberch, Jordi; Ginés, Silvia; Pérez-Navarro, Esther; Lucas, José J

    2015-09-01

    Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by brain atrophy particularly in striatum leading to personality changes, chorea and dementia. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase in the crossroad of many signaling pathways that is highly pleiotropic as it phosphorylates more than hundred substrates including structural, metabolic, and signaling proteins. Increased GSK-3 activity is believed to contribute to the pathogenesis of neurodegenerative diseases like Alzheimer's disease and GSK-3 inhibitors have been postulated as therapeutic agents for neurodegeneration. Regarding HD, GSK-3 inhibitors have shown beneficial effects in cell and invertebrate animal models but no evident efficacy in mouse models. Intriguingly, those studies were performed without interrogating GSK-3 level and activity in HD brain. Here we aim to explore the level and also the enzymatic activity of GSK-3 in the striatum and other less affected brain regions of HD patients and of the R6/1 mouse model to then elucidate the possible contribution of its alteration to HD pathogenesis by genetic manipulation in mice. We report a dramatic decrease in GSK-3 levels and activity in striatum and cortex of HD patients with similar results in the mouse model. Correction of the GSK-3 deficit in HD mice, by combining with transgenic mice with conditional GSK-3 expression, resulted in amelioration of their brain atrophy and behavioral motor and learning deficits. Thus, our results demonstrate that decreased brain GSK-3 contributes to HD neurological phenotype and open new therapeutic opportunities based on increasing GSK-3 activity or attenuating the harmful consequences of its decrease. PMID:26082469

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

    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 μM 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

  5. Effects of nitric oxide synthase inhibition on sympathetically-mediated tachycardia

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aim of the present study was to determine whether inhibition of nitric oxide (NO) synthesis directly alters the tachycardia produced by sympathetically-derived norepinephrine. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 50 micromol/kg, i.v.), produced a marked rise in mean arterial blood pressure. This pressor response was associated with a fall in heart rate which involved the withdrawal of cardiac sympathetic nerve activity. The NO-donor, sodium nitroprusside (5 microg/kg, i.v.), produced a pronounced fall in mean arterial blood pressure but only a minor increase in heart rate. The beta-adrenoceptor agonist, isoproterenol (0.5 micromol/kg, i.v.), and the membrane-permeable cAMP analogue, 8-(4-chlorophenylthiol)-cAMP (10 micromol/kg, i.v.), produced falls in mean arterial blood pressure and pronounced increases in heart rate. The indirectly acting sympathomimetic agent, tyramine (0.5 mg/kg, i.v.), produced a pressor response and a tachycardia. The effects of sodium nitroprusside, tyramine, isoproterenol and 8-(4-chlorophenylthiol)-cAMP on mean arterial blood pressure were not markedly affected by L-NAME. However, the tachycardia produced by these agents was considerably exaggerated in the presence of this NO synthesis inhibitor. These findings suggest that L-NAME potentiates the tachycardia produced by sympathetically-derived norepinephrine. The increased responsiveness to norepinephrine may involve (i) a rapid up-regulation of cardiac beta1-adrenoceptors and cAMP signaling in cardiac pacemaker cells due to the loss of the inhibitory influence of cardiac NO, and (ii) the up-regulation of beta1-adrenoceptor-mediated signal transduction processes in response to the L-NAME-induced withdrawal of cardiac sympathetic nerve activity.

  6. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    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.

  7. Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

    Magwamba, Clement Chedza; Rukseree, Kamolchanok; Palittapongarnpim, Prasit

    2016-05-01

    The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 μM, IC50 = 267.4 μM; Ki = 0.84 μM, IC50 = 9.28 μM; and Ki = 0.592 μM, IC50 = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS. PMID:27156617

  8. Packed red blood cells are an abundant and proximate potential source of nitric oxide synthase inhibition.

    Charles F Zwemer

    Full Text Available We determined, for packed red blood cells (PRBC and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS inhibitors asymmetric dimethylarginine (ADMA and monomethylarginine (LNMMA.ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.We measured total (free and protein incorporated ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM and LNMMA (58.9 ± 28.9 μM that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.The compelling physiological ramifications are that regardless of storage age, 1 PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2 PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon

  9. Inhibition of Mycobacterium tuberculosis dihydrodipicolinate synthase by alpha-ketopimelic acid and its other structural analogues.

    Shrivastava, Priyanka; Navratna, Vikas; Silla, Yumnam; Dewangan, Rikeshwer P; Pramanik, Atreyi; Chaudhary, Sarika; Rayasam, GeethaVani; Kumar, Anuradha; Gopal, Balasubramanian; Ramachandran, Srinivasan

    2016-01-01

    The Mycobacterium tuberculosis dihydrodipicolinate synthase (Mtb-dapA) is an essential gene. Mtb-DapA catalyzes the aldol condensation between pyruvate and L-aspartate-beta-semialdehyde (ASA) to yield dihydrodipicolinate. In this work we tested the inhibitory effects of structural analogues of pyruvate on recombinant Mtb-DapA (Mtb-rDapA) using a coupled assay with recombinant dihydrodipicolinate reductase (Mtb-rDapB). Alpha-ketopimelic acid (α-KPA) showed maximum inhibition of 88% and IC50 of 21 μM in the presence of pyruvate (500 μM) and ASA (400 μM). Competition experiments with pyruvate and ASA revealed competition of α-KPA with pyruvate. Liquid chromatography-mass spectrometry (LC-MS) data with multiple reaction monitoring (MRM) showed that the relative abundance peak of final product, 2,3,4,5-tetrahydrodipicolinate, was decreased by 50%. Thermal shift assays showed 1 °C Tm shift of Mtb-rDapA upon binding α-KPA. The 2.4 Å crystal structure of Mtb-rDapA-α-KPA complex showed the interaction of critical residues at the active site with α-KPA. Molecular dynamics simulations over 500 ns of pyruvate docked to Mtb-DapA and of α-KPA-bound Mtb-rDapA revealed formation of hydrogen bonds with pyruvate throughout in contrast to α-KPA. Molecular descriptors analysis showed that ligands with polar surface area of 91.7 Å(2) are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA. PMID:27501775

  10. Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine β-synthase.

    Vicente, João B; Malagrinò, Francesca; Arese, Marzia; Forte, Elena; Sarti, Paolo; Giuffrè, Alessandro

    2016-08-01

    Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H2S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H2S-producing human enzyme cystathionine β-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:27039165

  11. Indazole, Pyrazole, and Oxazole Derivatives Targeting Nitric Oxide Synthases and Carbonic Anhydrases.

    Maccallini, Cristina; Di Matteo, Mauro; Vullo, Daniela; Ammazzalorso, Alessandra; Carradori, Simone; De Filippis, Barbara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Supuran, Claudiu T; Amoroso, Rosa

    2016-08-19

    Nitric oxide (NO) is an essential endogenous mediator with a physiological role in the central nervous system as neurotransmitter and neuromodulator. A growing number of studies have demonstrated that abnormal nitrergic signaling is a crucial event in the development of neurodegeneration. In particular, the uncontrolled production of NO by neuronal nitric oxide synthase (nNOS) is observed in several neurodegenerative diseases. Moreover, it is well recognized that specific isoforms of human carbonic anhydrase (hCA) physiologically modulate crucial pathways of signal processing and that low expression of CA affects cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive impairments. In light of this, dual agents that are able to target both NOS (inhibition) and CA (activation) could be useful drug candidates for the treatment of Alzheimer's disease, aging, and other neurodegenerative diseases. In the present work, we show the design, synthesis, and in vitro biological evaluation of new nitrogen-based heterocyclic compounds. Among the tested molecules, 2-amino-3-(4-hydroxyphenyl)-N-(1H-indazol-5-yl)propanamide hydrochloride (10 b) was revealed to be a potent dual agent, able to act as a selective nNOS inhibitor and activator of the hCA I isoform. PMID:27377568

  12. Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom.

    Jeandroz, Sylvain; Wipf, Daniel; Stuehr, Dennis J; Lamattina, Lorenzo; Melkonian, Michael; Tian, Zhijian; Zhu, Ying; Carpenter, Eric J; Wong, Gane Ka-Shu; Wendehenne, David

    2016-01-01

    Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals. PMID:26933064

  13. Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis.

    Djaout, Kamel; Singh, Vinayak; Boum, Yap; Katawera, Victoria; Becker, Hubert F; Bush, Natassja G; Hearnshaw, Stephen J; Pritchard, Jennifer E; Bourbon, Pauline; Madrid, Peter B; Maxwell, Anthony; Mizrahi, Valerie; Myllykallio, Hannu; Ekins, Sean

    2016-01-01

    There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb. PMID:27283217

  14. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    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.

  15. Glycogen synthase kinase 3 phosphorylates RBL2/p130 during quiescence.

    Litovchick, Larisa; Chestukhin, Anton; DeCaprio, James A

    2004-10-01

    Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit. PMID:15456871

  16. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use

    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.

  17. [Role of nitric oxide synthase in the etiopathogenesis of hypertrophic pyloric stenosis in infants

    Barbosa, I M; Ferrante, S M; Mandarim-De-Lacerda, C A

    2001-01-01

    OBJECTIVE: To experimentally reproduce, in rats, the findings corresponding to the histopathology of infantile hypertrophic pyloric stenosis (IHPS), using nitric oxide synthase (NOS) inhibitor (L-NAME). METHODS: L-NAME was administered to pregnant rats (L-NAME group), from the 14th gestational day on in order to reproduce the model of NOS inhibition in the production of IHPS. This group was then compared to control animals. After birth, all the animals in the L-NAME group were maintained under NOS inhibition until the 42nd day of life, when they were sacrificed. The control animals, which did not receive any kind of drug, were also sacrificed on the 42nd day of life. The animals and their internal organs were analyzed and weighed. The pyloric region was technically prepared and observed through light microscopy. RESULTS: The L-NAME group presented lower body and intestinal weight and higher gastric weight than the control group. Light microscopy revealed hypertrophy of the circular smooth muscle layer of the pyloric muscle in L-NAME animals. CONCLUSIONS: This work reproduced an experimental model of an IHPS study, confirming the effect of NOS blockade on the pyloric musculature. PMID:14647863

  18. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits.

    Hoshida, S; Yamashita, N; Igarashi, J; Nishida, M; Hori, M; Kamada, T; Kuzuya, T; Tada, M

    1995-07-01

    The role of nitric oxide (NO) in myocardial ischemia-reperfusion injury is still controversial. To determine the role of NO in the propagation of myocardial injury in a coronary artery occlusion-reperfusion model, we examined the effect of a competitive NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), with and without L-arginine, on the size of the infarct resulting from coronary artery occlusion (30 min) followed by reperfusion (48 hr) in rabbits. L-NAME (300 micrograms/kg, as a bolus, and 100 micrograms/kg/min, i.v.) with and without L-arginine (30 mg/kg, as a bolus, and 10 mg/kg/min, i.v.) was administered immediately before coronary occlusion to 60 min after reperfusion. The infarct size in the L-NAME-treated rabbits (75.1% +/- 5.0%, n = 7), assessed as a percentage of infarcted region/ischemic region, was significantly larger than that of control rabbits (51.2% +/- 7.4%, n = 7; P pressure products, as an index of myocardial oxygen consumption, were comparable in all the groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7542338

  19. The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs.

    Veldman, Robert Jan; Mita, Alain; Cuvillier, Olivier; Garcia, Virginie; Klappe, Karin; Medin, Jeffrey A; Campbell, John D; Carpentier, Stéphane; Kok, Jan Willem; Levade, Thierry

    2003-06-01

    Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short-chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival. Furthermore, N-(n-butyl)deoxynojirimycin, a potent and non-toxic GCS inhibitor, had no chemosensitizing effect on wild-type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug-resistance mechanisms. PMID:12692077

  20. Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

    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 3β (GSK3β, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl or specific GSK3β 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, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  1. Resveratrol- and α-viniferin-induced alterations of acetylcholinesterase and nitric oxide synthase in Raillietina echinobothrida.

    Giri, Bikash Ranjan; Roy, Bishnupada

    2015-10-01

    Phytostilbenes, like resveratrol and α-viniferin, which occur mainly in the plants and belong to the families Cyperaceae, Vitaceae, and Gnetaceae are extensively popular for their medicinal and nutritional properties. In Northeast India, the Jaintia tribes consume these phytochemicals through aqueous extract of the medicinal plant Carex baccans to control helminthiasis. The present study aimed to investigate the inhibitory effect of the phytochemicals on neurotransmitters and its related enzymes in helminth parasite Raillietina echinobothrida. Viability of the parasites exposed to the phytostilbenes and extent of inhibition of cholinergic and nitrergic enzymes were evaluated in comparison to reference anthelmintic drug praziquantel and two known enzyme inhibitors, namely Nω-nitro-L-arginine and pyridostigmine. On exposure to resveratrol, α-viniferin, and reference drug praziquantel, the parasites ceased movement at 9.37, 11.38, and 0.24 h followed by death at 23.65, 34.13, and 1.87 h, respectively. Exposed parasites also showed a significant decrease in the activity of acetylcholinesterase (46.101, 65.935, and 63.645%) and nitric oxide synthase (61.241, 55.046, and 29.618%) in comparison to the controls. In addition, a decreased trend in nitric oxide (NO) level was also detected in the tissue of different phytochemical-exposed parasites compared to control. The present study suggests that anthelmintic potential of both the phytochemicals is mediated through inhibition of two vital enzymes which play diverse role in intracellular communications through neuromuscular system. PMID:26141435

  2. Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

    Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.

  3. Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-{kappa}B

    Leung, Kin Chung; Li, Ming-Yue; Leung, Billy C.S.; Hsin, Michael K.Y. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Mok, Tony S.K. [Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Underwood, Malcolm J. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Chen, George G., E-mail: gchen@cuhk.edu.hk [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong)

    2010-12-10

    Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-{kappa}B activity was determined in human lung cancer cells. The roles of ROS and NF-{kappa}B in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-{kappa}B by reducing phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-{kappa}B. Antioxidants can significantly block the inhibitory effect of 1-BI.

  4. Insights into the structure activity relationship of mPGES-1 inhibitors: Hints for better inhibitor design.

    Gupta, Ashish; Aparoy, Polamarasetty

    2016-07-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane protein which plays crucial role in arachidonic acid metabolism, in the catalysis of PGH2 to PGE2. It is a potential drug target involved in variety of human cancers and inflammatory disorders. In the present study we made an attempt to identify crucial amino acid residues involved in the effective binding of its inhibitors at the active site. Molecular docking and Structure Activity Relationship (SAR) studies were performed. In the present study 127 inhibitors having significant variability in parent scaffold were considered. The results clearly indicated that in the GSH and PGH2 binding site Arg70, Arg73, Asn74, Glu77, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 consistently form crucial interactions with inhibitors of different classes/scaffolds. These findings are consistent with that of existing reports on the active site residues pivotal at mPGES-1 active site. Further analysis suggested that out of all important amino acid residues identified; Arg73, Asn74, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 play a crucial role in hydrogen and π-π interactions. The identified amino acid residues can act as target sites for the design and development of drug candidates against mPGES-1. PMID:27012893

  5. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

    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...

  6. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

  7. Defining the Product Chemical Space of Monoterpenoid Synthases.

    Tian, Boxue; Poulter, C Dale; Jacobson, Matthew P

    2016-08-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  8. Structure and Mechanism of Human UDP-xylose Synthase

    Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

    2012-01-01

    UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with ...

  9. Nitric oxide synthase is induced in sporulation of Physarum polycephalum

    Golderer, Georg; Werner, Ernst R.; Leitner, Stefan; Gröbner, Peter; Werner-Felmayer, Gabriele

    2001-01-01

    The myxomycete Physarum polycephalum expresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares < 39% amino acid identity with known NOSs but contains conserved binding motifs for all NOS cofactors. It lacks the sequence insert responsible for calcium dependence in the calcium-dependent NOS isoenzymes. NOS expression was strongly up-r...

  10. The Domain Responsible for Sphingomyelin Synthase (SMS) Activity

    Yeang, Calvin; Varsheny, Shweta; Wang, Renxiao; ZHANG, YA; Ye, Deyong; Jiang, Xian-Cheng

    2008-01-01

    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. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this s...

  11. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Roč. 183, č. 2 (2012), s. 189-192. ISSN 0166-6851 R&D Projects: GA ČR(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

  12. Identification of a family of animal sphingomyelin synthases

    Huitema, K.R.; van den Dikkenberg, J.; Brouwers, J.F.H.M.; Holthuis, J.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...

  13. The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms

    Dashuang Shi; Allewell, Norma M.; Mendel Tuchman

    2015-01-01

    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,...

  14. Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

    Escartin, Frédéric; Skouloubris, Stéphane; Liebl, Ursula; Myllykallio, Hannu

    2008-01-01

    We have investigated the hitherto unexplored possibility that differences in the catalytic efficiencies of thymidylate synthases ThyX and ThyA, enzymes that produce the essential DNA precursor dTMP, have influenced prokaryotic genome evolution. We demonstrate that DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10-fold decreased compared with species that contain the catalytically more efficient ThyA. Our statistical studies of >400 genomes ind...

  15. Unexpected link between polyketide synthase and calcium carbonate biomineralization

    Hojo, Motoki; Omi, Ai; Hamanaka, Gen; Shindo, Kazutoshi; Shimada, Atsuko; Kondo, Mariko; Narita, Takanori; Kiyomoto, Masato; Katsuyama, Yohei; Ohnishi, Yasuo; Irie, Naoki; Takeda, Hiroyuki

    2015-01-01

    Introduction Calcium carbonate biominerals participate in diverse physiological functions. Despite intensive studies, little is known about how mineralization is initiated in organisms. Results We analyzed the medaka spontaneous mutant, ha, defective in otolith (calcareous ear stone) formation. ha lacks a trigger for otolith mineralization, and the causative gene was found to encode polyketide synthase (pks), a multifunctional enzyme mainly found in bacteria, fungi, and plant. Subsequent expe...

  16. The Origins of Specificity in Polyketide Synthase Protein Interactions

    Thattai, Mukund; Burak, Yoram; Shraiman , Boris I.

    2007-01-01

    Polyketides, a diverse group of heteropolymers with antibiotic and antitumor properties, are assembled in bacteria by multiprotein chains of modular polyketide synthase (PKS) proteins. Specific protein–protein interactions determine the order of proteins within a multiprotein chain, and thereby the order in which chemically distinct monomers are added to the growing polyketide product. Here we investigate the evolutionary and molecular origins of protein interaction specificity. We focus on t...

  17. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar t...... into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein....

  18. Conservation and Role of Electrostatics in Thymidylate Synthase

    Divita Garg; Stephane Skouloubris; Julien Briffotaux; Hannu Myllykallio; Wade, Rebecca C.

    2015-01-01

    International audience Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs fr...

  19. Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene

    Shaik, Abjal Pasha; Alsaeed, Abbas H; Sultana, Asma

    2012-01-01

    The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther's disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref NP_0003...

  20. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Hyun Jo Koo

    Full Text Available 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-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-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 α-zingiberene/β-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, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  1. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases. PMID:26799780

  2. Rotation and structure of FoF1-ATP synthase.

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background. PMID:21524994

  3. From bacterial to human dihydrouridine synthase: automated structure determination

    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 β-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.

  4. From bacterial to human dihydrouridine synthase: automated structure determination

    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 β-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

  5. Role of nitric oxide synthases in early blood-brain barrier disruption following transient focal cerebral ischemia.

    Zheng Jiang

    Full Text Available The role of nitric oxide synthases (NOSs in early blood-brain barrier (BBB disruption was determined using a new mouse model of transient focal cerebral ischemia. Ischemia was induced by ligating the middle cerebral artery (MCA at its M2 segment and reperfusion was induced by releasing the ligation. The diameter alteration of the MCA, arterial anastomoses and collateral arteries were imaged and measured in real time. BBB disruption was assessed by Evans Blue (EB and sodium fluorescein (Na-F extravasation at 3 hours of reperfusion. The reperfusion produced an extensive vasodilation and a sustained hyperemia. Although expression of NOSs was not altered at 3 hours of reperfusion, L-NAME (a non-specific NOS inhibitor abolished reperfusion-induced vasodilation/hyperemia and significantly reduced EB and Na-F extravasation. L-NIO (an endothelial NOS (eNOS inhibitor significantly attenuated cerebral vasodilation but not BBB disruption, whereas L-NPA and 7-NI (neuronal NOS (nNOS inhibitors significantly reduced BBB disruption but not cerebral vasodilation. In contrast, aminoguanidine (AG (an inducible NOS (iNOS inhibitor had less effect on either cerebral vasodilation or BBB disruption. On the other hand, papaverine (PV not only increased the vasodilation/hyperemia but also significantly reduced BBB disruption. Combined treatment with L-NAME and PV preserved the vasodilation/hyperemia and significantly reduced BBB disruption. Our findings suggest that nNOS may play a major role in early BBB disruption following transient focal cerebral ischemia via a hyperemia-independent mechanism.

  6. The ubiquitin proteasome system and efficacy of proteasome inhibitors in diseases.

    Chitra, Selvarajan; Nalini, Ganesan; Rajasekhar, Gopalakrishnan

    2012-06-01

    In eukaryotes the ubiquitin proteasome pathway plays an important role in cellular homeostasis and also it exerts a critical role in regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription and immune response. Defects in these pathways have been implicated in a number of human pathologies. Inhibition of the ubiquitin proteasome pathway by proteasome inhibitors may be a rational therapeutic approach for various diseases, such as cancer and inflammatory diseases. Many of the critical cytokine and chemokine mediators of the progression of rheumatoid arthritis are regulated by nuclear factor kappa B (NF-κB). In peptidoglycan/polysaccharide-induced polyarthritis, proteasome inhibitors limit the overall inflammation, reduce NF-κB activation, decrease cellular adhesion molecule expression, inhibit nitric oxide synthase, attenuate circulating levels of proinflammatory cytokine interleukin-6 and reduce the arthritis index and swelling in the joints of the animals. Since proteasome inhibitors exhibit anti-inflammatory and anti proliferative effects, diseases characterized by both of these processes such as rheumatoid arthritis might also represent clinical opportunities for such drugs. The regulation of the proteasomal complex by proteasome inhibitors also has implications and potential benefits for the treatment of rheumatoid arthritis. This review summarizes the ubiquitin proteasome pathway, the structure of 26S proteasomes and types of proteasome inhibitors, with their actions, and clinical applications of proteasome inhibitors in various diseases. PMID:22709487

  7. [JAK2 inhibitors].

    Hernández Boluda, Juan Carlos; Gómez, Montse; Pérez, Ariadna

    2016-07-15

    Pharmacological inhibition of the kinase activity of JAK proteins can interfere with the signaling of immunomodulatory cytokines and block the constitutive activation of the JAK-STAT pathway that characterizes certain malignancies, including chronic myeloproliferative neoplasms. JAK inhibitors may, therefore, be useful to treat malignancies as well as inflammatory or immune disorders. Currently, the most significant advances have been made in the treatment of myelofibrosis, where these drugs may lead to a remarkable improvement in the control of hyperproliferative manifestations. However, available data suggest that this treatment is not curative of myelofibrosis. In general, JAK2 inhibition induces cytopaenias, with this being considered a class side-effect. By contrast, the extrahaematologic toxicity profile varies significantly among the different JAK inhibitors. At present, there are several clinical trials evaluating the combination of ruxolitinib with other drugs, in order to improve its therapeutic activity as well as reducing haematologic toxicity. PMID:27033437

  8. Benzoylurea Chitin Synthesis Inhibitors.

    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

  9. Alpha glucosidase inhibitors.

    Kalra, Sanjay

    2014-04-01

    Alpha glucosidase inhibitors (AGIs) are a unique class of anti-diabetic drugs. Derived from bacteria, these oral drugs are enzyme inhibitors which do not have a pancreato -centred mechanism of action. Working to delay carbohydrate absorption in the gastrointestinal tract, they control postprandial hyperglycaemia and provide unquestioned cardiovascular benefit. Specially suited for a traditional Pakistani carbohydrate-rich diet, AGIs have been termed the 'untapped diamonds' of diabetology. The use of these oral antidiabetic drugs (OADs) that target pathophysiology in the early stages of type 2 diabetes, notably to reduce postprandial hyperglycaemia and hyperinsulinaemia will inevitably increase with time. This review describes the history of their development, mechanism of action, basic and clinical pharmacology, and suggests practical, evidence-based guidance for their optimal use. PMID:24864650

  10. Update on Aromatase Inhibitors

    Seifert-Klauss V

    2015-01-01

    Full Text Available Aromatase inhibitors (AI block the last phase of estrogen production in many types of tissues which express the enzym aromatase, among them muscle, liver, adrenal, brain and fat. The enzyme catalyzes the last step of the biosynthesis of the estrogens, i. e. the aromatisation of testosterone to estradiol and of androstendion to estrone. Aromatase is localized in the membrane of the endoplasmatic reticulum and is also produced in the placenta and the gonads. Mutations in the gene CYP19A1, which codes for aromatase, can lead either to lack or excess of aromatase. Gene polymorphisms also influence the amount of bioavailable estrogen and bone density.br Indications: AI are approved for the treatment of postmenopausal women with hormone receptor positive breast cancer, both in the adjuvant setting as well as after recurrence and in progressive disease. In premenopausal and in perimenopausal women AI cause an increased sensitivity of the ovaries to follicle stimulating hormone (FSH and can thereby lead to a boosted estrogen answer – this effect is particularly pronounced in early perimenopausal women – so that these situations demand a combination with GnRH-analogue if AI treatment is to be initiated. Alternatively, tamoxifene may be used in premenopausal patients, with or without GnRH analogues. Treatment of premenopausal patients with hormone receptor positive breast cancer with aromatase inhibiting therapy alone constitutes an absolute contraindication. Aromatase inhibitors do not lead to estrogen receptor downregulation or block the receptor such as tamoxifene. An exceptional application is the application in reproductive medicine in women who do not have hormone receptor positive breast cancer: because of the higher sensitivity induced by AI-co-therapy, FSH-doses and -costs for assisted reproduction are reduced, and ovarian hyperstimulation syndrome (OHSS may be avoided. For premenopausal diseases which are said to be positively affected by

  11. Specific MAPK inhibitors prevent hyperglycemia-induced renal diseases in type 1 diabetic mouse model.

    Hong, Zhe; Hong, Zongyuan; Wu, Denglong; Nie, Hezhongrong

    2016-08-01

    Mitogen-activated protein kinase (MAPK) and renin-angiotensin system (RAS) play critical roles in the process of renal diseases, but their interaction has not been comprehensively discussed. In the present studies, we investigated the renoprotective effects of MPAK inhibitors on renal diseases in type 1 diabetic mouse model, and clarify the crosstalk among MAPK signaling. Type 1 diabetic mouse model was established in male C57BL/6 J mice, and treated with or without 10 mg/kg MAPK blockers, including ERK inhibitor PD98059, p38 inhibitor SB203850, and JNK inhibitor SP600125 for four weeks. Hyperglycemia induced renal injuries, but treating them with MAPK inhibitors significantly decreased glomerular volume and glycogen in renal tissues. Although slightly changed body weight and fasting blood glucose levels, MAPK inhibitors attenuated blood urea nitrogen, urea protein, and microalbuminuria. Administration also reduced the diabetes-induced RAS activation, including angiotensin II converting enzyme (c) and Ang II, which contributed to its renal protective effects in the diabetic mice. In addition, the anti-RAS of MAPK inhibitor treatment markedly reduced gene expression of tumor necrosis factor-α, interleukin-6, and inducible nitric oxide synthase, fibrotic accumulation, and transforming growth factor-β1 levels in renal tissues. Furthermore, chemical inhibitors and genetic siRNA results identified the crosstalk among the three MAPK signaling, and proved JNK signaling played a critical role in MAPK-mediated ACE pathway in hyperglycemia state. Collectively, these results support the therapeutic effects of MAPK-specific inhibitors, especially JNK inactivation, on hyperglycemia-induced renal damages. PMID:27389030

  12. Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase[S

    Lim, Sing Mei; Chen, Dan; Teo, Hsiangling; Roos, Annette; Jansson, Anna Elisabet; Nyman, Tomas; Trésaugues, Lionel; Pervushin, Konstantin; Nordlund, Pär

    2013-01-01

    Lipocalin prostaglandin D synthase (L-PGDS) regulates synthesis of an important inflammatory and signaling mediator, prostaglandin D2 (PGD2). Here, we used structural, biophysical, and biochemical approaches to address the mechanistic aspects of substrate entry, catalysis, and product exit of this enzyme. Structure of human L-PGDS was solved in a complex with a substrate analog (SA) and in ligand-free form. Its catalytic Cys 65 thiol group was found in two different conformations, each making...

  13. Comparative study of Chalcone synthase promoters across plant families

    Francisco Buitrago

    2009-10-01

    Full Text Available Estudio comparativo de promotores de la Chalcón Sintasa en diferentes familias de plantas In the post – genomic era the understanding of gene regulation has become a challenge and a research priority. In this research, we performed a comparative study of the regulator sequences of the chalcone synthase gene across plant families. Twenty-two sequences of chalcone synthase promoters were compared considering three regulator Cis elements: G-Box, H-Box and TATA Box. Our results show that these Cis elements are conserved among species and even at the family level. However, in some species all of the Cis elements were not found, showing that the expression and regulation of these promoters via the Cis elements can be variable. Additionally, a comparison between promoters from a species with a chalcone synthase multigene family showed that the duplicate genes are variable in the composition of the Cis elements, suggesting that these genes could be expressing in different ways. Key Words: Promoter; Chalcone synthase; Cis elements; Floral expression. Resumen En la era post-genómica, el entendimiento de la regulación génica se ha convertido en un reto y una prioridad de investigación. En este trabajo realizamos un estudio comparativo de las secuencias reguladoras del gen de la chalcón sintetasa de varias familias botánicas. Veintidós secuencias de promotores de Chalcone Synthase fueron comparados teniendo en cuenta tres elementos Cis reguladores: Caja-G, Caja-H y Caja-TATA, que podrían estar actuando como una sola unidad cooperativa. Nuestra comparación muestra que estos elementos puede que se conserven en algunas especies e inclusive que se conserven a nivel de familia. Sin embargo, en algunas especies no todos los elementos Cis fueron encontrados, mostrando que no todas las especies se regulan bajo los mismos parámetros. Adicionalmente, una comparación entre promotores de una misma especie con una familia de multigenes Chs, mostró que los

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

    Ramani, Shilpa; Patil, Nandadevi; Jayabaskaran, Chelliah

    2010-01-01

    The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accum...

  15. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host

    YAMADA, YUUKI; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin’ya, Kazuo; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match any known compounds in the spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowe...

  16. Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

    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...

  17. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

    Fliedner, Stephanie MJ; Yang, Chunzhang; Thompson, Eli; Abu-Asab, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S; Wesley, Robert; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the α and β subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase β (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs. PMID:26101719

  18. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    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. An Update of Microsomal Prostaglandin E Synthase-1 and PGE2 Receptors in Cardiovascular Health and Diseases.

    Yang, Guangrui; Chen, Lihong

    2016-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) selective inhibitors, are among the most widely used drugs to treat pain and inflammation. However, clinical trials have revealed that these inhibitors predisposed patients to a significantly increased cardiovascular risk, consisting of thrombosis, hypertension, myocardial infarction, heart failure, and sudden cardiac death. Thus, microsomal prostaglandin E (PGE) synthase-1 (mPGES-1), the key terminal enzyme involved in the synthesis of inflammatory prostaglandin E2 (PGE2), and the four PGE2 receptors (EP1-4) have gained much attention as alternative targets for the development of novel analgesics. The cardiovascular consequences of targeting mPGES-1 and the PGE2 receptors are substantially studied. Inhibition of mPGES-1 has displayed a relatively innocuous or preferable cardiovascular profile. The modulation of the four EP receptors in cardiovascular system is diversely reported as well. In this review, we highlight the most recent advances from our and other studies on the regulation of PGE2, particularly mPGES-1 and the four PGE2 receptors, in cardiovascular function, with a particular emphasis on blood pressure regulation, atherosclerosis, thrombosis, and myocardial infarction. This might lead to new avenues to improve cardiovascular disease management strategies and to seek optimized anti-inflammatory therapeutic options. PMID:27594972

  20. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  1. Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei.

    Vega, Claudia; Moreno-Carranza, Bibiana; Zamorano, Miriam; Quintanar-Stéphano, Andrés; Méndez, Isabel; Thebault, Stéphanie; Martínez de la Escalera, Gonzalo; Clapp, Carmen

    2010-12-01

    Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction. PMID:20943859

  2. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    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.

  3. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    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.

  4. Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP pathway enzyme expression in Catharanthus roseus.

    Mei Han

    Full Text Available In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS, a new (type I DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR, respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms, DXR, and hydroxymethylbutenyl diphosphate synthase (HDS were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

  5. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  6. Assembly line polyketide synthases: mechanistic insights and unsolved problems.

    Khosla, Chaitan; Herschlag, Daniel; Cane, David E; Walsh, Christopher T

    2014-05-13

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology. PMID:24779441

  7. Adhesion Development and the Expression of Endothelial Nitric Oxide Synthase

    David M. Svinarich

    2001-01-01

    Full Text Available Objective: This study was conducted to determine whether nitric oxide (NO, a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions.

  8. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.; Chorna V. I.

    2011-01-01

    The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic anal...

  9. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  10. Molecular similarity of MDR inhibitors

    Simon Gibbons; Mire Zloh

    2004-01-01

    Abstract: The molecular similarity of multidrug resistance (MDR) inhibitors was evaluated using the point centred atom charge approach in an attempt to find some common features of structurally unrelated inhibitors. A series of inhibitors of bacterial MDR were studied and there is a high similarity between these in terms of their shape, presence and orientation of aromatic ring moieties. A comparison of the lipophilic properties of these molecules has also been conducted suggesting that this ...

  11. Isolation and characterization of galactinol synthases from hybrid poplar

    Unda, Faride; Canam, Thomas; Preston, Lindsay; Mansfield, Shawn D

    2011-01-01

    The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 2.4.1.123), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba×grandi...

  12. Alternatively spliced neuronal nitric oxide synthase mediates penile erection

    Hurt, K. Joseph; Sezen, Sena F.; Champion, Hunter C.; Crone, Julie K.; Palese, Michael A.; Huang, Paul L; Sawa, Akira; Luo, Xiaojiang; Musicki, Biljana; Snyder, Solomon H.; Burnett, Arthur L.

    2006-01-01

    A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOS−/− animals might suffice for normal physiologic function. We show that the β splice variant of nNOS elicits normal erec...

  13. Modelling the evolution of the archaeal tryptophan synthase

    Merkl Rainer

    2007-01-01

    Abstract Background Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate. Results In order to...

  14. Isoflavone synthase genes in legumes and non-leguminous plants

    Pičmanová, Martina; Koblovská, R.; Lapčík, 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 ČR GA525/09/0994; GA ČR(CZ) GAP501/11/1462; GA MŠk(CZ) OC10054 Institutional support: RVO:61389030 Keywords : legumes * non-leguminous plants * isoflavone synthase Subject RIV: EF - Botanics

  15. Role of interferon regulatory factor-1 and mitogen-activated protein kinase pathways in the induction of nitric oxide synthase-2 in retinal pigmented epithelial cells.

    Faure, V; Hecquet, C; Courtois, Y; Goureau, O

    1999-02-19

    Bovine retinal pigmented epithelial cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Experiments were performed to investigate the involvement of interferon regulatory factor-1 (IRF-1) on NOS-2 induction and its regulation by NOS-2 inhibitors such as pyrrolidine dithiocarbamate (PDTC), an antioxidant, or protein kinase inhibitors. Analysis by transitory transfections showed that LPS, alone or with IFN-gamma, stimulated activity of the murine NOS-2 promoter fragment linked upstream of luciferase and its suppression by PDTC and by the different protein kinase inhibitors, genistein (tyrosine kinase inhibitor), PD98059 (mitogen-actived protein (MAP) kinase kinase inhibitor), and SB 203580 (p38 MAP inhibitor). Using specific antibodies, we have confirmed that extracellular signal-regulated kinases and p38 MAP kinase were activated by LPS and IFN-gamma in retinal pigmented epithelial cells. Analysis by reverse transcriptase-polymerase chain reaction, Western blot, and electrophoretic mobility shift assay demonstrated that IFN-gamma alone or combined with LPS induced an accumulation of IRF-1 mRNA and protein and IRF-1 DNA binding. Transfections assays with the IRF-1 promoter showed an induction of this promoter with IFN-gamma, potentiated by LPS. The decrease of LPS/IFN-gamma-induced IRF-1 promoter activity, IRF-1 synthesis, and IRF-1 activation, by PDTC, genistein, PD98059, and SB 203580, could explained in part the inhibition of the NOS-2 induction by these compounds. Our results demonstrate that IRF-1 is necessary for NOS-2 induction by LPS and IFN-gamma and that its synthesis requires the involvement of a redox-sensitive step, the activation of tyrosine kinases, and extracellular signal-regulated kinases 1/2 and p38 MAP kinases. PMID:9988718

  16. ACE INHIBITORS: A COMPREHENSIVE REVIEW

    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, α-blockers, β-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.

  17. Synthesis of Lysine Methyltransferase Inhibitors

    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.

  18. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  19. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

    Culbertson, Justin E; Chung, Dong hee; Ziebart, Kristin T; Espiritu, Eduardo; Toney, Michael D

    2015-04-14

    The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS. PMID:25710100

  20. Ethanol withdrawal induces anxiety-like effects: Role of nitric oxide synthase in the dorsal raphe nucleus of rats.

    Gonzaga, Natália Almeida; Batistela, Melissa Resende; Padovan, Diego; de Martinis, Bruno Spinosa; Tirapelli, Carlos Renato; Padovan, Cláudia Maria

    2016-05-01

    Nitric oxide (NO) mediated transmission in the dorsal raphe nucleus (DRN) has been shown to be involved in the modulation of anxiety-like behaviors. We investigated whether inhibition of nitric oxide synthase (NOS) in the DRN would prevent anxiety-like behavior induced by ethanol withdrawal. Male Wistar rats were treated with ethanol 2-6% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 h after ethanol discontinuation. Rats with a guide cannula aimed at the DRN received intra-DRN injections of the non-selective NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME), selective neuronal NOS (nNOS) inhibitor N(ω)-propyl-l-arginine (NPLA), or selective inhibitor of inducible NOS (iNOS) N-([3-(aminomethyl)phenyl] methyl) ethanimidamidedihydrochloride (1400W). Five minutes later, the animals were tested in the elevated plus maze (EPM). Plasma ethanol levels were determined by gas chromatography. There was a reduction in plasma ethanol levels 48 h after ethanol withdrawal. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the EPM with no change in the exploration of enclosed arms. Intra-DRN treatment with l-NAME (100 nmoles/0.2 μL) and 1400W (1 nmol/0.2 μL), but not NPLA (10 nmoles/0.2 μL) in the DRN attenuated the decrease in the exploration of the open arms of the EPM induced by ethanol withdrawal. The major new finding of the present study is that iNOS in the DRN plays a role in the anxiety-like behavior induced by ethanol withdrawal. PMID:27139232

  1. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    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.

  2. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny; Henriksen, Anette

    2007-01-01

    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...... 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...

  3. Tryptophan synthase of Phaeophyceae originated from the secondary host nucleus

    ZHANG Yalan; CHI Shan; WU Shuangxiu; LIU Cui; YU Jun; WANG Xumin; CHEN Shengping; LIU Tao

    2014-01-01

    Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In pro-karyotes, tryptophan synthase is a multi-enzyme complex, and it consists ofαandβsubunit which forms anα-ββ-αcomplex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophy-ceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rho-dophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect withαsubunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

  4. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  5. Polyketide synthases from poison hemlock (Conium maculatum L.).

    Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

    2015-11-01

    Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. PMID:26260860

  6. Chromosomal localization of the human and mouse hyaluronan synthase genes

    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.

  7. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa. Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P21212 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed

  8. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency

    GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methane sulfonate, and three clones deficient in thymidylate synthase activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS-c1 and TS-c3), whereas one clone (TS-c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS-c1 and TSminusc2 but decreased levels in TS-c3. Clone TSminusc3 was 32-, 750-, and >100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo

  9. Insights into the subunit in-teractions of the chloroplast ATP synthase

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  10. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    Jørgensen, Anne; Nielsen, John E; Nielsen, Betina Frydenlund;

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found in...

  11. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    Jørgensen, Anne; Nielsen, John E.; Nielsen, Betina F.;

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E-2 synthase (pges) and especially the lipocalin-type prostaglandin D-2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found ...

  12. Attachment of fatty acid substrate fragments to prostaglandin (PG) H synthase during reaction with arachidonate

    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

  13. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.;

    2012-01-01

    resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99...

  14. Inhibitors of the Hydrolytic Enzyme Dimethylarginine Dimethylaminohydrolase (DDAH): Discovery, Synthesis and Development.

    Murphy, Rhys B; Tommasi, Sara; Lewis, Benjamin C; Mangoni, Arduino A

    2016-01-01

    Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (l-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed. PMID:27187323

  15. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    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 C3 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

  16. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  17. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    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.

  18. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    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.

  19. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    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

  20. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are appl...

  1. Oxidized phospholipids induce ceramide accumulation in RAW 264.7 macrophages: role of ceramide synthases.

    Lingaraju M Halasiddappa

    Full Text Available Oxidized phospholipids (OxPLs, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs. These OxPLs are factors contributing to pro-atherogenic effects of oxidized LDLs (OxLDLs, including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C16-, C22-, and C24:0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.

  2. Acid sphingomyelinase gene knockout ameliorates hyperhomocysteinemic glomerular injury in mice lacking cystathionine-β-synthase.

    Krishna M Boini

    Full Text Available Acid sphingomyelinase (ASM has been implicated in the development of hyperhomocysteinemia (hHcys-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs and Asm mouse gene by cross breeding Cbs(+/- and Asm(+/- mice. Given that the homozygotes of Cbs(-/-/Asm(-/- mice could not survive for 3 weeks. Cbs(+/-/Asm(+/+, Cbs(+/-/Asm(+/- and Cbs(+/-/Asm(-/- as well as their Cbs wild type littermates were used to study the role of Asm(-/- under a background of Cbs(+/- with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs(+/- mice with different copies of Asm gene compared to Cbs(+/+ mice with different Asm gene copies. Cbs(+/-/Asm(+/+ mice had significantly increased renal Asm activity, ceramide production and O(2.(- level compared to Cbs(+/+/Asm(+/+, while Cbs(+/-/Asm(-/- mice showed significantly reduced renal Asm activity, ceramide production and O(2.(- level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs(+/-/Asm(-/- mice compared to Cbs(+/-/Asm(+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs(+/-/Asm(-/- mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs(+/-/Asm(-/- mice compared to Cbs(+/-/Asm(+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O(2.(- production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme

  3. In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

    YAN; Song; NIU; RongLi; WANG; Zheng; LIN; XiuKun

    2007-01-01

    Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

  4. Glycogen synthase kinase-3 in the etiology and treatment of mood disorders

    Richard Scott Jope

    2011-08-01

    Full Text Available The mood disorders major depressive disorder and bipolar disorder are prevalent, are inadequately treated, and little is known about their etiologies. A better understanding of the causes of mood disorders would benefit from improved animal models of mood disorders, which now rely on behavioral measurements. This review considers the limitations in relating measures of rodent behaviors to mood disorders, and the evidence from behavioral assessments indicating that glycogen synthase kinase-3 (GSK3 dysregulation promotes mood disorders and is a potential target for treating mood disorders. The classical mood stabilizer lithium was identified by studying animal behaviors and later was discovered to be an inhibitor of GSK3. Several mood-relevant behavioral effects of lithium in rodents have been identified, and most have now been shown to be due to its inhibition of GSK3. An extensive variety of pharmacological and molecular approaches for manipulating GSK3 are discussed, the results of which strongly support the proposal that inhibition of GSK3 reduces both depression-like and manic-like behaviors. Studies in human postmortem brain and peripheral cells also have identified correlations between alterations in GSK3 and mood disorders. Evidence is reviewed that depression may be associated with impaired inhibitory control of GSK3, and mania by hyper-stimulation of GSK3. Taken together, these studies provide substantial support for the hypothesis that inhibition of GSK3 activity is therapeutic for mood disorders. Future research should identify the causes of dysregulated GSK3 in mood disorders and the actions of GSK3 that contribute to these diseases.

  5. Metabolism of phenol and hydroquinone to reactive products by macrophage peroxidase or purified prostaglandin H synthase

    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

  6. Nuclear glycogen synthase kinase-3 β (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    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 β 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 β 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 β isoform probably is involved in gene transcription factors during R. micro plus embryo development

  7. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

    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.

  8. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  9. Nuclear glycogen synthase kinase-3 {beta} (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    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.

  10. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-06-01

    Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases. PMID:17557809

  11. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata. PMID:26750479

  12. Endothelium negatively modulates the vascular relaxation induced by nitric oxide donor, due to uncoupling NO synthase.

    Bonaventura, Daniella; Lunardi, Claure N; Rodrigues, Gerson J; Neto, Mário A; Vercesi, Juliana A; de Lima, Renata G; da Silva, Roberto S; Bendhack, Lusiane M

    2009-10-01

    Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO](3+)-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD(2)) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD(2): 6.17+/-0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD(2): 6.65+/-0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD(2): 6.46+/-0.10), by the superoxide anion (O(2)(-)) scavenger TIRON (pD(2): 6.49+/-0.08), and by an NOS cofactor BH(4) (pD(2): 6.80+/-0.10). The selective dye for O(2)(-) (DHE) shows that TERPY enhances O(2)(-) concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00+/-317.75) compared with the basal concentration (IF: 7760.67+/-381.50), and this enhancement is blocked by L-NAME (IF: 8892.33+/-1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63+/-0.17% and after TERPY+L-NAME: -4.63+/-0.14%). Considering that TERPY could induce uncoupling NOS, thus producing O(2)(-), we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA(2)) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD(2) INDO: 6.80+/-0.17 and SQ29548: 6.85+/-0.15, respectively). However, a selective prostaglandin F(2alpha) receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA(2) stable metabolite (TXB(2)), but this effect is blocked by L-NAME and TIRON. The

  13. Glycogen synthase kinase 3α and 3β have distinct functions during cardiogenesis of zebrafish embryo

    Sun Chi-Kuang

    2007-08-01

    Full Text Available Abstract Background Glycogen synthase kinase 3 (GSK3 encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3α (51 kDa and GSK3β (47 kDa. In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3α and GSK3β, making it difficult to identify an inhibitor that can be selective against GSK3α or GSK3β. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3α or GSK3β and uncover the isoform-specific roles that GSK3 plays during cardiogenesis. Results We blocked gsk3α and gsk3β translations by injection of morpholino antisense oligonucleotides (MO. Both gsk3α- and gsk3β-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3α- and gsk3β-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3α morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3β morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3β morphants. bmp4 expression in gsk3β morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3β morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3β might play a role in cardiac valve development through the Wnt/β-catenin pathway. Finally, the phenotypes of gsk3α mutant embryos cannot be rescued by gsk3β mRNA, and vice versa, demonstrating that GSK3α and GSK3β are

  14. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  15. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg. PMID:23467831

  16. Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase

    The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in λgt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases

  17. 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

    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

  18. Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases.

    Zhen, R. G.; Baykov, A. A.; Bakuleva, N. P.; Rea, P. A.

    1994-01-01

    The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) > dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum. PMID:12232069

  19. Atorvastatin enhance efficacy of mesenchymal stem cells treatment for swine myocardial infarction via activation of nitric oxide synthase.

    Lei Song

    Full Text Available BACKGROUND: In a swine model of acute myocardial infarction (AMI, Statins can enhance the therapeutic efficacy of mesenchymal stem cell (MSCs transplantation. However, the mechanisms remain unclear. This study aims at assessing whether atorvastatin (Ator facilitates the effects of MSCs through activation of nitric oxide synthase (NOS, especially endothelial nitric oxide synthase (eNOS, which is known to protect against ischemic injury. METHODS AND RESULTS: 42 miniswines were randomized into six groups (n = 7/group: Sham operation; AMI control; Ator only; MSC only, Ator+MSCs and Ator+MSCs+NG-nitrol-L-arginine (L-NNA, an inhibitor of NOS. In an open-heart surgery, swine coronary artery ligation and reperfusion model were established, and autologous bone-marrow MSCs were injected intramyocardium. Four weeks after transplantation, compared with the control group, Ator+MSCs animals exhibited decreased defect areas of both "perfusion" defined by Single-Photon Emission Computed Tomography (-6.2±1.8% vs. 2.0±5.1%, P = 0.0001 and "metabolism" defined by Positron Emission Tomography (-3.00±1.41% vs. 4.20±4.09%, P = 0.0004; Ejection fraction by Magnetic Resonance Imaging increased substantially (14.22±12.8% vs. 1.64±2.64%, P = 0.019. In addition, indices of inflammation, fibrosis, and apoptosis were reduced and survivals of MSCs or MSC-derived cells were increased in Ator+MSCs animals. In Ator or MSCs alone group, perfusion, metabolism, inflammation, fibrosis or apoptosis were reduced but there were no benefits in terms of heart function and cell survival. Furthermore, the above benefits of Ator+MSCs treatment could be partially blocked by L-NNA. CONCLUSIONS: Atorvastatin facilitates survival of implanted MSCs, improves function and morphology of infarcted hearts, mediated by activation of eNOS and alleviated by NOS inhibitor. The data reveal the cellular and molecular mechanism for anti-AMI therapy with a combination of statin and

  20. Reactivation of latent tuberculosis by an inhibitor of inducible nitric oxide synthase in an aerosol murine model

    Botha, Tania; Ryffel, Bernhard

    2002-01-01

    Exposure to Mycobacterium tuberculosis results in clinical tuberculosis only in a small percentage of healthy individuals. In most instances the bacilli are controlled by the immune system and survive in a latent state within granuloma. Immunosuppression, however, may result in reactivation of infection, resulting in clinical disease. Using a low-dose aerosol infection (30 colony-forming units) in mice, we describe a short-duration model for studying spontaneous and drug-induced reactivation of anti-tuberculous drug-treated, latent tuberculosis infection. Although a 4-week treatment with rifampicin and isoniazid reduced the number of bacilli to undetectable levels, the infection spontaneously reactivated following therapy. By contrast, an 8-week treatment period induced a state of latent infection, requiring immunosuppression to reactivate infection. Finally, a 12-week treatment period eliminated the bacilli completely and aminoguanidine did not induce reactivation of infection. In view of the fact that therapy in the selected protocol reduces the mycobacterial load to undetectable levels, the data suggest that an 8-week treatment period is necessary and sufficient to mount protective immunity in mice. PMID:12423311

  1. microRNA and human inducible nitric oxide synthase.

    Guo, Zhong; Geller, David A

    2014-01-01

    Regulation of human inducible nitric oxide synthase (iNOS) expression involves both transcriptional and posttranscriptional mechanisms. Human iNOS gene transcription is controlled in a cell type-specific manner by extracellular cytokines. Transcriptional regulation of human iNOS gene involves transcription factors NF-κB, Stat-1, AP-1, C/EBPβ, KLF6, Oct 1, and NRF. Important posttranscriptional mechanisms also regulate human iNOS mRNA stability through RNA binding proteins HuR, TTP, KSRP, and PABP. Recently, there are several miRNAs that were validated to regulate human and rodent iNOS gene expression. Among them, miR-939 and miR-26a were identified to bind with the human iNOS 3'-UTR and exert a translational blockade of human iNOS protein synthesis. PMID:25189382

  2. The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms

    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.

  3. The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms.

    Shi, Dashuang; Allewell, Norma M; Tuchman, Mendel

    2015-01-01

    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. PMID:26068232

  4. Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

    Kholodar, Svetlana A; Kohen, Amnon

    2016-07-01

    Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the de novo biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead. PMID:27327197

  5. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content...... expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review will...... discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  6. Urease Inhibitor Drug Treatment for Urea Cycle Disorders

    2016-01-28

    Ornithine Transcarbamylase Deficiency; Argininosuccinate Synthetase Deficiency (Citrullinemia); Argininosuccinic Acid Lyase Deficiency (Argininosuccinic Aciduria); Carbamyl-Phosphate Synthase I Deficiency

  7. Differential behaviour of four plant polysaccharide synthases in the presence of organic solvents.

    Kerry, M E; Gregory, A C; Bolwell, G P

    2001-08-01

    The behaviour of four membrane-bound glycosyl transferases involved in cell wall polysaccharide synthesis has been studied in relation to the effects of a graded series of organic solvents on their activity and type of product formed. Relative enzyme inhibition observed for some solvents was in direct relationship to the hydrophilicity of the product. This was in the order of arabinan synthase > callose synthase> xylan synthase > beta-1,4-glucan synthase. The former two were always inhibited, the xylan synthase rather less so. However, the beta-1,4-glucan synthase showed significant increases in substrate incorporation in the presence of solvents. A graded series of primary alcohols were much more effective in enhancing activity than acetone, ethyl acetate and dimethyl formamide. In the presence of the most effective solvent, methanol, there was considerable activation of beta-1,4-glucan production. This reciprocal nature of the behaviour of the beta-1,4- and beta-1,3-glucan synthases in organic solvent is supportive of recent molecular data that the two types of glucans are catalysed by separate enzyme systems. However, the results reported here do not totally negate the proposition that either enzyme is capable of synthesising the other linkage in minor amounts in vitro. PMID:11430978

  8. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. PMID:23911730

  9. Exposure to Diflubenzuron Results in an Up-Regulation of a Chitin Synthase 1 Gene in Citrus Red Mite, Panonychus citri (Acari: Tetranychidae

    Wen-Kai Xia

    2014-02-01

    Full Text Available Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor, which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult. When larvae were exposed to diflubenzuron (DFB for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.

  10. Exposure to diflubenzuron results in an up-regulation of a chitin synthase 1 gene in citrus red mite, Panonychus citri (Acari: Tetranychidae).

    Xia, Wen-Kai; Ding, Tian-Bo; Niu, Jin-Zhi; Liao, Chong-Yu; Zhong, Rui; Yang, Wen-Jia; Liu, Bin; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB. PMID:24590130

  11. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms. PMID:26941091

  12. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    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.

  13. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  14. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    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.

  15. Selective inhibition of inducible nitric oxide synthase reduces neurological deficit but not cerebral edema following traumatic brain injury.

    Louin, G; Marchand-Verrecchia, C; Palmier, B; Plotkine, M; Jafarian-Tehrani, M

    2006-02-01

    The role of inducible nitric oxide synthase (iNOS) in cerebral edema and neurological deficit following traumatic brain injury (TBI) is not yet clear-cut. Therefore, the aim of this study was to investigate the effect of three different iNOS inhibitors on cerebral edema and functional outcome after TBI. First, the time courses of blood--brain barrier (BBB) breakdown, cerebral edema, and neurological deficit were studied in a rat model of fluid percussion-induced TBI. The permeability of BBB to Evans blue was increased from 1 h to 24 h after TBI. Consistently, a significant increase in brain water content (BWC) was observed at 6 and 24 h post-TBI. A deficit in sensorimotor neurological functions was also observed from 6 h to 7 days with a maximum 24 h after TBI. Second, a single dose of aminoguanidine (AG; 100 mg/kg, i.p.), L-N-iminoethyl-lysine (L-NIL; 20 mg/kg, i.p.), or N-[3-(aminomethyl)benzyl]acetamide (1400W; 20 mg/kg, s.c.) was administered at 6 h post-TBI. Treatment with AG reduced by 71% the increase in BWC evaluated at 24 h, while L-NIL and 1400W had no effect. In contrast, the three iNOS inhibitors reduced the neurological deficit from 30% to 40%. Third, 1400W (20 mg/kg, s.c.) was administered at 5 min, 8 and 16 h post-TBI. Although this treatment paradigm had no effect on cerebral edema evaluated at 24 h, it significantly reduced the neurological deficit and iNOS activity. In conclusion, iNOS contributes to post-TBI neurological deficit but not to cerebral edema. The beneficial effect of iNOS inhibitors is not due to their anti-edematous effect, and the reduction of cerebral edema by AG is unlikely related to iNOS inhibition. The 6 h therapeutic window of iNOS inhibitors could allow their use in the treatment of functional deficit at the acute phase of TBI. PMID:16242164

  16. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.;

    2008-01-01

    The possible associations between microsatellite instability, homocysteine and thymidylate synthase were investigated in tumors and plasma from 130 patients with colorectal cancer. Other analyses included thymidylate synthase and 5,10-methylene-tetrahydrofolate reductase gene polymorphisms......, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  17. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-01-01

    ? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a...... sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears...

  18. [Pharmacology of bone resorption inhibitor].

    Menuki, Kunitaka; Sakai, Akinori

    2015-10-01

    Currently, bone resorption inhibitor is mainly used for osteoporosis. A number of these agents have been developed. These pharmacological action are various. Bisphosphonate inhibit functions of the osteoclasts by inducing apoptosis. On the one hand, RANK-ligand inhibitor and selective estrogen receptor modulator inhibit formation of osteoclasts. It is important to understand these pharmacological action for the selection of the appropriate medicine. PMID:26529923

  19. Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

    Glintborg, Dorte; Højlund, Kurt; Andersen, Nicoline Resen;

    2008-01-01

    CONTEXT: Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. SUBJECTS AND METHODS: We...... investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were...... metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry. RESULTS: Reduced insulin-mediated glucose disposal (P < 0.05) was associated with a lower insulin-stimulated GS activity in PCOS patients (P < 0.05), compared with controls. This was, in part, explained by absent insulin...

  20. Reduced nitric oxide-mediated relaxation and endothelial nitric oxide synthase expression in the tail arteries of streptozotocin-induced diabetic rats.

    Mokhtar, Siti Safiah; Vanhoutte, Paul M; Leung, Susan Wai Sum; Suppian, Rapeah; Yusof, Mohd Imran; Rasool, Aida Hanum Ghulam

    2016-02-15

    Diabetes is associated with endothelial dysfunction, which is characterized by impaired endothelium-dependent relaxations. The present study aimed to examine the role of nitric oxide (NO), prostacyclin and endothelium-dependent hyperpolarization (EDH), in the relaxation of ventral tail arteries of rats under diabetic conditions. Relaxations of tail arteries of control and diabetic rats were studied in wire myograph. Western blotting and immunostaining were used to determine the presence of proteins. Acetylcholine-induced relaxations were significantly smaller in arteries of diabetic compared to control rats (Rmax; 70.81±2.48% versus 85.05±3.15%). Incubation with the combination of non-selective cyclooxygenase (COX) inhibitor, indomethacin and potassium channel blockers, TRAM 34 and UCL 1684, demonstrated that NO-mediated relaxation was attenuated significantly in diabetic compared to control rats (Rmax; 48.47±5.84% versus 68.39±6.34%). EDH-type (in the presence of indomethacin and NO synthase inhibitor, LNAME) and prostacyclin-mediated (in the presence of LNAME plus TRAM 34 and UCL 1684) relaxations were not significantly reduced in arteries of diabetic compared to control rats [Rmax: (EDH; 17.81±6.74% versus 34.16±4.59%) (prostacyclin; 15.85±3.27% versus 17.23±3.75%)]. Endothelium-independent relaxations to sodium nitroprusside, salbutamol and prostacyclin were comparable in the two types of preparations. Western blotting and immunostaining indicated that diabetes diminished the expression of endothelial NO synthase (eNOS), while increasing those of COX-1 and COX-2. Thus, since acetylcholine-induced NO-mediated relaxation was impaired in diabetes because of reduced eNOS protein expression, pharmacological intervention improving NO bioavailability could be useful in the management of diabetic endothelial dysfunction. PMID:26825543

  1. Expression characteristics of CS-ACS1, CS-ACS2 and CS-ACS3, three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in cucumber (Cucumis sativus L.) fruit under carbon dioxide stress.

    Mathooko, F M; Mwaniki, M W; Nakatsuka, A; Shiomi, S; Kubo, Y; Inaba, A; Nakamura, R

    1999-02-01

    We investigated the expression pattern of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes, CS-ACS1, CS-ACS2 and CS-ACS3 in cucumber (Cucumis sativus L.) fruit under CO2 stress. CO2 stress-induced ethylene production paralleled the accumulation of only CS-ACS1 transcripts which disappeared upon withdrawal of CO2. Cycloheximide inhibited the CO2 stress-induced ethylene production but superinduced the accumulation of CS-ACS1 transcript. At higher concentrations, cycloheximide also induced the accumulation of CS-ACS2 and CS-ACS3 transcripts. In the presence of CO2 and cycloheximide, the accumulation of CS-ACS2 transcript occurred within 1 h, disappeared after 3 h and increased greatly upon withdrawal of CO2. Inhibitors of protein kinase and types 1 and 2A protein phosphatases which inhibited and stimulated, respectively, CO2 stress-induced ethylene production had little effect on the expression of these genes. The results presented here identify CS-ACS1 as the main ACC synthase gene responsible for the increased ethylene biosynthesis in cucumber fruit under CO2 stress and suggest that this gene is a primary response gene and its expression is under negative control since it is expressed by treatment with cycloheximide. The results further suggest that the regulation of CO2 stress-induced ethylene biosynthesis by reversible protein phosphorylation does not result from enhanced ACC synthase transcription. PMID:10202812

  2. l-Cysteine enhances nutrient absorption via a cystathionine-β-synthase-derived H2 S pathway in rodent jejunum.

    Xiao, Ailin; Li, Jing; Liu, Tianjian; Liu, Zhuxi; Wei, Chuanfei; Xu, Xiaomeng; Li, Qin; Li, Jingxin

    2016-05-01

    Hydrogen sulphide (H2 S) is generated endogenously from l-cysteine (l-Cys) by the enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). In addition, l-Cys is commonly used as a precursor in the food and pharmaceutical industries. The aim of the present study is to determine whether l-Cys regulates intestinal nutrient transport. To that end, the presence of CBS and CSE in the jejunum epithelium was assessed by immunohistochemistry, Western blotting and the methylene blue assay. In addition, in vivo l-Cys (100 mg/kg, administered immediately after the glucose load) significantly increased blood glucose levels 30 min after the oral administration of glucose to mice. This effect of l-Cys was completely blocked by amino-oxyacetic acid (AOA; 50 mg/kg; administered at the same time as l-Cys) an inhibitor of CBS. Measurements of the short-circuit current (Isc ) in the rat jejunum epithelium revealed that l-Cys (1 mmol/L; 6 min before the administration of l-alanine) enhances Na(+) -coupled l-alanine or glucose transport, and that this effect is inhibited by AOA (1 mmol/L;10 min before the administration of l-Cys), but not d,l-propargylglycine (PAG;1 mmol/L; 10 min before the administration of l-Cys), a CSE inhibitor. Notably, l-Cys-evoked enhancement of nutrient transport was alleviated by glibenclamide (Gli;0.1 mmol/L; 10 min before the administration of l-Cys), a K(+) channel blocker. Together, the data indicate that l-Cys enhances jejunal nutrient transport, suggesting a new approach to future treatment of nutrition-related maladies, including a range of serious health consequences linked to undernutrition. PMID:26901099

  3. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression. PMID:16651461

  4. Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells.

    Shen, Jiangang; Lee, Waisin; Li, Yue; Lau, Chi Fai; Ng, Kwong Man; Fung, Man Lung; Liu, Ke Jian

    2008-10-01

    Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells. PMID:18717816

  5. De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy.

    Masini, T.; Pilger, J.; Kroezen, B.; Illarionov, B.; Lottmann, P.; Fischer, M.; Griesinger, C.; Hirsch, A.

    2014-01-01

    We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans D...

  6. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. PMID:25447532

  7. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

    Jung, W; Yu, O; Lau, S M; O'Keefe, D P; Odell, J; Fader, G; McGonigle, B

    2000-02-01

    Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes. PMID:10657130

  8. Molecular size estimation of plasma membrane β-glucan synthase from red beet root

    Cellulose and cell wall β-D-glucans in higher plants are thought to be synthesized by the plasma membrane enzyme, β-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

  9. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael (MSU); (NWU)

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  10. Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes.

    Heider, Johann; Szaleniec, Maciej; Martins, Berta M; Seyhan, Deniz; Buckel, Wolfgang; Golding, Bernard T

    2016-01-01

    The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield (R)-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting p-cresol, 2-methylnaphthalene or n-alkanes. PMID:26959246

  11. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  12. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

    Cathy W C Bi

    Full Text Available Fo Shou San (FSS is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong and Angelicae Sinensis Radix (ASR; Danggui in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs, and it also stimulated the production of nitric oxide (NO as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+ levels in HUVECs, and the Ca(2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+ and a reduction of ROS.

  13. The nopaline synthase (nos) promoter is inducible by UV-B radiation through a pathway dependent on reactive oxygen species

    The molecular mechanism of plant response to UV-B radiation was studied using the nopaline synthase (nos) promoter, which has been shown to be inducible by methyl jasmonate (MJ) and reactive oxygen species (ROS). In the leaves of transgenic tobacco (Nicotiana tabacum L.) plants that carried a fusion between the nos promoter and the chloramphenicol acetyltransferase (cat) gene, 2 h of UV-B treatment resulted in a transient increase in the level of cat mRNA, a maximum being reached at 6 h after the UV-B treatment. It was also found that MJ and UV-B enhance nos promoter expression via separate pathways. Diethyldithiocarbamic acid, a potent inhibitor of jasmonate production, had little effect on UV-B stimulation of the nos promoter. In contrast, antioxidants, such as dimethylthiourea, reduced glutathione, cysteine, N-acetylcysteine and DTT, blocked UV-B induction of the nos promoter, but did not affect MJ induction of the nos promoter. These results suggest that UV-B induction of the nos promoter is mediated via a pathway that requires reactive oxygen species and is distinct from the jasmonate or MJ mediating pathway. (author)

  14. GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway.

    Li, Qiong; Leija, Christopher; Rijo-Ferreira, Filipa; Chen, Jun; Cestari, Igor; Stuart, Kenneth; Tu, Benjamin P; Phillips, Margaret A

    2015-09-01

    The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5'-monophosphate (GMP) formation: conversion from xanthosine-5'-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche. PMID:26043892

  15. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    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.

  16. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    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.

  17. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    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

  18. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    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

  19. Curcumin inhibits intracellular fatty acid synthase and induces apoptosis in human breast cancer MDA-MB-231 cells.

    Fan, Huijin; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; Sun, Jia; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-05-01

    High levels of fatty acid synthase (FAS) expression have been found in many tumors, including prostate, breast, and ovarian cancers, and inhibition of FAS has been reported to obstruct tumor growth in vitro and in vivo. Curcumin is one of the major active ingredients of Curcuma longa, which has been proven to inhibit the growth of cancer cells. In the present study, we investigated the potential activity of curcumin as a FAS inhibitor for chemoprevention of breast cancer. As a result, curcumin induced human breast cancer MDA-MB-231 cell apoptosis with the half-inhibitory concentration value of 3.63 ± 0.26 µg/ml, and blocked FAS activity, expression and mRNA level in a dose-dependent manner. Curcumin also regulated B-cell lymphoma 2 (Bcl-2), Bax and p-Akt protein expression in MDA-MB-231 cells. Moreover, FAS knockdown showed similar effect as curcumin. All these results suggested that curcumin may induce cell apoptosis via inhibiting FAS. PMID:26985864

  20. Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

    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.