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The localization of nitrite reductase, glutamate synthase and malate metabolism enzymes in Pisum arvense L. roots  

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Full Text Available Centrifugation of a homogenate made from Pisum arvense L. roots in a sucrose density gradient enabled the separation of the plastid fraction from mitochondria and microsomes. The presence of nitrite reductase and glutamate synthase was demonstrated in the plastids. Malic enzyme activity was not linked with any organelle fraction and was found only in the cytosol. High malate dehydrogenase activity was found in the mitochondria fraction, although its activity was also determined in plastids. The results suggest that malic acid metabolism in plastids may be the source of reduced pyridine nucleotides for reactions catalysed by nitrite reductase and glutamate synthase.

Genowefa Kubik-Dobosz

1985-03-01

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The glcB locus of Rhizobium leguminosarum VF39 encodes an arabinose-inducible malate synthase.  

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In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia. PMID:12489782

García-de los Santos, Alejandro; Morales, Alejandro; Baldomá, Laura; Clark, Scott R D; Brom, Susana; Yost, Christopher K; Hernández-Lucas, Ismael; Aguilar, Juan; Hynes, Michael F

2002-10-01

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Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon  

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Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH4)SO4, glyoxylate and high concentration of MgCl2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

1989-01-01

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Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis.  

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The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction. PMID:21332878

Nakazawa, Masami; Nishimura, Masaaki; Inoue, Kengo; Ueda, Mitsuhiro; Inui, Hiroshi; Nakano, Yoshihisa; Miyatake, Kazutaka

2011-01-01

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Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G  

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Full Text Available Abstract Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA comprises ~530 residues, the G isoform (MSG is ~730 residues, and this third isoform (MSH-halophilic is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a ?8/?8 (TIM barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved. Sequence analysis in light of the structure indicates that additional members of isoform H likely exist in the databases but have been misannotated.

Thomas Geoffrey C

2011-05-01

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Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).  

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Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible. PMID:12493858

Pua, Eng-Chong; Chandramouli, Sumana; Han, Ping; Liu, Pei

2003-01-01

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Long-range effects in anion-? interactions: their crucial role in the inhibition mechanism of Mycobacterium tuberculosis malate synthase.  

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The glyoxylate shunt is an anaplerotic bypass of the traditional Krebs cycle. It plays a prominent role in Mycobacterium tuberculosis virulence, so it can be exploited for the development of antitubercular therapeutics. The shunt involves two enzymes: isocitrate lyase (ICL) and malate synthase (GlcB). The shunt bypasses two steps of the tricarboxylic acid cycle, allowing the incorporation of carbon, and thus, refilling oxaloacetate under carbon-limiting conditions. The targeting of ICL is complicated; however, GlcB, which accommodates the pantothenate tail of acetyl-CoA in the active site, is easier to target. A catalytic Mg(2+) unit is located at the bottom of the cavity, and plays a very important role. Recently, the development of effective antituberculosis drugs based on phenyldiketo acids (PDKAs) has been reported. Interestingly, all the crystal structures of GlcB-inhibitor complexes exhibit close contact between the carboxylate of Asp633 and the face of the aromatic ring of the inhibitor. Remarkably, the replacement of the phenyl ring in PDKA by aliphatic moieties yields inactive inhibitors, suggesting that the aromatic moiety is crucial for inhibition. However, the aromatic ring of PDKA is not electron-deficient, and consequently, the anion-? interaction is expected to be very weak (dominated only by polarization effects). Herein, through a combination analysis of the recent X-ray structures of GlcB-PDKA complexes retrieved from the protein data bank (PDB) and computational ab?initio studies (RI-MP2/def2-TZVP level of theory), we demonstrate the prominent role of the Mg(2+) ion in the active site, which promotes long-range enhancement of the anion-? interaction. PMID:24740694

Bauzá, Antonio; Quiñonero, David; Deyà, Pere M; Frontera, Antonio

2014-06-01

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Anaerobic induction of isocitrate lyase and malate synthase in submerged rice seedlings indicates the important metabolic role of the glyoxylate cycle.  

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The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2 compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings, we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment. The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions. PMID:15944756

Lu, Ying; Wu, Yong-Rui; Han, Bin

2005-06-01

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Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G  

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Abstract Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ...

Bracken Colten D; Neighbor Amber M; Lamlenn Kenneth K; Thomas Geoffrey C; Schubert Heidi L; Whitby Frank G; Howard Bruce R

2011-01-01

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The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin  

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Full Text Available Abstract Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM. This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.

Pereira Maristela

2009-12-01

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Malate synthase gene AoMls in the nematode-trapping fungus Arthrobotrys oligospora contributes to conidiation, trap formation, and pathogenicity.  

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Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the ?AoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the ?AoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the ?AoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the ?AoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora. PMID:24323290

Zhao, Xinying; Wang, Yunchuan; Zhao, Yong; Huang, Yan; Zhang, Ke-Qin; Yang, Jinkui

2014-03-01

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MALATE DEHYDROGENASE ACTIVITY POST EXPOSURE RECOVERY FROM LEAD INTOXICATED FRESHWATER FISH ANABAS TESTUDINEUS  

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Full Text Available Malate dehydrogenase activity are important amongst the several enzymes available in the cells, Carbohydrates play an important role in the cellular process  Under extreme stress conditions, carbohydrate enzyme such as Malate dehydrogenase  have been known to act as the energy supplier in metabolic pathways and biochemical reactions. In the present investigation fish  treated with an equitoxic dose of 10 ppm of lead nitrate and lead acetate intoxicated fish After a period of 15 days of exposure a batch from lead nitrate exposed fish and a batch from lead acetate exposed fish were transfered to lead-free water. Fishes were scarified on 1, 4, 8, 12 and 15 days for the analysis of of recovery pattern  in tissues viz. liver, muscle, kidney, gill and brain .It is found that lead toxicated fishes were recovered after 15 days depends upon physical condition of the fish.

Afsar Shaikh

2012-03-01

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Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase  

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Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10?3 M and 0.87?mM?L?1?min?1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism.

Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing

2014-01-01

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Transhydrogenase activities and malate dismutation linked to fumarate reductase system in the filarial parasite Setaria digitata.  

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Setaria digitata, a cattle filarial parasite, similar to human filarial parasites, possesses significant activities of the 4 transhydrogenases namely NADH-NAD+, NADPH-NAD+, NADH-NADP+, and NADPH-NADP+ in the sonicated mitochondria like particles. The transhydrogenases appear to regulate the metabolic pathways of the parasite in response to the presence of adenyl nucleotides and are non-energy linked. Observations on the transhydrogenase and fumarate reductase activities show the existence of a protein bound NAD in the MLP and a linkage between the fumarate reductase system and malic enzyme through transhydrogenases. The malate dismutation reaction is the result of malic and fumarase enzyme activities. Fumarase and fumarate reductase activities result in succinate formation under anaerobic conditions showing major energy production at the fumarate reductase site. The existence of acetate kinase, phosphotransacetylase, pyruvate carboxylase, propionyl CoA carboxylase and CoA transferase enzymes in the mitochondrial system shows the presence of other energy producing sites in the parasite. The transhydrogenase system, NAD+/NADP+ malic enzyme, fumarase and fumarate reductase are the key enzymes of, production of reducing power for synthetic reactions and regulation of oxidative and reductive stages of the mitochondrial system. Hence, specific drugs targeted against this interconnected complex enzyme system, will be very effective in the control of filarial parasites. PMID:7558563

Unnikrishnan, L S; Raj, R K

1995-07-01

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The Membrane Topology of ALMT1, an Aluminum-Activated Malate Transport Protein in Wheat (Triticum aestivum)  

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The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used ...

Motoda, Hirotoshi; Sasaki, Takayuki; Kano, Yoshio; Ryan, Peter R.; Delhaize, Emmanuel; Matsumoto, Hideaki; Yamamoto, Yoko

2007-01-01

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Unique animal prenyltransferase with monoterpene synthase activity  

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

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

2009-06-01

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A Bacillus subtilis malate dehydrogenase gene.  

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A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.

Jin, S.; Jesu?s-berri?os, M.; Sonenshein, A. L.

1996-01-01

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Evidence of enhancement of malate-aspartate shuttle activity in beta cells of streptozotocin-induced non-insulin-dependent diabetic rats.  

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Glucose-induced insulin secretion is selectively impaired in beta cells from animals with non-insulin-dependent diabetes mellitus (NIDDM). This study was performed to clarify whether the malate-aspartate shuttle among the glucose metabolic pathways is intact in beta cells of NIDDM rats. The insulin secretory capacity of the islets and the K(ATP) channel activity in single beta cells were measured in control and NIDDM rats injected with streptozotocin (STZ) during the neonatal period, using a radioimmunoassay and patch-clamp technique. The increase of insulin secretion induced by 11.1 mmol/L glucose or 10 mmol/L dihydroxyacetone (DHA) was significantly reduced in NIDDM islets, suggesting an impaired glycerol-phosphate shuttle. The application of glyceraldehyde (10 mmol/L) in NIDDM or control islets elicited an increase in insulin secretion, but the difference between the 2 groups was indistinguishable. On the contrary, the increase of insulin secretion and the inhibition of K(ATP) channel activity induced by aspartate, which preferentially participates in the malate-aspartate shuttle, were significantly greater in NIDDM versus the control. However, intracellularly applied aspartate in the inside-out mode did not inhibit K(ATP) channel activity. These findings show that malate-aspartate shuttle activity is potentiated in pancreatic beta cells of NIDDM rats, suggesting the development of a compensatory mechanism for the reduced activity of the glycerol-phosphate shuttle in NIDDM. PMID:10647070

Song, D K; Ahn, Y H; Bae, J H; Park, W K; Hong, Y S; Ho, W K; Earm, Y E

2000-01-01

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

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

Muhitch, M J

1988-01-01

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Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.  

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The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.

Homann, M. J.; Henry, S. A.; Carman, G. M.

1985-01-01

 
 
 
 
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Profile of Enzyme Activity And Growth of Wood Rotting Fungi In Metal Ion Containing Media  

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Key enzyme of the glyoxylate cycle (isocilrate lyase and malate synthase), tricarboxylic acid cycle (isocitrate dehydrogenase), and GABA route (glutamate dehydrogenase) were measured in mycelia of Fomitopsis palustris grown on metal ion containing media. A higher isocitrate lyase activity was observed on the initial stage of cultivation at all linds of media tested. while malate synthase was constant throughout the incubation period. The activity of isocitrate dehydrogenase and glutamate dehy...

2008-01-01

22

Oxidase Activity of a Flavin-Dependent Thymidylate Synthase  

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Flavin-dependent thymidylate synthases (FDTSs) catalyze the production of 2?-deoxythymidine-5?-monophosphate (dTMP) from 2?-deoxyuridine-5?-monophosphate (dUMP) and N5, N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a flavin adenine dinucleotide (FAD) coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an...

Wang, Zhen; Chernyshev, Anatoly; Koehn, Eric M.; Manuel, Antonio; Lesley, Scott A.; Kohen, Amnon

2009-01-01

23

Malate and fumarate extend lifespan in Caenorhabditis elegans.  

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Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in the nematode C. elegans. Malate can be synthesized from fumarate by the enzyme fumarase and further oxidized to oxaloacetate by malate dehydrogenase with the accompanying reduction of NAD. Addition of fumarate also extended lifespan, but succinate addition did not, although all three intermediates activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The glyoxylate shunt, an anabolic pathway linked to lifespan extension in C. elegans, reversibly converts isocitrate and acetyl-CoA to succinate, malate, and CoA. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductase F48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Reduction of fumarate to succinate is coupled with the oxidation of FADH2 to FAD. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Malate also increased NADPH, NAD, and the NAD/NADH ratio. Fumarate reduction, glyoxylate shunt activity, and mild mitochondrial uncoupling likely contribute to the lifespan extension induced by malate and fumarate by increasing the amount of oxidized NAD and FAD cofactors. PMID:23472183

Edwards, Clare B; Copes, Neil; Brito, Andres G; Canfield, John; Bradshaw, Patrick C

2013-01-01

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Activated glycogen synthase-3? suppresses cardiac hypertrophy in vivo  

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The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) ph...

Antos, Christopher L.; Mckinsey, Timothy A.; Frey, Norbert; Kutschke, William; Mcanally, John; Shelton, John M.; Richardson, James A.; Hill, Joseph A.; Olson, Eric N.

2002-01-01

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Changes in Activities of Sucrose Synthase and Sucrose Phosphate Synthase and Sugar Content During Postharvest Senescence in Two Broccoli Cultivars  

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Changes in sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities and sugar content during postharvest senescence in broccoli (Brassica oleracea L. cvs. `Hartland` and `Sairin`) were studied. The first change in color became apparent between 2 and 3 d but whole head yellowed after 5 d of storage. The SS activity increased gradually in all portions although with few fluctuations in branchlets of both cultivars at the end of the storage time. In case of SPS, there w...

Bimal Kumar Pramanik; Toshiyuki Matsui; Haruo Suzuki; Yusuke Kosugi

2004-01-01

26

Nitric oxide synthase expression and enzymatic activity in multiple sclerosis.  

DEFF Research Database (Denmark)

We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant iNOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age and sex showed no such changes. Our data support the hypothesis that NO is a pathogenic factor in MS, and that NOS IR is strongly expressed in brain regions appearing normal by MRI

Broholm, H; Andersen, B

2004-01-01

27

Oxidase activity of a flavin-dependent thymidylate synthase.  

Science.gov (United States)

Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O(2) to H(2)O(2), using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O(2) compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 microm), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex. PMID:19459936

Wang, Zhen; Chernyshev, Anatoly; Koehn, Eric M; Manuel, Tony D; Lesley, Scott A; Kohen, Amnon

2009-05-01

28

CDP-diacylglycerol synthase activity in Clostridium perfringens.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) ...

Carman, G. M.; Zaniewski, R. L.; Cousminer, J. J.

1982-01-01

29

Derepression of nitrogenase by addition of malate to cultures of Rhodospirillum rubrum grown with glutamate as the carbon and nitrogen source.  

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Rhodospirillum rubrum grown in continuous culture with glutamate as the sole fixed C and N source produced no nitrogenase, and the cultures were characterized by high extracellular ammonium concentrations. Addition of organic acids derepressed nitrogenase. Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, malate dehydrogenase, nitrogenase, and ammonium were assayed before and after malate addition.

Hoover, T. R.; Ludden, P. W.

1984-01-01

30

Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.  

Science.gov (United States)

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing. PMID:24188189

Ligaba, Ayalew; Dreyer, Ingo; Margaryan, Armine; Schneider, David J; Kochian, Leon; Piñeros, Miguel

2013-12-01

31

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase.  

Science.gov (United States)

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. PMID:24584238

Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

2014-05-01

32

Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex.  

Science.gov (United States)

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate. PMID:1899027

Kirschner, K; Lane, A N; Strasser, A W

1991-01-15

33

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

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

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

1988-01-01

34

Regulation of glycogen synthase and phosphorylase activities by glucose and insulin in human skeletal muscle.  

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We examined the insulin dose-response characteristics of human muscle glycogen synthase and phosphorylase activation. We also determined whether increasing the rate of glucose disposal by hyperglycemia at a fixed insulin concentration activates glycogen synthase. Physiological increments in plasma insulin but not glucose increased the fractional activity of glycogen synthase. The ED50: s for insulin stimulation of whole body and forearm glucose disposal were similar and unaffected by glycemia...

1987-01-01

35

Structural basis for glucose-6-phosphate activation of glycogen synthase  

Energy Technology Data Exchange (ETDEWEB)

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D. (Indiana-Med)

2010-11-22

36

Activity of endothelial nitric oxide synthase: substrates, modulators and products  

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In this thesis, the activity of endothelial nitric oxide synthase (eNOS) and the effects of its substrates, modulators and products are described. eNOS produces nitric oxide (NO), which is involved in vascular biology. L-arginine and BH4 are both essential factors for adequate eNOS function. Reduced levels of the substrate or cofactor lead to the formation of superoxide by eNOS, a process referred to as eNOS uncoupling. In all studies described here, a microvascular endothelial cell line (bEn...

Bevers, L. M.

2006-01-01

37

Association of thymidylate synthase enhancer region polymorphisms with thymidylate synthase activity in vivo.  

Science.gov (United States)

Two known polymorphisms in the 5' enhancer region (ER) of the thymidylate synthase (TS) gene, a variable number of tandem repeats of a 28?bp sequence (2R/3R) and a further G>C single nucleotide substitution within the repeats, result in genotypes with 0-5 functional upstream stimulatory factor (USF) E-box consensus elements. However, the relationship between these polymorphisms, regulation of TS expression and patient response to fluoropyrimidine treatment has been inconsistent. In this study, seven possible TSER allele configurations showed similar patterns of luciferase gene expression regardless of cell type or USF-1 content, with no significant difference in promoter activity between the wild-type 2RGC and 3RGGC (1.40±0.37 vs 1.43±0.32, P=0.90), whereas the minor alleles, 2RCC and 3RGCC, were significantly reduced (0.84±0.17, P=0.01) and increased (3.19±0.72, P=0.001) respectively. Patient plasma levels of 2'-deoxyuridine, a surrogate marker of TS activity, were significantly different between genotypes (PTSER, is responsible for determining gene expression in vitro and TS activity in vivo. PMID:20531375

de Bock, C E; Garg, M B; Scott, N; Sakoff, J A; Scorgie, F E; Ackland, S P; Lincz, L F

2011-08-01

38

Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells.  

Science.gov (United States)

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes. PMID:10064135

Verschuur, A C; Van Gennip, A H; Muller, E J; Voûte, P A; Vreken, P; Van Kuilenburg, A B

1999-01-01

39

Glycosphingolipids in Plasmodium falciparum. Presence of an active glucosylceramide synthase.  

Science.gov (United States)

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[(14)C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [(14)C]palmitic acid and [(14)C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy. PMID:15153110

Couto, Alicia S; Caffaro, Carolina; Uhrig, M Laura; Kimura, Emilia; Peres, Valnice J; Merino, Emilio F; Katzin, Alejandro M; Nishioka, Masae; Nonami, Hiroshi; Erra-Balsells, Rosa

2004-06-01

40

Methylene blue inhibits hippocampal nitric oxide synthase activity in vivo  

DEFF Research Database (Denmark)

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

Volke, V; Wegener, Gregers

1999-01-01

 
 
 
 
41

CDP-diacylglycerol synthase activity in Clostridium perfingens  

Energy Technology Data Exchange (ETDEWEB)

CTP: phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimium between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition on the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C. (Refs. 24).

Carmen, G.M.; Zaniewski, R.L.; Cousminer, J.J.

1982-01-01

42

Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells  

International Nuclear Information System (INIS)

Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio [(activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)]. Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of "1"2"5I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms

1986-05-01

43

SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS  

Directory of Open Access Journals (Sweden)

Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

DURDI QUJEQ

1999-10-01

44

Chitin synthase II inhibitory activity of ursolic acid, isolated from Crataegus pinnatifida.  

Science.gov (United States)

Two triterpenoid compounds, ursolic acid and uvaol, were isolated from Crataegus pinnatifida Bunge leaves. Ursolic acid inhibits chitin synthase II from S. cerevisiae with an IC50 value of 0.84 microgram/ml and the inhibition appears to be selective for chitin synthase II, whereas uvaol has no inhibitory activity up to 280 micrograms/ml. Oleanolic acid, alpha-hederin hydrate, and betulic acid inhibited the chitin synthase II activity under the same conditions with an IC50 of 5.6, 64.3, and 98.7 micrograms/ml, respectively. PMID:10232075

Jeong, T S; Hwang, E I; Lee, H B; Lee, E S; Kim, Y K; Min, B S; Bae, K H; Bok, S H; Kim, S U

1999-04-01

45

Changes in Activities of Sucrose Synthase and Sucrose Phosphate Synthase and Sugar Content During Postharvest Senescence in Two Broccoli Cultivars  

Directory of Open Access Journals (Sweden)

Full Text Available Changes in sucrose synthase (SS and sucrose phosphate synthase (SPS activities and sugar content during postharvest senescence in broccoli (Brassica oleracea L. cvs. `Hartland` and `Sairin` were studied. The first change in color became apparent between 2 and 3 d but whole head yellowed after 5 d of storage. The SS activity increased gradually in all portions although with few fluctuations in branchlets of both cultivars at the end of the storage time. In case of SPS, there was no inclining or declining pattern of the activity in any portion of the two cultivars at the end of the storage period. In both cultivars, branchlets showed higher SS and SPS activity than florets. Sucrose content gradually decreased in both portions of the two cultivars with time. Fructose content was higher than glucose and sucrose in the florets as well as branchlets of both cultivars. There was a highly significant negative correlation observed between the SS activity and sucrose content in the florets and branchlets of both cultivars. There was no significant correlation between sugar contents and SPS activity in any portions of both cultivars.

Bimal Kumar Pramanik

2004-01-01

46

Screening for latent acute intermittent porphyria: the value of measuring both leucocyte delta-aminolaevulinic acid synthase and erythrocyte uroporphyrinogen-1-synthase activities.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder of haem biosynthesis characterised by reduced activity of the enzyme uroporphyrinogen-1-(URO) synthase and compensatory increased activity of the rate controlling enzyme delta-aminolaevulinic acid (ALA) synthase. Subjects with the disorder should be identified as they are at risk of developing severe porphyric attacks if exposed to a variety of drugs or chemicals. We have assessed the value of measuring the activ...

Mccoll, K. E.; Moore, M. R.; Thompson, G. G.; Goldberg, A.

1982-01-01

47

Long Noncoding RNA MALAT1 Controls Cell Cycle Progression by Regulating the Expression of Oncogenic Transcription Factor B-MYB  

Science.gov (United States)

The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle–regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.

Tripathi, Vidisha; Shen, Zhen; Chakraborty, Arindam; Giri, Sumanprava; Freier, Susan M.; Wu, Xiaolin; Zhang, Yongqing; Gorospe, Myriam; Prasanth, Supriya G.; Lal, Ashish; Prasanth, Kannanganattu V.

2013-01-01

48

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli  

Directory of Open Access Journals (Sweden)

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.

Bechard Matthew E.

2003-01-01

49

Changes in Carbohydrate Content and Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Broccoli During Short Term Storage at Low Temperature  

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This study investigated the changes in carbohydrate content and activities of invertase, sucrose synthase and sucrose phosphate synthase in two broccoli cultivar (Brassica oleracea L. Cvs. `Hartland` and `Sairin`) stored at 5?C. Respiration rate rapidly declined after 1 day of storage and gradually decreased at the end of the storage period. Color did not change significantly at the end of the storage period. In both cultivars and portions, the invertase activity increased gradually ...

Bimal Kumar Pramanik; Toshiyuki Matsui; Haruo Suzuki; Yusuke Kosugi

2004-01-01

50

Structural features conferring dual Geranyl/Farnesyl diphosphate synthase activity to an aphid prenyltransferase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In addition to providing lipid chains for protein prenylation, short-chain isoprenyl diphosphate synthases (scIPPSs) play a pivotal role in the biosynthesis of numerous mevalonate pathway end-products, including insect juvenile hormone and terpenoid pheromones. For this reason, they are being considered as targets for pesticide development. Recently, we characterized an aphid scIPPS displaying dual geranyl diphosphate (GPP; C10)/farnesyl diphosphate (FPP; C15) synthase activity in vitro. To i...

2009-01-01

51

Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases.  

Science.gov (United States)

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides. PMID:14568070

Eckermann, Christian; Matthes, Bernd; Nimtz, Manfred; Reiser, Verena; Lederer, Barbara; Böger, Peter; Schröder, Joachim

2003-11-01

52

Mechanism and active site residues of GDP-fucose synthase.  

Science.gov (United States)

L-fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewis(X) ligands. The biosynthesis of GDP-L-fucose begins with the action of a dehydratase that converts GDP-D-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose. The GFS reaction involves epimerizations at both C-3'' and C-5'' followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-D-altrose as its major product indicating that C-3'' epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3''-(2)H]-GDP-6-deoxy-4-keto-mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3''. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5'' position of GDP-fucose in the presence of NADP(+). This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3'' position of [3''-(2)H]-GDP-6-deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3'' followed by C-5'') and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps. PMID:19053199

Lau, Stephen T B; Tanner, Martin E

2008-12-24

53

Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Vegetable Soybean During Fruit Development  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This study investigated the changes in carbohydrate content and activities of acid invertase, sucrose synthase (SS) and sucrose phosphate synthase (SPS) in two vegetable soybean cultivars (Glycine max (L.) Merr. vars. Ajigen and Fuuki) during fruit development ranging from 28 to 63 days after anthesis. In both cultivars, sucrose was the predominant sugar while fructose and glucose were found in trace amounts. Sucrose accumulation was highest at 35 and 42 days after anthesis in Fuuki an...

Kassinee Sitthiwong; Toshiyuki Matsui; Nobuyuki Okuda; Haruo Suzuki

2005-01-01

54

Ethylene negatively regulates aluminium-induced malate efflux from wheat roots and tobacco cells transformed with TaALMT1.  

Science.gov (United States)

An important mechanism for Al(3+) tolerance in wheat is exudation of malate anions from the root apex through activation of malate-permeable TaALMT1 channels. Here, the effect of ethylene on Al(3+)-activated efflux of malate was investigated using Al(3+)-tolerant wheat genotype ET8, which has high expression of TaALMT1. Exposure of ET8 plants to Al(3+) enhanced ethylene evolution in root apices. Treatment with the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene gas suppressed Al(3+)-induced malate efflux from root apices, whereas the intracellular malate concentrations in roots were not affected. Malate efflux from root apices was enhanced in the presence of Al(3+) by two antagonists of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and 2-aminoisobutyric acid (AIB). An increase in Al accumulation in root apices was observed when treated with ACC, whereas AVG and AIB suppressed Al accumulation in root apices. Al(3+)-induced inhibition of root elongation was ameliorated by pretreatment with AIB. In addition, ethylene donor (Ethrel) also inhibited Al(3+)-induced malate efflux from tobacco cells transformed with TaALMT1. ACC and the anion-channel blocker niflumate had a similar and non-additive effect on Al-induced malate efflux from root apices. Treatment of ET8 plants with ACC enhanced expression of TaALMT1, suggesting that the inhibitory effect of ethylene on Al-induced malate efflux is unlikely to occur at the transcriptional level. These findings indicate that ethylene may behave as a negative regulator of Al(3+)-induced malate efflux by targeting TaALMT1-mediated malate efflux by an unknown mechanism. PMID:24668874

Tian, Qiuying; Zhang, Xinxin; Ramesh, Sunita; Gilliham, Matthew; Tyerman, Stephen D; Zhang, Wen-Hao

2014-06-01

55

Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0, and tobacco (BY-2, were permeabilized with the channel-forming peptide alamethicin. Results Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. Conclusion Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed, during which plasmodesmata are transiently left open.

Rasmusson Allan G

2009-03-01

56

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

Science.gov (United States)

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

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

2007-06-01

57

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active  

International Nuclear Information System (INIS)

Highlights: ? Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. ? Chitin synthase activity releases chitin, partly associated with the cell surface. ? Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. ? Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. ? This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA? cell lines are shown.

2011-12-02

58

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active  

Energy Technology Data Exchange (ETDEWEB)

Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

Schoenitzer, Veronika [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Eichner, Norbert [Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Clausen-Schaumann, Hauke [Munich University of Applied Sciences, Lothstrasse 34, D-80335 Muenchen, Germany, and Center for NanoScience (CeNS), Geschwister-Scholl-Platz 1, D-80539 Muenchen (Germany); Weiss, Ingrid M., E-mail: ingrid.weiss@inm-gmbh.de [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany)

2011-12-02

59

cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphosphodolichol synthase activity.  

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We have investigated the possible role of a cAMP-mediated protein-phosphorylation event(s) as the key regulatory mechanism in beta-adrenoreceptor-stimulated activation of mannosylphosphodolichol (Man-P-Dol) synthase (GDP-mannose:dolichyl-phosphate O-beta-D-mannosyltransferase, EC 2.4.1.83) in rat parotid acinar cells. Microsomal membranes isolated from these cells pretreated with 10 microM isoproterenol for 60 min showed approximately 40-80% enhanced Man-P-Dol synthase activity compared to th...

Banerjee, D. K.; Kousvelari, E. E.; Baum, B. J.

1987-01-01

60

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity.  

Science.gov (United States)

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme. PMID:18466770

Vandermoten, Sophie; Charloteaux, Benoit; Santini, Sébastien; Sen, Stephanie E; Béliveau, Catherine; Vandenbol, Micheline; Francis, Frédéric; Brasseur, Robert; Cusson, Michel; Haubruge, Eric

2008-06-11

 
 
 
 
61

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.  

Science.gov (United States)

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown. PMID:22079092

Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

2011-12-01

62

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase  

Energy Technology Data Exchange (ETDEWEB)

The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)

2011-08-17

63

Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I  

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The present study shows that rat liver and brain mitochondrial nitric oxide synthase (mtNOS) are functionally associated with mitochondrial respiratory chain complex I. When complex I is activated, mtNOS exerts high activity and generates nitric oxide, whereas inactivation of complex I leads mtNOS to abandon its NOS activity. Functional association of mtNOS with complex I is potentially important in regulating mtNOS activity and mitochondrial functions.

Parihar, Mordhwaj S.; Nazarewicz, Rafal R.; Kincaid, Erick; Bringold, Urs; Ghafourifar, Pedram

2008-01-01

64

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

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

Grimes, Carol A.; Jope, Richard S.

2001-01-01

65

Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms  

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Zinc-supplementation (20??M) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The ...

2012-01-01

66

Growth and sucrose synthase activity of developing chickpea (Cicer arietinum L. seeds under field conditions  

Directory of Open Access Journals (Sweden)

Full Text Available Seed growth characteristics and sucrose synthase activity in chickpea (Cicer arietinum L. were examined in a field experiment at Merredin, Western Australia. ‘Sona’, a small-seeded desi cultivar, and ‘Kaniva’, a large-seeded kabuli cultivar, were grown after flowering with irrigation and under a rainout shelter, used to induce terminal drought. Seed and pod wall dry weight followed a similar pattern in the two cultivars with terminal drought significantly reducing the dry weight of the pod wall and seed in both cultivars. The pod wall reached its maximum dry weight 21 days after podding (DAP in ‘Kaniva’ and 28 and 35 DAP in ‘Sona’ with terminal drought and irrigation, respectively. The dry weight of the pod wall decreased during seed filling, particularly in the plants subjected to terminal drought. The increase in seed dry weight followed a sigmoid curve with a lag phase of 14 DAP and 21 DAP in ‘Kaniva’ and ‘Sona’, respectively, followed by a rapid almost-linear phase until 35 DAP when the dry weight leveled off and even decreased slightly near maturity. Sucrose synthase activity peaked at 32 DAP in ‘Kaniva’ and 35 DAP in ‘Sona’ and then decreased to near zero at maturity. A significant and positive association was observed between seed dry weight at maturity and peak sucrose synthase activity in both cultivars and both treatments. We suggest that sucrose synthase is a good physiological indicator for use in breeding for improved seed size in chickpea.

Ashok Kumar

2009-01-01

67

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli  

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Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after...

2000-01-01

68

The metabolism of malate by cultured rat brain astrocytes  

International Nuclear Information System (INIS)

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-[U-14C]malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain

1990-01-01

69

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

Science.gov (United States)

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

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

2013-04-01

70

Anaerobic Toluene Activation by Benzylsuccinate Synthase in a Highly Enriched Methanogenic Culture  

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Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic photot...

Beller, Harry R.; Edwards, Elizabeth A.

2000-01-01

71

A Noncatalytic Domain of Glycogen Synthase Kinase-3 (GSK-3) Is Essential for Activity*  

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Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3? and GSK-3?, are serine/threonine kinases involved in numerous cellular processes and diverse diseases, including Alzheimer disease, cancer, and diabetes. GSK-3 isoforms function redundantly in some settings, while, in others, they exhibit distinct activities. Despite intensive investigation into the physiological roles of GSK-3 isoforms, the basis for their differential activities remains unresolved. A more comprehensive understanding of t...

Buescher, Jessica L.; Phiel, Christopher J.

2010-01-01

72

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp.  

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Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of no...

2000-01-01

73

Constitutive nitric oxide synthase (cNOS) activity in Langerhans islets from streptozotocin diabetic rats  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the p [...] resence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

T.M., Fonovich de Schroeder; M.D., Carattino; M., Frontera; O.L., Catanzaro.

74

Constitutive nitric oxide synthase (cNOS activity in Langerhans islets from streptozotocin diabetic rats  

Directory of Open Access Journals (Sweden)

Full Text Available Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

T.M. Fonovich de Schroeder

1998-05-01

75

Does Citrulline Malate Enhance Physical Performance.  

Science.gov (United States)

The purpose of this study was to investigate the ability of citrulline malate (CM) to enhance physical performance. Our hypothesis was that CM ingestion prior to exercise would result in increased maximal or peak oxygen consumption and time-to-exhaustion....

C. N. Vojta M. F. Zupan M. J. Rutter T. B. Walker

2010-01-01

76

Correlation of transcription of MALAT-1, a novel noncoding RNA, with deregulated expression of tumor suppressor p53 in small DNA tumor virus models  

Science.gov (United States)

Although metastasis-associated lung adenocarcinoma transcript (MALAT)-1 is known to be consistently upregulated in several epithelial malignancies, little is known about its function or regulation. We therefore examined the relationship between MALAT-1 expression and candidate modulators such as DNA tumor virus oncoproteins human papillomavirus (HPV)-16 E6 and E7, BK virus T antigen (BKVTAg), mouse polyoma virus middle T antigen (MPVmTAg) and tumor suppressor genes p53 and pRb. Using suppressive subtractive hybridization (SSH) and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays, MALAT-1 was shown to be increased in viral oncongene-expressing salivary gland biopsies from humans and mice. The results also indicated that MALAT-1 transcripts and promoter activity were increased in vitro when viral oncongene-expressing plasmids were introduced into different cell types. These same viral oncogenes in addition to increasing MALAT-1 transcription have also been shown to inhibit p53 and/or pRb function. In p53 mutant or inactive cell lines MALAT-1 was also shown to be highly upregulated. We hypothesize that there is a correlation between MALAT-1 over-expression and p53 deregulation. In conclusion, we show that disruption of p53, by both polyoma and papilloma oncoproteins appear to play an important role in the up-regulation of MALAT-1. MALAT-1 might therefore represent a biomarker for p53 deregulation within malignancies.

Jeffers, Liesl K.; Duan, Kaiwen; Ellies, Lesley G.; Seaman, William T.; Burger-Calderon, Raquel A.; Diatchenko, Luda B.; Webster-Cyriaque, Jennifer

2013-01-01

77

Malate-aspartate shuttle, cytoplasmic NADH redox potential, and energetics in vascular smooth muscle.  

Science.gov (United States)

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0. 4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2 consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism. PMID:9737943

Barron, J T; Gu, L; Parrillo, J E

1998-08-01

78

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

Science.gov (United States)

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

Steeves, Craig H; Bearne, Stephen L

2011-09-15

79

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity  

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Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3), a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and re...

Turenne Gaetan A; Price Brendan D

2001-01-01

80

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium  

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Abstract To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarea...

2003-01-01

 
 
 
 
81

Fumonisin concentration and ceramide synthase inhibitory activity of corn, masa, and tortilla chips.  

Science.gov (United States)

Nixtamalization removes fumonisins from corn and reduces their amounts in masa and tortilla products. Fumonisin concentrations and potential toxicity could be underestimated, however, if unknown but biologically active fumonisins are present. Therefore, the relative amounts of fumonisins in extracts of fumonisin-contaminated corn and its masa and tortilla chip nixtamalization products were determined with an in vitro ceramide synthase inhibition bioassay using increased sphinganine (Sa) and sphinganine to sphingosine ratio (Sa/So) as endpoints. African green monkey kidney cells (Vero cells ATCC CCL-81) were grown in 1-ml wells and exposed to 4 microl of the concentrated extracts for 48 h. The corn extract inhibited ceramide synthase as Sa (mean = 132 pmol/well) and Sa/So (mean = 2.24) were high compared to vehicle controls (Sa = 9 pmol/well; Sa/So = 0.10). Inhibitory activity (mean Sa = 14-24 pmol/well; mean Sa/So = 0.17-0.28) of the masa and tortilla chip extracts were reduced > or = 80% compared to the corn extract. Results were corroborated in a second experiment in which Sa and Sa/So of the wells treated with masa or tortilla chip extracts were reduced > or = 89% compared to those treated with the corn extract. Masa and tortilla chip FB1 concentrations (4-7 ppm) were reduced about 80-90% compared to the corn (30 ppm) when the materials were analyzed by high-performance liquid chromatography (HPLC). Therefore, nixtamalization reduced both the measured amount of FB1 and the ceramide synthase inhibitory activity of masa and tortilla chips extracts. The results further suggest that the masa and tortilla chip extracts did not contain significant amounts of unknown fumonisins having ceramide synthase inhibitory activity. PMID:16760143

Voss, Kenneth A; Norred, William P; Meredith, Filmore I; Riley, Ronald T; Stephen Saunders, D

2006-07-01

82

Growth and sucrose synthase activity of developing chickpea (Cicer arietinum L.) seeds under field conditions  

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Seed growth characteristics and sucrose synthase activity in chickpea (Cicer arietinum L.) were examined in a field experiment at Merredin, Western Australia. ‘Sona’, a small-seeded desi cultivar, and ‘Kaniva’, a large-seeded kabuli cultivar, were grown after flowering with irrigation and under a rainout shelter, used to induce terminal drought. Seed and pod wall dry weight followed a similar pattern in the two cultivars with terminal drought significantly reducing the dry weight of t...

2009-01-01

83

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala: Fabricius, 1794  

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Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of...

Faraldo, A. C.; Sa?-nunes, A.; Faccioli, L. H.; Del Bel, E. A.; Lello, E.

2007-01-01

84

Activity of nitric oxide synthase and concentration of nitric oxide end metabolites in the gingiva under experimental pathological conditions.  

Science.gov (United States)

Parameters of NO metabolism in the gingiva were studied during experimental periodontitis accompanied by alloxan diabetes and exogenous hypercholesterolemia. We measured activities of inducible and constitutive NO synthase and concentrations of stable NO end metabolites in rat gingival tissue (total contents of nitrite and nitrate). Under pathological conditions NO-metabolism significantly differed from the control. Treatment with mexidol for 14 days significantly decreased activity of inducible NO synthase in the gingiva of experimental animals. PMID:16671561

Popkov, V L; Fil'chukova, I A; Lapina, N V; Galenko-Yaroshevskii, V P; Dukhanin, A S

2005-10-01

85

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

Energy Technology Data Exchange (ETDEWEB)

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: tmiller2@utep.edu [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)

2010-04-09

86

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

International Nuclear Information System (INIS)

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [14C]-L-arginine to [14C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [14C]-L-arginine to [14C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

2010-04-09

87

Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2  

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The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC[subscript Cs]). PhaC[subscript Cs] showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV)...

2010-01-01

88

Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes  

Energy Technology Data Exchange (ETDEWEB)

Here we demonstrate that human keratinocytes possess a Ca{sup 2+}/ calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm{sup 2}) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[{sup 3}H]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm{sup 2}) of particulate NO synthase. A 5-fold increase in superoxide (O{sub 2}{sup -}) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO{sup -}) production by UVB (20 mJ/cm{sup 2})-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm{sup 2}) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO{sup -} released by UVB-irradiated human keratinocytes in skin erythema and inflammation. (Author).

Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C. [Athens Univ. (Greece). Medical School

1996-12-15

89

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

Directory of Open Access Journals (Sweden)

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

SA Teixeira

2005-03-01

90

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.  

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To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NID...

1991-01-01

91

Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase  

Energy Technology Data Exchange (ETDEWEB)

The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. (Univ. of California, Davis (USA)); Kenny, J.W.; Thompson, G.A. (Calgene Inc., Davis, CA (USA))

1990-10-01

92

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.  

Science.gov (United States)

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. PMID:24814295

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

93

Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP.  

Science.gov (United States)

The bacterial signaling molecule cyclic di-GMP (c-di-GMP) stimulates the synthesis of bacterial cellulose, which is frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the c-di-GMP-activated BcsA-BcsB complex. The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Additionally, the c-di-GMP-activated BcsA-BcsB complex contains a nascent cellulose polymer whose terminal glucose unit rests at a new location above BcsA's active site and is positioned for catalysis. Our mechanistic insights indicate how c-di-GMP allosterically modulates enzymatic functions. PMID:24704788

Morgan, Jacob L W; McNamara, Joshua T; Zimmer, Jochen

2014-05-01

94

An unusual plant triterpene synthase with predominant ?-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.  

Science.gov (United States)

The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. ?-Amyrin and ?-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of ?-amyrin to ?-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of ?-amyrin produced is > 80% of the total product and is, therefore, primarily an ?-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either ?-amyrin and/or ?-amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the ?-amyrin synthases. PMID:21575133

Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

2011-07-01

95

The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3-L1 adipocytes.  

Science.gov (United States)

The role of increased glucose transport in the hormonal regulation of glycogen synthase by insulin was investigated in 3T3-L1 adipocytes. Insulin treatment stimulated glycogen synthase activity 4-5-fold in these cells. Cytosolic glycogen synthase levels decreased by 75% in response to insulin, whereas, conversely, the glycogenolytic agent isoproterenol increased cytosolic enzyme levels by 200%. Removal of extracellular glucose reduced glycogen synthase activation by 40% and completely blocked enzymatic translocation. Addition of 5 mM 2-deoxyglucose did not restore glycogen synthase translocation but did augment dephosphorylation of the protein by insulin. The translocation event could be reconstituted in vitro only by the addition of UDP-glucose to basal cell lysates. Amylase pretreatment of the extracts suppressed glycogen synthase translocation, indicating that the enzyme was binding to glycogen. Incubation of 3T3-L1 adipocytes with 10 mM glucosamine induced a state of insulin resistance, blocked the translocation of glycogen synthase, and inhibited insulin-stimulated glycogen synthesis by 50%. Surprisingly, glycogen synthase activation by insulin was enhanced 4-fold, in part due to allosteric activation by a glucosamine metabolite. In vitro, glucosamine 6-phosphate and glucose 6-phosphate stimulated glycogen synthase activity with similar concentration curves. These results indicate that glucose metabolites have an impact on the regulation of glycogen synthase activation and localization by insulin. PMID:10488084

Brady, M J; Kartha, P M; Aysola, A A; Saltiel, A R

1999-09-24

96

Distinct parts of leukotriene C-4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein  

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Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence that LTC4S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC4S. FLAP bound to the N-terminal part/first hydrophobic region of LTC4S. This part did not bind 5-LO which bound to the second hydrophili...

2009-01-01

97

Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase†‡  

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The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with R- and S-azabisabolenes and PPi bind 3 Mg2+ ions and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change...

Vedula, L. Sangeetha; Cane, David E.; Christianson, David W.

2005-01-01

98

Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity.  

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The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells reveal...

1994-01-01

99

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p...

Grabin?ska, Kariona A.; Magnelli, Paula; Robbins, Phillips W.

2007-01-01

100

Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice  

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The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild-type (WT) and iNOS?/? mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS?/? mice revealed comparable fungal burden in their lungs and ...

Gonzalez, Angel; Hung, Chiung-yu; Cole, Garry T.

2011-01-01

 
 
 
 
101

Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase  

Energy Technology Data Exchange (ETDEWEB)

Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M. (UW)

2009-01-12

102

Structural features conferring dual geranyl/farnesyl diphosphate synthase activity to an aphid prenyltransferase.  

Science.gov (United States)

In addition to providing lipid chains for protein prenylation, short-chain isoprenyl diphosphate synthases (scIPPSs) play a pivotal role in the biosynthesis of numerous mevalonate pathway end-products, including insect juvenile hormone and terpenoid pheromones. For this reason, they are being considered as targets for pesticide development. Recently, we characterized an aphid scIPPS displaying dual geranyl diphosphate (GPP; C(10))/farnesyl diphosphate (FPP; C(15)) synthase activity in vitro. To identify the mechanism(s) responsible for this dual activity, we assessed the product selectivity of aphid scIPPSs bearing mutations at Gln107 and/or Leu110, the fourth and first residue upstream from the "first aspartate-rich motif" (FARM), respectively. All but one resulted in significant changes in product chain-length selectivity, effectively increasing the production of either GPP (Q107E, L110W) or FPP (Q107F, Q107F-L110A); the other mutation (L110A) abolished activity. Although some of these effects could be attributed to changes in steric hindrance within the catalytic cavity, molecular dynamics simulations identified other contributing factors, including residue-ligand Van der Waals interactions and the formation of hydrogen bonds or salt bridges between Gln107 and other residues across the catalytic cavity, which constitutes a novel product chain-length determination mechanism for scIPPSs. Thus the aphid enzyme apparently evolved to maintain the capacity to produce both GPP and FPP through a balance between these mechanisms. PMID:19720147

Vandermoten, Sophie; Santini, Sébastien; Haubruge, Eric; Heuze, Fabien; Francis, Frédéric; Brasseur, Robert; Cusson, Michel; Charloteaux, Benoit

2009-10-01

103

Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution  

Directory of Open Access Journals (Sweden)

Full Text Available One management practice of which the efficiency has not yet been scientifically tested is spraying coffee plants with diluted sucrose solutions as a source of carbon for the plant. This paper evaluates the effect of foliar spraying with sugar on the endogenous level of carbohydrates and on the activities of invertase and sucrose synthase in coffee (Coffea arabica L. seedlings with reduced (low and high (normal levels of carbon reserve. The concentrations used were 0.5 and 1.0% sucrose, and water as a control. The use of sucrose at 1.0% caused an increase in the concentration of total soluble sugars in depauperate plants, as well as increased the activity of the following enzymes: cell wall and vacuole acid invertase, neutral cytosol invertase and sucrose synthase. In plants with high level of carbon reserve, no increments in total soluble sugar levels or in enzymatic activity were observed. Regardless of treatments or plants physiological state, no differences in transpiration or stomatal conductance were observed, demonstrating the stomatal control of transpiration. Photosynthesis was stimulated with the use of 0.5 and 1.0 % sucrose only in depauperate plants. Coffee seedling spraying with sucrose is only efficient for depauperate plants, at the concentration of 1.0%.

Silva José Carlos da

2003-01-01

104

Insights into the phosphatase and the synthase activities of human bisphosphoglycerate mutase: a quantum mechanics/molecular mechanics simulation.  

Science.gov (United States)

Bisphosphoglycerate mutase (BPGM) is a multi-activity enzyme. Its main function is to synthesize the 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. This enzyme can also catalyze the 2,3-bisphosphoglycerate to the 3-phosphoglycerate. In this study, the reaction mechanisms of both the phosphatase and the synthase activities of human bisphosphoglycerate mutase were theoretically calculated by using the quantum mechanics/molecular mechanics method based on the metadynamics and umbrella sampling simulations. The simulation results not only show the free energy curve of the phosphatase and the synthase reactions, but also reveal the important role of some residues in the active site. Additionally, the energy barriers of the two reactions indicate that the activity of the synthase in human bisphosphoglycerate mutase is much higher than that of the phosphatase. The estimated reaction barriers are consistent with the experimental data. Therefore, our work can give important information to understand the catalytic mechanism of the bisphosphoglycerate mutase family. PMID:24441588

Chu, Wen-Ting; Zheng, Qing-Chuan; Zhang, Hong-Xing

2014-03-01

105

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

Science.gov (United States)

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

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

2010-07-15

106

Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Vegetable Soybean During Fruit Development  

Directory of Open Access Journals (Sweden)

Full Text Available This study investigated the changes in carbohydrate content and activities of acid invertase, sucrose synthase (SS and sucrose phosphate synthase (SPS in two vegetable soybean cultivars (Glycine max (L. Merr. vars. Ajigen and Fuuki during fruit development ranging from 28 to 63 days after anthesis. In both cultivars, sucrose was the predominant sugar while fructose and glucose were found in trace amounts. Sucrose accumulation was highest at 35 and 42 days after anthesis in Fuuki and Ajigen, respectively. On the other hand, fructose and glucose were almost maintained throughout the experimental period. The activity of soluble acid invertase was highest at the 42 days but was not maintained until the 63 days after anthesis. The acid invertase activity in cell wall-bound fraction was highest in young fruit (28 days after anthesis and gradually decreased throughout development. Ajigen had higher activity than Fuuki. SS activity showed a continuous increase with time while SPS activity did not show specific inclining or declining pattern. SS and SPS activities in Fuuki were higher than Ajigen. There was a highly significant negative correlation observed between the acid invertase activity in cell wall-bound fraction and sucrose content in Ajigen. A significant positive correlation was also found between the SS and SPS activities and sucrose content in Fuuki. However, a highly significant negative correlation was observed between SS activity and other soluble sugars (glucose and fructose in both cultivars except glucose content in Fuuki. No significant correlation was found between the SPS activity and other soluble sugars (glucose and fructose in both cultivars.

Kassinee Sitthiwong

2005-01-01

107

Aerobic exercise plus weight loss improves insulin sensitivity and increases skeletal muscle glycogen synthase activity in older men.  

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The purpose of this study was to determine the effects of 6-month aerobic exercise training + weight loss (AEX + WL) on basal and insulin activation of glycogen synthase, basal citrate synthase activity, and Akt and AS160 phosphorylation in older, overweight/obese insulin-resistant men (n = 14; 63 ± 2 years; body mass index, 32 ± kg/m(2)). Muscle samples of the vastus lateralis were collected before and during a 3-hour 80 mU/m(2)/min hyperinsulinemic-euglycemic clamp. AEX + WL increased VO2max by 11% (p skeletal muscle glycogen synthase activity that likely contributes to improved glucose utilization in older insulin-resistant men. PMID:24357038

Ryan, Alice S; Katzel, Leslie I; Prior, Steven J; McLenithan, John C; Goldberg, Andrew P; Ortmeyer, Heidi K

2014-07-01

108

Materials and methods for efficient succinate and malate production  

Energy Technology Data Exchange (ETDEWEB)

Genetically engineered microorganisms have been constructed to produce succinate and malate in mineral salt media in pH-controlled batch fermentations without the addition of plasmids or foreign genes. The subject invention also provides methods of producing succinate and malate comprising the culture of genetically modified microorganisms.

Jantama, Kaemwich; Haupt, Mark John; Zhang, Xueli; Moore, Jonathan C; Shanmugam, Keelnatham T; Ingram, Lonnie O' Neal

2014-04-08

109

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

Science.gov (United States)

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

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

2012-01-01

110

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

Scientific Electronic Library Online (English)

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

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

111

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax  

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Full Text Available Abstract Background Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. Results We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098 after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7, suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. Conclusion We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Nassar Marwa

2006-10-01

112

Methylene green electrodeposited on SWNTs-based "bucky" papers for NADH and l-malate oxidation.  

Science.gov (United States)

This research introduces a cavity anode design based on new single-walled nanotube (SWNTs) papers, "bucky" papers, used for the oxidation (and regeneration) of nicotinamide adenine dinucleotide (NADH) and the oxidation of l-malate. The materials designed are paper-like processed composites containing also additives: BP11 sample contains SWNTs and isopropanol (IPA); the BPMG sample contains SWNTs, IPA, and methylene green (MG). NADH/NAD(+) is the cofactor responsible for the oxidation of l-malate by malate dehydrogenase (MDH), in the Krebs' cycle. Because of the high overpotential of NADH oxidation, poly methylene green (PMG) was utilized as the electrocatalyst to produce NAD(+). The electrocatalyst was deposited on the surface of the "bucky" papers by electropolymerization by means of 10 voltammetric cycles in a range of -0.5 V and +1.3 V (vs Ag/AgCl reference electrode) at a scan rate of 5 mV/s. The catalytic performance of PMG was evaluated by chronoamperometric measurements of NADH oxidation at 0.3 V in phosphate buffer and l-malate oxidation at 0.1 V in the presence of MDH. For both "bucky" papers, the chronoamperometric curves of PMG, current vs NADH concentration, show a linear relationship demonstrating to have a first order Fick's law behavior for concentrations of NADH lower than 6 mM. The chronoamperometric curves in the presence of MDH, current against l-malate concentration, show a Michaelis-Menten behavior where no inhibition or competitive reaction are detected. Additionally, the anodic materials were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), the polymerization of MG is effectively observed in the form of particles nucleation. The anodes show an excellent electrocatalytic activity toward NADH oxidation. The electrode design is feasible, reproducible, and overall stable. PMID:21667995

Narváez Villarrubia, Claudia W; Rincón, Rosalba A; Radhakrishnan, Vinod K; Davis, Virginia; Atanassov, Plamen

2011-07-01

113

Human platelet nitric oxide synthase activity: an optimized method Atividade da óxido nítrico sintase em plaquetas humanas: um método otimizado  

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We investigated the kinetic analysis of human platelet Nitric Oxide Synthase (NOS) activity by the rate of conversion of [³H] arginine to [³H]-citrulline in unstimulated fresh platelets. NOS activity was present in the membrane fraction and cytosol, and was Ca2+- and calmodulin dependent which is a characteristic of endothelial NOS. NOS activity was also dependent of NADPH since the omission of this cofactor induced an important decrease (85,2%) in the enzyme activity. The kinetic varied wi...

2002-01-01

114

Analysis of polyhydroxyalkanoate (PHA) synthase gene and PHA-producing bacteria in activated sludge that produces PHA containing 3-hydroxydodecanoate.  

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Activated sludge is an alternative to pure cultures for polyhydroxyalkanoate (PHA) production due to the presence of many PHA-producing bacteria in activated sludge community. In this study, activated sludge was submitted to aerobic dynamic feeding in a sequencing batch reactor. During domestication, the changes of bacterial community structure were observed by terminal restriction fragment length polymorphism analysis. Furthermore, some potential PHA-producing bacteria, such as Thauera, Acinetobacter and Pseudomonas, were identified by denaturing gradient gel electrophoresis analysis. The constructed PHA synthase gene library was analyzed by DNA sequencing. Of the 80 phaC genes obtained, 76 belonged to the Class I PHA synthase, and four to the Class II PHA synthase. Gas chromatography-mass spectrometry analysis showed that PHA produced by activated sludge was composed of three types of monomers: 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxydodecanoate (3HDD). This is the first report of production of medium-chain-length PHAs (PHAMCL ) containing 3HDD by activated sludge. Further studies suggested that a Pseudomonas strain may play an important role in the production of PHAMCL containing 3HDD. Moreover, a Class II PHA synthase was found to have a correlation with the production of 3HDD-containing PHAMCL . PMID:23800349

Yang, Chao; Zhang, Wei; Liu, Ruihua; Zhang, Chi; Gong, Ting; Li, Qiang; Wang, Shufang; Song, Cunjiang

2013-09-01

115

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp.  

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Full Text Available Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW, the nodule fresh weight (FW, the nitrogenase activity, the nodule glutamine synthetase (GS and glutamate synthase (GOGAT activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of nodules was measured in L. pedunculatus. Nitrogenase activity in L. tenuis, L. pedunculatus and L. subbiflorus was six fold the activity in L. corniculatus. Nodule GS and GOGAT activities did not follow this same pattern. L. tenuis had the highest values of GS and GOGAT activities in the nodule, and a high nitrogenase activity which is consistent with its high plant DW. The four species of Lotus were compared and no correlation between nitrogen fixation parameters and ammonia assimilation enzymes was found, but the GS/GOGAT ratio has a positive and significant correlation (r²=0.82** with the amino acid content in stems.

GONNET SUSANA

2000-01-01

116

Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.  

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Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo. PMID:12423881

Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

2002-11-01

117

Effect of hydrogen peroxide on rabbit urinary bladder citrate synthase activity in the presence and absence of a grape suspension  

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Full Text Available PURPOSE: The etiology of obstructive bladder dysfunction includes free radical damage to mitochondria. Feeding rabbits a standardized grape suspension protects the ability of the bladder to contract and empty in part by preventing mitochondrial damage, thus maintaining smooth muscle and mucosal metabolism. The objective of the current study is to determine the direct effect of this grape suspension on the response of mitochondria to the oxidative effects of hydrogen peroxide. MATERIALS AND METHODS: Six male rabbits were anesthetized with sodium pentobarbital and the bladders excised. Four full thickness strips were obtained for contractile studies and the balance separated into smooth muscle and mucosa compartments by blunt dissection. The effect of hydrogen peroxide on the contractile response to field stimulation was quantitated. Each tissue was homogenized and the effects of increasing concentrations of hydrogen peroxide in the presence and absence of grape suspension on citrate synthase activity was determined. RESULTS: Citrate synthase activity was significantly higher in the mucosa than in the muscle. The grape suspension had no effect on control citrate synthase activity. However, the grape suspension provided significant protection of both smooth muscle and mucosal citrate synthase activity. CONCLUSIONS: These studies support the conclusion that the grape suspension provides direct protection of mitochondrial function.

Vijay Venugopal

2010-12-01

118

Obese Mice Lacking Inducible Nitric Oxide Synthase Are Sensitized to the Metabolic Actions of Peroxisome Proliferator–Activated Receptor-? Agonism  

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OBJECTIVE—Synthetic ligands for peroxisome proliferator–activated receptor-? (PPAR-?) improve insulin sensitivity in obesity, but it is still unclear whether inflammatory signals modulate their metabolic actions. In this study, we tested whether targeted disruption of inducible nitric oxide (NO) synthase (iNOS), a key inflammatory mediator in obesity, modulates the metabolic effects of rosiglitazone in obese mice.

Dallaire, Patrice; Bellmann, Kerstin; Laplante, Mathieu; Ge?linas, Ste?phanie; Centeno-baez, Carolina; Penfornis, Patrice; Peyot, Marie-line; Latour, Martin G.; Lamontagne, Julien; Trujillo, Maria E.; Scherer, Philipp E.; Prentki, Marc; Deshaies, Yves; Marette, Andre?

2008-01-01

119

Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity  

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GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase domain

Willemoës, Martin; Mølgaard, Anne

2005-01-01

120

Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity.  

Science.gov (United States)

GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase domain. PMID:15670165

Willemoës, Martin; Mølgaard, Anne; Johansson, Eva; Martinussen, Jan

2005-02-01

 
 
 
 
121

Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels  

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BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, Parginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (PArginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Berkowitz, Dan E.; White, Ron; Li, Dechun; Minhas, Khalid M.; Cernetich, Amy; Kim, Soonyul; Burke, Sean; Shoukas, Artin A.; Nyhan, Daniel; Champion, Hunter C.; Hare, Joshua M.

2003-01-01

122

Systemic administration of pramiracetam increases nitric oxide synthase activity in the cerebral cortex of the rat.  

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The effect of systemic administration of pramiracetam on neuronal type nitric oxide synthase (NOS) activity and NOS mRNA expression were studied in the hippocampus and cerebral cortex in rats. A dose of 300 mg/kg (i.p.) of this nootropic produced an approximately 20% increase in NOS activity in rat brain cortical homogenates but not in hippocampal homogenates; no significant changes were observed in NOS mRNA expression in the cortex and hippocampus. A lower dose of pramiracetam (100 mg/kg i.p.) was ineffective on NOS mRNA expression and enzyme activity. Interestingly, administration of pramiracetam (300 mg/kg i.p.) in rats pretreated (24 h before) with lithium chloride (LiCl) (3 mEq/kg i.p.) yielded a 40% increase in cortical NOS activity. However, in LiCl-pretreated rats this nootropic failed to affect cortical NOS mRNA expression; LiCl (3 mEq/kg i.p.) given alone produced no effect. In conclusion, the present data demonstrate that pramiracetam given alone or in combination with LiCl increases NOS activity in brain cortical homogenates of rats and this may contribute to the mechanisms underlying learning and memory improvement produced by this nootropic. PMID:8557218

Corasaniti, M T; Paoletti, A M; Palma, E; Granato, T; Navarra, M; Nisticò, G

1995-01-01

123

Mutual inhibition between YAP and SRSF1 maintains long non-coding RNA, Malat1-induced tumourigenesis in liver cancer.  

Science.gov (United States)

Emerging studies have revealed that Malat1 is overexpressed in many malignant diseases, including liver cancer, and contributes to enhancing cell migration or facilitating proliferation. However, the mechanism underlying its regulation has largely remained elusive. Here, we characterised the oncoprotein Yes-associated protein (YAP), which up-regulated metastasis-associated lung adenocarcinoma transcript 1 (Malat1) expression at both transcriptional and post-transcriptional levels, whereas serine/arginine-rich splicing factor 1 (SRSF1) played an opposing role. SRSF1 inhibited YAP activity by preventing its co-occupation with TCF/?-catenin on the Malat1 promoter. In contrast, overexpression of YAP impaired the nuclear retention of both SRSF1 and itself via an interaction with Angiomotin (AMOT). This effect removed the inhibitory role of SRSF1 on Malat1 in the nucleus. Furthermore, higher expression of YAP was consistent with a lower SRSF1 nuclear accumulation in human liver cancer tissues. We also revealed that overexpression of YAP combined with a knockdown of SRSF1 resulted in conspicuously enhanced transwell cell mobility, accelerated tumour growth rate, and loss of body weight in a tail vein-injected mouse models. Taken together, these data provided a novel mechanism underlying the balance between SRSF1, YAP and Malat1 and uncovered a new role of YAP in regulating long non-coding RNA (lncRNA). Thus, disrupting the interaction between YAP and SRSF1 may serve as a crucial therapeutic method in liver cancer. PMID:24468535

Wang, Jiayi; Wang, Hongmei; Zhang, Yue; Zhen, Ni; Zhang, Li; Qiao, Yongxia; Weng, Wenhao; Liu, Xiangfan; Ma, Lifang; Xiao, Weifan; Yu, Wenjun; Chu, Qinghua; Pan, Qiuhui; Sun, Fenyong

2014-05-01

124

Site-directed mutations of the gatekeeping loop region affect the activity of Escherichia coli spermidine synthase.  

Science.gov (United States)

Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis. PMID:23001854

Lee, Mon-Juan; Yang, Ya-Ting; Lin, Vivian; Huang, Haimei

2013-06-01

125

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

Science.gov (United States)

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

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

2007-07-01

126

Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis.  

Science.gov (United States)

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione. PMID:10871602

Pinzar, E; Miyano, M; Kanaoka, Y; Urade, Y; Hayaishi, O

2000-10-01

127

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg [...] neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P

A.M., Herculano; M.E., Crespo-López; S.M.A., Lima; D.L.W., Picanço-Diniz; J.L.M. Do, Nascimento.

128

A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity  

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Full Text Available Abstract Background Prostaglandin H2 synthase (PGHS is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2 prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2 are the target for nonsteroidal anti-inflammatory drugs (NSAIDs. Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel in vitro electron paramagnetic resonance (EPR-based assay for measuring the activity of PGHS-1. Methods We validated a novel in vitro PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH to 3-carboxy-proxy (CP under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR spectrum. Results The assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 ?M, 1 mM caused significant inhibition of spin-adduct formation (72 ± 11 and 100 ± 16% inhibition of control respectively; P 0.05. Conclusion We have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays.

Rossi Adriano G

2006-09-01

129

l-Malate Production by Metabolically Engineered Escherichia coli? †  

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Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactiv...

2011-01-01

130

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala: Fabricius, 1794  

Scientific Electronic Library Online (English)

Full Text Available SciELO Argentina | Language: English Abstract in english Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate [...] many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

A. C., Faraldo; A, Sá-Nunes; L. H., Faccioli; E. A., Del Bel; E, Lello.

131

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells  

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Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3?,5?-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

2014-01-01

132

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala: Fabricius, 1794  

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Full Text Available Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL, yeast-injected (YIL, and saline-injected (SIL larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

A. C. Faraldo

2007-08-01

133

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794).  

Science.gov (United States)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects. PMID:17902268

Faraldo, A C; Sá-Nunes, A; Faccioli, L H; Del Bel, E A; Lello, E

2007-08-01

134

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

International Nuclear Information System (INIS)

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

1991-01-01

135

Increased nitric oxide synthase in the lung after ozone inhalation is associated with activation of NF-kappa B.  

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Acute inhalation of ozone is associated with a inflammatory response characterized by the accumulation of macrophages at sites of tissue injury. These cells, along with resident alveolar epithelial cells, become activated and release cytotoxic and proinflammatory mediators, such as nitric oxide (.NO), that we speculate contribute to toxicity. In these studies we analyzed mechanisms regulating increased .NO synthase activity in lung macrophages and type II cells after ozone inhalation. Brief e...

Laskin, D. L.; Sunil, V.; Guo, Y.; Heck, D. E.; Laskin, J. D.

1998-01-01

136

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

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The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu c...

Leiper, James; Murray-rust, Judith; Mcdonald, Neil; Vallance, Patrick

2002-01-01

137

Transdermal oestradiol replacement therapy enhances platelet constitutive nitric oxide synthase activity in postmenopausal women with type 2 diabetes mellitus.  

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OBJECTIVE: To determine whether treatment with transdermal oestrogen replacement therapy (TERT) in postmenopausal women with type 2 diabetes mellitus was able to increase the activity of constitutive nitric oxide synthase (cNOS) in platelets. RESEARCH DESIGN AND METHODS: Eighteen postmenopausal women with type 2 diabetes mellitus (group A) were studied in basal conditions (A1) and on the 10th day of the third month of a sequential opposed TERT (A2) evaluating platelet cNOS activity measured b...

Pescarmona, Gianpiero; Martina, Valentino

2002-01-01

138

High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation  

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NO propagates a number of antiatherogenic effects in the endothelium, and diminished availability has been associated with vascular disease. Recently it has been reported that phosphorylation of endothelial NO synthase (eNOS) at Ser-1179 is required to increase activity in response to stimuli, including high-density lipoprotein (HDL). The current study was undertaken to further examine the mechanism by which HDL activates eNOS and to specifically determine the role of the major apolipoprotein...

Drew, Brian G.; Fidge, Noel H.; Gallon-beaumier, Gabrielle; Kemp, Bruce E.; Kingwell, Bronwyn A.

2004-01-01

139

Exercise with calorie restriction improves insulin sensitivity and glycogen synthase activity in obese postmenopausal women with impaired glucose tolerance  

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Our objective was to compare the effects of in vivo insulin on skeletal muscle glycogen synthase (GS) activity in normal (NGT) vs. impaired glucose-tolerant (IGT) obese postmenopausal women and to determine whether an increase in insulin activation of GS is associated with an improvement in insulin sensitivity (M) following calorie restriction (CR) and/or aerobic exercise plus calorie restriction (AEX + CR) in women with NGT and IGT. We did a longitudinal, clinical intervention study of CR co...

Ryan, Alice S.; Ortmeyer, Heidi K.; Sorkin, John D.

2012-01-01

140

Reductive heme-dependent activation of the n-oxide prodrug AQ4N by nitric oxide synthase.  

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Anaerobic reduction of anticancer prodrugs is a promising route to achieve targeting and selectivity in anticancer drug design. Most reductive prodrug activations involve simple electron transfer from a flavoprotein and are not amenable to specific targeting. Here, we report that the N-oxide AQ4N is reduced by a nitric oxide synthase. This reduction involves interaction with the heme iron atom in the active site and is thus subject to specific protein constraints. PMID:18681417

Nishida, Clinton R; Ortiz de Montellano, Paul R

2008-08-28

 
 
 
 
141

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium  

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Full Text Available Abstract To investigate the possible role of nitric oxide (NO produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS activity was measured in samples from first trimester (villous, and non villous-trophoblast, term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation whereas placenta and myometrium, from the same patient, at deliveries by Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of 14C-citrulline from 14C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS and neuronal (nNOS isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.

Laurini Ricardo

2003-06-01

142

PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.  

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Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-?-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules. PMID:24212577

Pfeiffer, Daniel; Jendrossek, Dieter

2014-01-01

143

Piceatannol-3'-O-?-D-glucopyranoside as an active component of rhubarb activates endothelial nitric oxide synthase through inhibition of arginase activity  

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Arginase competitively inhibits nitric oxide synthase (NOS) via use of the common substrate L-arginine. Arginase II has recently reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. Here, we demonstrate that piceatannol-3'-O-?-D-glucopyranoside (PG), a potent component of stilbenes, inhibits the activity of arginase I and II prepared from mouse liver and kidney lysates, respectively, in a dose-dependent manner. In human umbilical vein e...

2010-01-01

144

Molecular mechanism of endothelial nitric-oxide synthase activation by Platycodon grandiflorum root-derived saponins.  

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Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) has antithrombotic and antiatherosclerotic properties in the vasculature. Previously, we demonstrated that saponins derived from the roots of Platycodon grandiflorum (CKS) inhibited the tumor necrosis factor-alpha-induced expression of adhesion molecules in human endothelial cells. In this study, we found that CKS increased eNOS phosphorylation and NO production in human endothelial cells. Treatment with CKS increased the phosphorylation of Akt, p38/MAPK, AMP-activated protein kinase (AMPK), and calmodulin-dependent protein kinase II (CaMK II) leading to increased NO production in human endothelial cells. Moreover, inhibitors of Akt (LY294002), p38/MAPK (SB203580), AMPK (compound C), and CaMK II (W7) failed to suppress CKS-induced eNOS phosphorylation. In addition, CKS-induced eNOS phosphorylation was inhibited by the overexpression of a dominant-negative mutant form of AMPK (DN-AMPK). Taken together, these results indicate that CKS stimulates eNOS phosphorylation and NO production via the activation of PI3K/Akt, p38/MAPK, AMPK, and CaMK II. PMID:20230881

Kim, Hyung Gyun; Hien, Tran Thi; Han, Eun Hee; Chung, Young Chul; Jeong, Hye Gwang

2010-06-01

145

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells1[S  

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The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

Martin, Maria Laura; Liebisch, Gerhard; Lehneis, Stefan; Schmitz, Gerd; Alonso-Sande, Maria; Bestard-Escalas, Joan; Lopez, Daniel H.; Garcia-Verdugo, Jose Manuel; Soriano-Navarro, Mario; Busquets, Xavier; Escriba, Pablo V.; Barcelo-Coblijn, Gwendolyn

2013-01-01

146

Insulin activation of mouse diaphragm glycogen synthase (GS) involves generation of electrophoretically distinct subunit species  

International Nuclear Information System (INIS)

Glycogen synthase, the rate limiting enzyme for glycogen synthesis, was analyzed in mouse diaphragm extracts both by immunoprecipitation and immunoblotting using specific antibodies raised to the rabbit muscle enzyme. Diaphragms, with the supporting ribs attached, were incubated either with or without ["3"2P]P/sub i/ in the medium. In extracts from unincubated, rapidly frozen diaphragms, immunoblotting indicated the presence of 3 distinct species, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, phosphorylation of immunoprecipitated GS with the kinase F/sub A//GSK-3 converted the higher mobility forms into the low mobility species. In diaphragms incubated with ["3"2P]P/sub i/, "3"2P was incorporated only into one of the GS species, that of lowest mobility, indicating differential labelling among the multiple subunit forms. Insulin action, which increased the -/+ glucose-6-P activity ratio from 0.2 to 0.4, converted the low mobility species to the two higher mobility forms. The authors propose that this effect of insulin can be explained by dephosphorylation in the proline/serine rich site 3 region of GS, which has potent influence on both mobility on SDS-PAGE and activity

1986-05-01

147

Activation of macrophage nuclear factor-?B and induction of inducible nitric oxide synthase by LPS  

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Full Text Available Abstract Background Chronic lung disease (CLD of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS to stimulate rat alveolar macrophages to produce nitric oxide (NO, express inducible nitric oxide synthase (iNOS and activate nuclear factor-?B (NF-?B in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes. Methods Griess reaction was used to measure the nitrite level. Western blot and a semi-quantitative RT-PCR were performed to detect iNOS expression. Electrophoretic mobility shift assay (EMSA was performed to analyze the activation of NF-?B. Results We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (?10 ng/ml and time dependent manner (p -4–10-10 M and dexamethasone (10-4–10-6 M (p Conclusion Our findings imply that Gram-negative bacterial infection and the inflammatory responses are important factors in the development of CLD. The down-regulatory effect of steroids on iNOS expression and NO production might explain the beneficial effect of steroids in neonates with CLD.

Yan Zhong-Qun

2002-09-01

148

UDP-[14C]glucose-labelable polypeptides from pea: Possible components of glucan synthase I activity  

International Nuclear Information System (INIS)

A membrane-bound polypeptide doublet of about 40 kD can be rapidly labeled with UDP-[14C]glucose under the assay conditions for glucan synthase I (GS-I). Label seems covalently bound, and chases when unlabeled UDPG is added; it might represent a covalent intermediate in polysaccharide synthesis. Labeling and GS-I activity show several common features: they co-sediment with Golgi membranes in sucrose gradients; they depend similarly on Mg2+ or Mn2+ (not Ca2+); they decrease dramatically from stem apex to base, and are higher in epidermis than internal tissue; they show similar sensitivities to several inhibitors. But the doublet still labels after polysaccharide-synthesizing activity has been destroyed by Triton X-100. The doublet polypeptides might be glucosyl tranferases whose ability to transfer glucose units to a glucan chain is detergent-sensitive, but to accept glucose from UDPG is not; or they might be detergent-insensitive primary glucose acceptors, from which a distinct, detergent-sensitive transferase(s) move(s) these units to glucan chains

1989-01-01

149

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX  

Science.gov (United States)

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.

Laptenok, Sergey P.; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Myllykallio, Hannu; Liebl, Ursula; Vos, Marten H.

2013-01-01

150

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

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

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

2011-04-01

151

Mitochondrial-associated nitric oxide synthase activity inhibits cytochrome c oxidase: implications for breast cancer.  

Science.gov (United States)

Nitric oxide (NO) is produced and nitric oxide synthase (NOS) activity is expressed in many types of tumor cells, but their precise role in tumor proliferation has not been clearly elucidated. Recently, it has been observed that patients with triple-negative breast tumors expressing NOS have a significantly worse prognosis compared to those that do not express any NOS. We observed that NOS activity was associated with the mitochondria in two breast cancer cell lines, ZR-75-30 and BT-474, compared with another NO-producing benign breast epithelial cell line, MCF-12F, in which no significant mitochondrial-associated NOS activity was detected. The rate of proliferation of the malignant cells expressing mitochondrial-associated NOS was decreased in the presence of an inhibitor of NO synthesis, but it had no effect on the normal breast epithelial cells, MCF-12F, which also expressed NOS, but not associated with mitochondria. The basal rate of proliferation was not affected by ODQ, an inhibitor of soluble guanylate cyclase, indicating that the effects of the endogenous NO produced by the malignant cell lines on proliferation are cGMP independent. Our results indicate that mitochondrial-associated NOS activity exhibited by the cancer cell lines ZR-75-30 and BT-474 inhibited cytochrome c oxidase, resulting in increased production of hydrogen peroxide (H2O2), which inhibited protein phosphatase 2A activity. This resulted in the maintenance of Akt and ERK1/2 in a phosphorylated state, leading to cell proliferation. PMID:23089229

Sen, Suvajit; Kawahara, Brian; Chaudhuri, Gautam

2013-04-01

152

Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae.  

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Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad. PMID:18533686

Qian, Jinghua; Khandogin, Jana; West, Ann H; Cook, Paul F

2008-07-01

153

The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial nitric-oxide (NO) synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester.  

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We have previously shown that estrogen exerts a vasoprotective effect by accelerating reendothelialization after perivascular artery injury through activation of the estrogen receptor alpha. Because 17beta-estradiol (E2) is known to increase the bioavailability of nitric oxide, in this study, we used the same perivascular model to characterize the role of the endothelial nitric oxide synthase (eNOS) pathway in reendothelialization. Surprisingly, we found that the stimulatory effect of E2 on r...

2008-01-01

154

The Estrogen Effects on Endothelial Repair and Mitogen-Activated Protein Kinase Activation Are Abolished in Endothelial Nitric-Oxide (NO) Synthase Knockout Mice, but Not by NO Synthase Inhibition by N-Nitro-l-arginine Methyl Ester  

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We have previously shown that estrogen exerts a vasoprotective effect by accelerating reendothelialization after perivascular artery injury through activation of the estrogen receptor ?. Because 17?-estradiol (E2) is known to increase the bioavailability of nitric oxide, in this study, we used the same perivascular model to characterize the role of the endothelial nitric oxide synthase (eNOS) pathway in reendothelialization. Surprisingly, we found that the stimulatory effect of E2 on reendo...

2008-01-01

155

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture  

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Full Text Available The visual system is a potential target for methylmercury (MeHg intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05. Maximum values (P < 0.05 were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates. MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

A.M. Herculano

2006-03-01

156

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer  

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Full Text Available Abstract Background The enzyme porphobilinogen synthase (PBGS, which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. Results The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of ~6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. Conclusion The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Fairman Robert

2004-11-01

157

Distinct parts of leukotriene C4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein  

International Nuclear Information System (INIS)

Leukotriene C4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C4 synthase (LTC4S) participate in its biosynthesis. We report evidence that LTC4S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC4S. FLAP bound to the N-terminal part/first hydrophobic region of LTC4S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC4S. Fluorescent FLAP- and LTC4S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC4S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC4S. Direct interaction of 5-LO and LTC4S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC4S.

2009-04-17

158

Comparative computational analysis of active and inactive cofactors of nitric oxide synthase.  

Science.gov (United States)

Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide from L-Arg in the presence of oxygen. Of the two electrons required for the first step of the reaction, the second is primarily donated by the tetrahydrobiopterin (H4B) cofactor bound adjacent to the heme, which is eventually reduced back to resting state by the ultimate electron source of the reaction, the flavins of the NOS reductase domain. Density functional theory calculations were carried out to identify those protonation states of different cofactor molecules that best support radicalization of the cofactor and the coupled increase in the electron density of the heme-bound oxygen molecule. Three cofactor molecules were studied, native H4B, an active analogue, 5-methyl-H4B, and the inactive 4-amino-H4B. Findings support the emerging model where H4B and 5-methyl-H4B are coupled proton/electron sources of NOS catalysis, while 4-amino-H4B is an inhibitor due to its inability to donate the catalytically required proton. PMID:19708267

Menyhárd, Dóra K

2009-03-12

159

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling  

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Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a ?-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3? (Ser-21/? (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of ?-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

Lu Frances Fangjia

2012-11-01

160

Suppression of pathogen-inducible NO synthase (iNOS) activity in tomato increases susceptibility to Pseudomonas syringae  

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Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and Arabidopsis plants. It was shown recently that a variant form of the Arabidopsis P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and sus...

Chandok, Meena R.; Ekengren, Sophia K.; Martin, Gregory B.; Klessig, Daniel F.

2004-01-01

 
 
 
 
161

Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product  

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Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vit...

Beller, Harry R.; Spormann, Alfred M.

1998-01-01

162

Important differences in nitric oxide synthase activity and predominant isoform in reproductive tissues from human and rat  

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Abstract For the extrapolation of data obtained from experimental animals to the human situation, it is important to know the similarities and differences between human and animal species. Some important characteristics of nitric oxide synthase (NOS) in myometrium and vagina from human and rat were compared. NOS-activity was measured by the formation of 14C-citrulline from 14C-arginine and the expression of NOS isoforms was examined by Western blotting. NOS act...

2003-01-01

163

Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase  

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Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new appro...

Gupta, Vineet; Patwardhan, Gauri A.; Zhang, Qian-jin; Cabot, Myles C.; Jazwinski, S. Michal; Liu, Yong-yu

2010-01-01

164

The effect of intermittent cryotherapy on the activities of citrate synthase and lactate dehydrogenase in regenerating skeletal muscle  

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This study examined the effect of three sessions of cryotherapy (three sessions of 30 minutes applied each 2 h) and muscle compression in the regenerating skeletal muscle of the rats. The middle belly of tibialis anterior muscle was injured by a frozen iron bar and received one of the following intervention: injury + cryotherapy (treated with cryotherapy); injury + placebo (sand pack), and injury (I).The enzymatic activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measur...

Nuno Miguel Lopes de Oliveira; João Luiz Quagliotti Durigan; Flávia Simone Munin; Maria Luiza Barcelos Schwantes; Tania de Fátima Salvini

2013-01-01

165

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

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The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StA...

Wang, Xingjia; Yin, Xiangling; Schiffer, Randolph B.; King, Steven R.; Stocco, Douglas M.; Grammas, Paula

2008-01-01

166

Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene.  

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We began characterization of the protein--DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression expe...

Schulze-lefert, P.; Dangl, J. L.; Becker-andre?, M.; Hahlbrock, K.; Schulz, W.

1989-01-01

167

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

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

2010-01-01

168

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

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

2011-01-01

169

Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen1[W  

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Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants u...

Bermu?dez, Mª A?ngeles; Galme?s, Jeroni; Moreno, Inmaculada; Mullineaux, Philip M.; Gotor, Cecilia; Romero, Luis C.

2012-01-01

170

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

Science.gov (United States)

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

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

2000-01-01

171

The histone demethylase JMJD1A induces cell migration and invasion by up-regulating the expression of the long noncoding RNA MALAT1  

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Patients with neuroblastoma due to N-Myc oncogene amplification have a high frequency of tumor metastasis. However, it is not clear how N-Myc induces cell migration, invasion and metastasis. The histone demethylase JMJD1A activates gene transcription by demethylating the lysine 9 residue of histone H3 (H3K9) at target gene promoters. The long noncoding RNA MALAT1 induces lung cancer cell migration and plays a pivotal role in lung cancer metastasis. Here we demonstrated that N-Myc up-regulated the expression of JMJD1A in N-Myc oncogene-amplified human neuroblastoma cells by directly binding to the JMJD1A gene promoter. Affymetrix microarray studies revealed that the gene second most significantly up-regulated by JMJD1A was MALAT1. Consistent with this finding, RT-PCR and chromatin immunoprecipitation assays showed that JMJD1A bound to the MALAT1 gene promoter and demethylated histone H3K9 at the MALAT1 gene promoter. Moreover, JMJD1A and MALAT1 induced, while the small molecule JMJD1A inhibitor DMOG suppressed, neuroblastoma cell migration and invasion. Taken together, our data identify a novel pathway through which N-Myc causes neuroblastoma cell migration and invasion, and provide important evidence for further development of more potent JMJD1A/MALAT1 inhibitors for the prevention of tumor metastasis.

Tee, Andrew E.; Ling, Dora; Nelson, Charlotte; Atmadibrata, Bernard; Dinger, Marcel E.; Xu, Ning; Mizukami, Tamio; Liu, Pei Y.; Liu, Bing; Cheung, Belamy; Pasquier, Eddy; Haber, Michelle; Norris, Murray D.; Suzuki, Takayoshi; Marshall, Glenn M.; Liu, Tao

2014-01-01

172

Dual Regulation of Muscle Glycogen Synthase during Exercise by Activation and Compartmentalization  

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Glycogen synthase (GS) is considered the rate-limiting enzyme in glycogenesis but still today there is a lack of understanding on its regulation. We have previously shown phosphorylation-dependent GS intracellular redistribution at the start of glycogen re-synthesis in rabbit skeletal muscle (Prats, C., Cadefau, J. A., Cusso, R., Qvortrup, K., Nielsen, J. N., Wojtaszewki, J. F., Wojtaszewki, J. F., Hardie, D. G., Stewart, G., Hansen, B. F., and Ploug, T. (2005) J. Biol. Chem. 280, 23165-23172). In the present study we investigate the regulation of human muscle GS activity by glycogen, exercise, and insulin. Using immunocytochemistry we investigate the existence and relevance of GS intracellular compartmentalization during exercise and during glycogen re-synthesis. The results show that GS intrinsic activity is strongly dependent on glycogen levels and that such regulation involves associated dephosphorylation at sites 2 + 2a, 3a, and 3a + 3b. Furthermore, we report the existence of several glycogen metabolismregulatory mechanisms based on GS intracellular compartmentalization. After exhausting exercise, epinephrine-induced protein kinase A activation leads to GS site 1b phosphorylation targeting the enzyme to intramyofibrillar glycogen particles, which are preferentially used during muscle contraction. On the other hand, when phosphorylated at sites 2 + 2a, GS is preferentially associated with subsarcolemmal and intermyofibrillar glycogen particles. Finally, we verify the existence in human vastus lateralis muscle of the previously reported mechanism of glycogen metabolism regulation in rabbit tibialis anterior muscle. After overnight low muscle glycogen level and/or in response to exhausting exercise-induced glycogenolysis, GS is associated with spherical structures at the I-band of sarcomeres.

Prats, Clara; Helge, Jorn W.

2009-01-01

173

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity  

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Full Text Available Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3, a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. Results The 2 isoforms of GSK3, GSK3? and GSK3?, phosphorylate the sequence Ser-X-X-X-Ser(P when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3? phosphorylation at serine 33 in vitro. GSK3? did not phosphorylate p53 under any condition. GSK3? increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3? to regulate p53 transcriptional activity. GSK3? is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3? kinase activity was inhibited in response to DNA damage, suggesting that GSK3? regulation of p53 is not involved in the p53-DNA damage response. Conclusions GSK3? can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3? does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3? may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

Price Brendan D

2001-07-01

174

ACTIVATION OF A CYCLIC AMP-GUANINE EXCHANGE FACTOR IN HEPATOCYTES DECREASES NITRIC OXIDE SYNTHASE EXPRESSION  

Science.gov (United States)

Adenosine 3?, 5?-cyclic adenosine monophosphate (cAMP) activates intracellular signaling by regulating Protein Kinase A (PKA), calcium influx, and cAMP-binging guanine nucleotide exchange factors (Epac or cAMP-GEF). cAMP inhibits cytokine-induced expression of nitric oxide synthase (iNOS) in hepatocytes by a PKA-independent mechanism. We hypothesized that Epac mediates this effect. A cyclic AMP analogue that specifically activates Epac, 8-(4-methoxyphenylthio)-2?-O-methyladenosine-3?,5?-cyclic monophosphate (OPTmecAMP) and overexpression of liver specific Epac2 both inhibited IL-1?/IFN?–induced iNOS expression and nitrite production. OPTmecAMP inactivated Raf1/MEK/ERK signaling but ERK had no effect on iNOS expression. OPTmecAMP induced a persistent Akt phosphorylation in hepatocytes that lasted up to 8 hours. Overexpression of a dominant negative Akt blocked the inhibitory effect of OPTmecAMP on iNOS production. A specific PI3K inhibitor, LY294002, attenuated the inhibition of nitrite production and iNOS expression produced by overexpressing a liver specific Epac2 (LEpac2). OPTmecAMP also induced c-Jun N-terminal kinase (JNK) phosphorylation in hepatocytes. Overexpression of dominant negative JNK enhanced cytokine- induced iNOS expression and nitrite production and reversed the inhibitory effects of LEpac2 on nitrite production and iNOS expression. We conclude that Epac regulates hepatocyte iNOS expression through an Akt- and JNK- mediated signaling mechanism.

Zhang, Baochun; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G.

2012-01-01

175

Activity-dependent heterogeneous populations of nitric oxide synthase neurons in the rat dorsal raphe nucleus.  

Science.gov (United States)

The brainstem dorsal raphe nucleus (DRN) contains an abundant distribution of nitric oxide (NO) synthase (NOS)-containing neuronal profiles in two distinct populations: faint- and intense-immunoreactive cells in midline (ventromedial and dorsomedial) and lateral wing subregions, respectively. This study tested the hypothesis that different functional dynamics underlie the topography of NOS-containing cells in the DRN rostrocaudal and mediolateral neuraxis by using a capsaicin challenge paradigm (50 mg/kg, subcutaneous). Compared with vehicle, capsaicin significantly and preferentially increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, an index of constitutive NOS) reactivity in the rostral midline and caudal lateral wing subregions. Furthermore, capsaicin activated more Fos-positive cells than vehicle within all subregions of the DRN but with a caudal versus rostral predominance in activation pattern. In addition, a high proportion of capsaicin-induced Fos cells in the midline but almost none in lateral wing stained for NADPH-d. These observations suggest the existence of two functionally distinct populations of NOS neurons in the DRN. Furthermore, capsaicin increased galanin immunoreactivity with predominant staining in cell soma and fiber processes in midline and lateral wing subregions of the nucleus, respectively. The total capsaicin-induced galanin immunoreactivity was higher in rostral versus caudal DRN, and a high proportion of galanin-positive cells in the midline also contained NADPH-d and neuronal NOS, thus suggesting a potential NO-galanin interaction in these neurons. The differential pattern of Fos/NADPH-d colocalization across the nucleus suggests that midline and lateral wing NOS neurons of the DRN express their neuromodulatory actions on discrete efferent targets via different intracellular mechanisms. PMID:16616732

Okere, Chuma O; Waterhouse, Barry D

2006-05-01

176

Linolenate 9R-dioxygenase and allene oxide synthase activities of Lasiodiplodia theobromae.  

Science.gov (United States)

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2 mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-(2)H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200 mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants. PMID:22048860

Jernerén, Fredrik; Eng, Felipe; Hamberg, Mats; Oliw, Ernst H

2012-01-01

177

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

International Nuclear Information System (INIS)

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

1985-06-18

178

Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis  

Energy Technology Data Exchange (ETDEWEB)

Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. The authors now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolated the wild-type hemA gene encoding delta-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizobium meliloti hemA gene as a hybridization probe. A deletion of the hemA gene region, generated in vitro, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no delta-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless delta-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial delta-aminolevulinic acid synthase activity as the exclusive source of delta-aminolevulinic acid for heme formation in soybean nodules.

Guerinot, M.L.; Chelm, B.K.

1986-03-01

179

Cutaneous constitutive nitric oxide synthase activation in postural tachycardia syndrome with splanchnic hyperemia.  

Science.gov (United States)

Models of microgravity are linked to excessive constitutive nitric oxide (NO) synthase (NOS), splanchnic vasodilation, and orthostatic intolerance. Normal-flow postural tachycardia syndrome (POTS) is a form of chronic orthostatic intolerance associated with splanchnic hyperemia. To test the hypothesis that there is excessive constitutive NOS in POTS, we determined whether cutaneous microvascular neuronal NO and endothelial NO are increased. We performed two sets of experiments in POTS and control subjects aged 21.4 ± 2 yr. We used laser-Doppler flowmetry to measure the cutaneous response to local heating as an indicator of bioavailable neuronal NO. To test for bioavailable endothelial NO, we infused intradermal acetylcholine through intradermal microdialysis catheters and used the selective neuronal NOS inhibitor l-N(?)-nitroarginine-2,4-L-diamino-butyric amide (N(?), 10 mM), the selective inducible NOS inhibitor aminoguanidine (10 mM), the nonspecific NOS inhibitor nitro-l-arginine (NLA, 10 mM), or Ringer solution. The acetylcholine dose response and the NO-dependent plateau of the local heating response were increased in POTS compared with those in control subjects. The local heating plateau was significantly higher, 98 ± 1%maximum cutaneous vascular conductance (%CVC(max)) in POTS compared with 88 ± 2%CVC(max) in control subjects but decreased to the same level with N(?) (46 ± 5%CVC(max) in POTS compared with 49 ± 4%CVC(max) in control) or with NLA (45 ± 3%CVC(max) in POTS compared with 47 ± 4%CVC(max) in control). Only NLA blunted the acetylcholine dose response, indicating that NO produced by endothelial NOS was released by acetylcholine. Aminoguanidine was without effect. This is consistent with increased endothelial and neuronal NOS activity in normal-flow POTS. PMID:21642500

Stewart, Julian M; Nafday, Abhinav; Ocon, Anthony J; Terilli, Courtney; Medow, Marvin S

2011-09-01

180

NADP-malate dehydrogenase from Chlamydomonas: prediction of new structural determinants for redox regulation by homology modelling.  

Science.gov (United States)

The function of a gene closely linked to nitrate assimilation loci from Chlamydomonas reinhardtii has been investigated. Gene expression analysis shows that its mRNA accumulation is modulated by light, carbon source and adaptation to light/dark cyclic conditions of growth. A full-length cDNA was isolated for the light-regulated transcript, and sequence characterization indicates that it encodes the NADP-malate dehydrogenase from C. reinhardtii (NADP-MDH;Cr). The primary structure of NADP-MDH;Cr is closely related to plant, mossfern and algal NADP-malate dehydrogenases, and shares structural determinants for chloroplast targeting, cofactor binding and catalysis. Sequence conservation extends to the carboxy end of the protein, where plant and mossfern enzymes have two cysteines and an acidic C-terminus with a critical role for regulation of NADP-MDH activity by the thioredoxin/ferredoxin system. Accordingly, incubation with DTT activates NADP-MDH enzyme in cell-free extracts from C. reinhardtii. Like NADP-malate dehydrogenases from two other green algae, the N-terminal extension of NADP-MDH;Cr lacks two thiol residues whose reduction constitutes the rate-limiting step in the activation reaction of plant enzymes. Homology-based 3D modelling of NADP-MDH;Cr, the first structure predicted for NADP-malate dehydrogenase from a lower eukaryote, evidences close positioning of two new cysteines in an accessible region of the protein surface. These results suggest that the algal enzyme has a different arrangement of regulatory disulfide bridges, which might involve the existence of new mechanisms that control functioning of the malate valve, the main system to export reducing power from the chloroplast of plant cells. PMID:11855723

Gómez, Ia; Merchán, Faustino; Fernández, Emilio; Quesada, Alberto

2002-02-01

 
 
 
 
181

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter  

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Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN- gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 wa...

1995-01-01

182

Inducible Nitric Oxide Synthase Deficiency Impairs Matrix Metalloproteinase-9 Activity and Disrupts Leukocyte Migration in Hepatic Ischemia/Reperfusion Injury  

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Matrix metalloproteinase 9 (MMP-9) is a critical mediator of leukocyte migration in hepatic ischemia/reperfusion (I/R) injury. To test the relevance of inducible nitric oxide synthase (iNOS) expression on the regulation of MMP-9 activity in liver I/R injury, our experiments included both iNOS-deficient mice and mice treated with ONO-1714, a specific iNOS inhibitor. The inability of iNOS-deficient mice to generate iNOS-derived nitric oxide (NO) profoundly inhibited MMP-9 activity and depressed...

Hamada, Takashi; Duarte, Sergio; Tsuchihashi, Seiichiro; Busuttil, Ronald W.; Coito, Ana J.

2009-01-01

183

Structural basis for reduced activity of 1-aminocyclopropane-1-carboxylate synthase affected by a mutation linked to andromonoecy.  

Science.gov (United States)

1-aminocyclopropane-1-carboxylate synthase (ACS) is a key enzyme in the biosynthesis of the plant hormone ethylene. Recently, a new biological role for ACS has been found in Cucumis melo where a single point mutation (A57V) of one isoform of the enzyme, causing reduced activity, results in andromonoecious plants. We present here a straightforward structural basis for the reduced activity of the A57V mutant, based on our work on Malus domestica ACS, including a new structure of the unliganded apple enzyme at 1.35Å resolution. PMID:21075107

Schärer, Martin A; Eliot, Andrew C; Grütter, Markus G; Capitani, Guido

2011-01-01

184

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

International Nuclear Information System (INIS)

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

1986-05-01

185

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1986-05-01

186

Helicobacter pylori Induces Disturbances in Gastric Mucosal Akt Activation through Inducible Nitric Oxide Synthase-Dependent S-Nitrosylation: Effect of Ghrelin  

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Gastric mucosal inflammatory response to H. pylori and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in apoptosis and the disturbances in NO signaling pathways. Here, we report that H. pylori LPS-induced enhancement in the mucosal inducible nitric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive nitric oxide synthase (cNOS) phosphorylation. Further, we demonstrate that the LPS effect o...

Slomiany, Bronislaw L.; Slomiany, Amalia

2011-01-01

187

Expression and Bactericidal Activity of Nitric Oxide Synthase in Brucella suis-Infected Murine Macrophages  

Science.gov (United States)

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-?)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-? enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-?) mRNAs was induced in both c-Brucella- and ops-Brucella-infected cells and was strongly potentiated by IFN-?. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-?-treated cells infected with c-Brucella or ops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-?-treated cells infected with ops-Brucella. These discrepencies between iNOS mRNA and protein levels were not due to differences in TNF-? production. The iNOS inhibitor N?-nitro-l-arginine methyl ester increased B. suis multiplication specifically in IFN-?-treated cells infected with ops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fc? receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-? and antibrucella antibodies are present, i.e., following expression of acquired immunity.

Gross, Antoine; Spiesser, Sandra; Terraza, Annie; Rouot, Bruno; Caron, Emmanuelle; Dornand, Jacques

1998-01-01

188

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.  

Science.gov (United States)

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA. PMID:23645258

De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

189

Role of endothelial nitric oxide synthase and vagal activity in the endothelial protection of atorvastatin in ischemia/reperfusion injury.  

Science.gov (United States)

Vascular endothelial dysfunction plays a pivotal role in the development and maintenance of ischemia/reperfusion (I/R) injury. Statins, developed as lipid-lowering drugs, partially restore vagal activity and exhibit pleiotropic effects. This study was aimed at determining the effect of atorvastatin (ATV) on endothelial dysfunction in peripheral resistance arteries after I/R injury. After pretreatment with ATV (10 mg·kg·d) or its vehicle for 3 days, the superior mesenteric artery was occluded for 60 minutes and reperfusion for 90 minutes or the rats were anesthetized without being subjected to ischemia. In the ATV-treated I/R group, the increased contractions to KCl and 5-hydroxytryptamine induced by I/R were ameliorated, and attenuated endothelium-dependent relaxations to acetylcholine (ACh) were normalized. The restored relaxation to ACh was abolished by N-nitro-L-arginine methyl ester. ATV prevented the structural damage of vascular endothelial cells. Furthermore, the activities of phosphatidylinositol-3-kinase, Akt, and endothelial nitric oxide synthase were elevated in mesenteric arteries after ATV treatment. In addition, I/R-induced increment of endothelial cells apoptosis was also attenuated by ATV. Intriguingly, ATV also increased baroreflex sensitivity and serum ACh content after I/R. In conclusion, the endothelial protective effect of ATV in peripheral arteries is associated with the activated phosphatidylinositol-3-kinase/Akt/endothelial nitric oxide synthase pathway and restored vagal activity. PMID:23364605

Bi, Xue-Yuan; He, Xi; Zhao, Ming; Yu, Xiao-Jiang; Zang, Wei-Jin

2013-05-01

190

Pertussis toxin-sensitive G proteins influence nitric oxide synthase III activity and protein levels in rat heart.  

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Inhibitory G protein activity (Gi) and nitric oxide (NO) modulate muscarinic-cholinergic (MC) inhibition of cardiac beta-adrenergic inotropic responses. We hypothesized that Gi mediates MC-NO synthase (NOS) signal transduction. Isoproterenol (0.2-0.8 microg/min) and acetylcholine (1 microM) were administered to isolated perfused rat hearts pretreated with saline (controls; n = 8) or pertussis toxin (PT; 30 microg/kg intraperitoneally 3 d before study; n = 20). PT abrogated in vitro ADP-ribosy...

Hare, J. M.; Kim, B.; Flavahan, N. A.; Ricker, K. M.; Peng, X.; Colman, L.; Weiss, R. G.; Kass, D. A.

1998-01-01

191

The effect of intermittent cryotherapy on the activities of citrate synthase and lactate dehydrogenase in regenerating skeletal muscle  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english This study examined the effect of three sessions of cryotherapy (three sessions of 30 minutes applied each 2 h) and muscle compression in the regenerating skeletal muscle of the rats. The middle belly of tibialis anterior muscle was injured by a frozen iron bar and received one of the following inte [...] rvention: injury + cryotherapy (treated with cryotherapy); injury + placebo (sand pack), and injury (I).The enzymatic activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the presence of 1mM or 10mM pyruvate. The ANOVA and Tukey's test (p

Nuno Miguel Lopes de, Oliveira; João Luiz Quagliotti, Durigan; Flávia Simone, Munin; Maria Luiza Barcelos, Schwantes; Tania de Fátima, Salvini.

192

Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus.  

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1. In the presence of atropine (1 microM) and guanethidine (5 microM), electrical field stimulation (EFS, 120 mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM)-precontracted longitudinal muscle strips from the rat gastric fundus. 2. L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adre...

1996-01-01

193

Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition  

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Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS–nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20–30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in u...

Galijasevic, Semira; Saed, Ghassan M.; Diamond, Michael P.; Abu-soud, Husam M.

2003-01-01

194

Malate Synthesis by Dark Carbon Dioxide Fixation in Leaves 1  

Science.gov (United States)

The rates of dark CO2 fixation and the label distribution in malate following dark 14CO2 fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark 14CO2 fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO2 at rates of 1.4, 3.4, 0.23, and 1.0 ?moles of CO2/mg of chlorophyll· hour, respectively. Net CO2 fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO2 for the duration of the 23-hour experiment. A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the ?-carboxyl (C4) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C4 as much as 15 to 20%. The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO2 fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix 14CO2 more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

Levi, Carolyn; Perchorowicz, John T.; Gibbs, Martin

1978-01-01

195

Determination of amino-acidic positions important for Ocimum basilicum geraniol synthase activity  

Directory of Open Access Journals (Sweden)

Full Text Available Terpenes are one of the largest and most diversified families of natural compounds. Although they have found numerous industrial applications, the molecular basis of their synthesis in plants has, until now, not been fully understood. Plant genomes have been shown to contain dozens of terpene synthase (TPS genes, however knowledge of their amino-acidic protein sequence in not sufficient to predict which terpene(s will be produced by a particular enzyme. In order to investigate the structural basis of a TPS specificity, we performed site directed mutations in the geraniol synthase from Ocimum basilicum. The results obtained suggest that a specific region on the catalytic site plays an important role in GPP transformation, either by stabilizing the GPP substrate on the catalytic site, or by enabling its transformation into a monoterpenol via an intermediate carbocation.

Marc Bergdoll

2013-02-01

196

Cutaneous constitutive nitric oxide synthase activation in postural tachycardia syndrome with splanchnic hyperemia  

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Models of microgravity are linked to excessive constitutive nitric oxide (NO) synthase (NOS), splanchnic vasodilation, and orthostatic intolerance. Normal-flow postural tachycardia syndrome (POTS) is a form of chronic orthostatic intolerance associated with splanchnic hyperemia. To test the hypothesis that there is excessive constitutive NOS in POTS, we determined whether cutaneous microvascular neuronal NO and endothelial NO are increased. We performed two sets of experiments in POTS and con...

Stewart, Julian M.; Nafday, Abhinav; Ocon, Anthony J.; Terilli, Courtney; Medow, Marvin S.

2011-01-01

197

Central nervous system nitric oxide synthase activity regulates insulin secretion and insulin action.  

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Systemic inhibition of nitric oxide synthase (NOS) with NG-monomethyl-L-arginine (L-NMMA) causes acute insulin resistance (IR), but the mechanism is unknown. We tested whether L-NMMA-induced IR occurs via NOS blockade in the central nervous system (CNS). Six groups of Sprague-Dawley rats were studied after chronic implantation of an intracerebroventricular (ICV) catheter into the lateral ventricle and catheters into the carotid artery and jugular vein. Animals were studied after overnight foo...

Shankar, R.; Zhu, J. S.; Ladd, B.; Henry, D.; Shen, H. Q.; Baron, A. D.

1998-01-01

198

Hepatitis B Virus X Protein Regulates Hepatic Glucose Homeostasis via Activation of Inducible Nitric Oxide Synthase*  

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Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). H...

Shin, Hye-jun; Park, Young-ho; Kim, Sun-uk; Moon, Hyung-bae; Park, Do Sim; Han, Ying-hao; Lee, Chul-ho; Lee, Dong-seok; Song, In-sung; Lee, Dae Ho; Kim, Minhye; Kim, Nam-soon; Kim, Dae-ghon; Kim, Jin-man; Kim, Sang-keun

2011-01-01

199

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)  

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Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relati...

Durbin, Mary L.; Denton, Amy L.; Clegg, Michael T.

2001-01-01

200

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal mic...

Rumbo, Martin; Courjault-gautier, Franc?oise; Sierro, Fre?de?ric; Sirard, Jean-claude; Felley-bosco, Emanuela

2005-01-01

 
 
 
 
201

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exist as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated to the particulate fraction of cell extracts. Confocal micros...

Rumbo, Martin; Courjault-gautier, Franc?oise; Sierro, Fre?de?ric; Sirard, Jean-claude; Felley-bosco, Emanuela

2005-01-01

202

The anticytomegaloviral activity of raltitrexed is abrogated in quiescent mouse fibroblasts that overexpress thymidylate synthase  

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Cytomegalovirus (CMV) replication in non-proliferating cells requires the coordinated expression of the host enzymes responsible for deoxyribonucleotide synthesis. Thymidylate synthase (TS) is an essential cellular enzyme that catalyzes de novo synthesis of thymidylic acid (dTMP). In this report we show that murine CMV (MCMV) replication and DNA synthesis are inhibited in quiescent 3T6 fibroblasts by raltitrexed, a quinazoline-based folate analog that specifically inhibits TS. This antiviral ...

Landolfo, Santo; Riera, Ludovica; Gribaudo, Giorgio; Lembo, David; Andrea, Marco

2001-01-01

203

Amino acid sequence of an active site peptide of avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase.  

Science.gov (United States)

Hydroxymethylglutaryl-CoA synthase is irreversibly inhibited by the active site-directed inhibitor 3-chloropropionyl-CoA. Enzyme modification has been postulated to involve alkylation of an active site cysteinyl sulfhydryl group. DEAE-Sephadex chromatography of tryptic digests prepared from enzyme inactivated using chloro[14C]propionyl-CoA suggested that bound radioactivity is localized on one peptide. Specificity of the modification was further demonstrated by reverse-phase high pressure liquid chromatography, which was used to isolate the radioactively labeled peptide in a chemically homogeneous form. Automated gas-phase Edman degradation techniques have been employed to confirm the assignment of cysteine as the inhibitor's target residue and to elucidate the sequence of amino acids which flank the 14C-carboxyethylated cysteine: Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-(Thr)- Asn-Ala-S-[14C]carboxyethylcysteine-Tyr-Gly-Gln-Thr-(Ala). These data represent the first assignment of active site structure for hydroxymethyl-glutaryl-CoA synthase. PMID:2865259

Miziorko, H M; Behnke, C E

1985-11-01

204

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

Science.gov (United States)

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

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

2012-12-14

205

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

2003-01-01

206

Selenoprotein-dependent up-regulation of hematopoietic prostaglandin D2 synthase in macrophages is mediated through the activation of peroxisome proliferator-activated receptor (PPAR) gamma.  

Science.gov (United States)

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-?(12,14)-prostaglandin J(2) (15d-PGJ(2)) and decreases the proinflammatory PGE(2). Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD(2) metabolites, ?(12)-PGJ(2) and 15d-PGJ(2). A dose-dependent increase in the expression of hematopoietic-PGD(2) synthase (H-PGDS) by selenium and a corresponding increase in ?(12)-PGJ(2) and 15d-PGJ(2) in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ(2) production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor ? ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-?B-dependent thromboxane synthase and microsomal PGE(2) synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ(2). These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD(2) metabolites, which may have clinical implications. PMID:21669866

Gandhi, Ujjawal H; Kaushal, Naveen; Ravindra, Kodihalli C; Hegde, Shailaja; Nelson, Shakira M; Narayan, Vivek; Vunta, Hema; Paulson, Robert F; Prabhu, K Sandeep

2011-08-01

207

Selenoprotein-dependent Up-regulation of Hematopoietic Prostaglandin D2 Synthase in Macrophages Is Mediated through the Activation of Peroxisome Proliferator-activated Receptor (PPAR) ?*  

Science.gov (United States)

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-?12,14-prostaglandin J2 (15d-PGJ2) and decreases the proinflammatory PGE2. Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD2 metabolites, ?12-PGJ2 and 15d-PGJ2. A dose-dependent increase in the expression of hematopoietic-PGD2 synthase (H-PGDS) by selenium and a corresponding increase in ?12-PGJ2 and 15d-PGJ2 in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ2 production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor ? ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-?B-dependent thromboxane synthase and microsomal PGE2 synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ2. These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD2 metabolites, which may have clinical implications.

Gandhi, Ujjawal H.; Kaushal, Naveen; Ravindra, Kodihalli C.; Hegde, Shailaja; Nelson, Shakira M.; Narayan, Vivek; Vunta, Hema; Paulson, Robert F.; Prabhu, K. Sandeep

2011-01-01

208

[Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)].  

Science.gov (United States)

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen. PMID:22513468

Zhu, Rui-Juan; Rao, Xin-Feng; Wei, Deng-Bang; Wang, Duo-Wei; Wei, Lian; Sun, Sheng-Zhen

2012-04-25

209

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity  

International Nuclear Information System (INIS)

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3? (GSK-3?) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-? (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

2008-04-01

210

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.  

Science.gov (United States)

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-?-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-10-29

211

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

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

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

2004-10-22

212

The Cytoplasmic Tail of GM3 Synthase Defines Its Subcellular Localization, Stability, and In Vivo Activity  

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GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH2-terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was e...

2009-01-01

213

Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression.  

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Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDMØs) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage act...

Connelly, L.; Jacobs, A. T.; Palacios-callender, M.; Moncada, S.; Hobbs, A. J.

2003-01-01

214

Involvement of glutamate, oxidative stress and inducible nitric oxide synthase in the convulsant activity of ciprofloxacin in mice.  

Science.gov (United States)

This study investigated the potential convulsive activity of ciprofloxacin in mice and the possible mechanism(s) of this activity. Intraperitoneal (i.p.) administration of ciprofloxacin into mice resulted in convulsive seizures in a dose-dependent manner. The clonic median convulsant dose (CD(50)) of ciprofloxacin in mice was increased by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine, not changed by pretreatment with 7-nitroindazole and decreased by pretreatment with L-arginine and fenbufen. The increase in nitric oxide (NO) production and malondialdehyde (MDA) level as well as the decrease in intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity induced by the estimated clonic CD(50) of ciprofloxacin in mice brain was inhibited by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine. These biochemical alterations were not changed by pretreatment with 7-nitroindazole but enhanced by pretreatment with L-arginine. The elevation induced by the clonic CD(50) of ciprofloxacin in brain glutamate level was not changed by pretreatment with MK-801, alpha-lipoic acid, aminoguanidine or L-arginine. Combined treatment of mice with fenbufen and ciprofloxacin produced elevation of brain NO production and glutamate and MDA levels as well as inhibition of brain intracellular GSH level and GSH-Px activity. In addition, i.p. administration of the clonic CD(50) of ciprofloxacin produced an increase in inducible but not in neuronal NO synthase mRNA and protein expressions in mice brain. These results suggest that elevation of brain glutamate levels with consequent oxidative stress and increase in the expression and activity of brain inducible NO synthase may play a pivotal role in ciprofloxacin-induced convulsive seizures. PMID:22542655

Abdel-Zaher, Ahmed O; Afify, Abdel-Halim M; Kamel, Sohair M; Farghaly, Hanan M; El-Osely, Gehan M; El-Awaad, Ehab A M

2012-06-15

215

Malate dehydrogenase from the thermophilic green bacterium Chloroflexus aurantiacus: purification, molecular weight, amino acid composition, and partial amino acid sequence.  

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Malate dehydrogenase (MDH; EC 1.1.1.37) from the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus was purified by a two-step procedure involving affinity chromatography and gel filtration. The enzyme consists of identical subunits which had molecular weights of approximately 35,000. In its active form at 55 degrees C, it formed tetramers. At lower temperatures, inactive dimers and trimers existed. Antibodies against the purified enzyme were produced, and immunotitration and enz...

Rolstad, A. K.; Howland, E.; Sireva?g, R.

1988-01-01

216

Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.  

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The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains...

1994-01-01

217

The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation.  

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Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages. PMID:16873379

Molle, Virginie; Brown, Alistair K; Besra, Gurdyal S; Cozzone, Alain J; Kremer, Laurent

2006-10-01

218

Formulation and evaluation of almotriptan malate nasal drops  

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Nasal solutions of almotriptan malate were prepared in phosphate buffer containing different proportions of HPMC E15. In vitro permeation studies were performed using Franz diffusion cell with dialysis membrane and ex vivo permeation studies were carried out using sheep nasal mucosal layer. The formulations were radiolabeled with 99mTc and the nasal residence time was studied in rabbits. Nasal irritation was evaluated in rats. Formulations prepared with HPMC E15 5% w/v di...

2009-01-01

219

Formulation and Evaluation of Almotriptan Malate Nasal Drops  

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Nasal solutions of almotriptan malate were prepared in phosphate buffer containing different proportions of HPMC E15. In vitro permeation studies were performed using Franz diffusion cell with dialysis membrane and ex vivo permeation studies were carried out using sheep nasal mucosal layer. The formulations were radiolabeled with 99mTc and the nasal residence time was studied in rabbits. Nasal irritation was evaluated in rats. Formulations prepared with HPMC E15 5% w/v did not retard the rele...

2009-01-01

220

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica  

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Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme...

Kabran, Philome?ne; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-marc; Neuve?glise, Ce?cile

2012-01-01

 
 
 
 
221

The effect of intermittent cryotherapy on the activities of citrate synthase and lactate dehydrogenase in regenerating skeletal muscle  

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Full Text Available This study examined the effect of three sessions of cryotherapy (three sessions of 30 minutes applied each 2 h and muscle compression in the regenerating skeletal muscle of the rats. The middle belly of tibialis anterior muscle was injured by a frozen iron bar and received one of the following intervention: injury + cryotherapy (treated with cryotherapy; injury + placebo (sand pack, and injury (I.The enzymatic activities of citrate synthase (CS and lactate dehydrogenase (LDH were measured in the presence of 1mM or 10mM pyruvate. The ANOVA and Tukey's test (p<0.05 were performed for the statistical analysis. In summary, the intermittent sessions of cryotherapy, associated to muscle compression and applied immediately after the primary muscle injury minimized the CS and LDH activity at 4h30 and 24h periods post-lesion, which could be related to the reduction in the secondary muscle injury inherent to cryotherapy treatment.

Nuno Miguel Lopes de Oliveira

2013-02-01

222

Enzymatic Changes in Phenylalanine Ammonia-lyase, Cinnamic-4-hydroxylase, Capsaicin Synthase, and Peroxidase Activities in Capsicum under Drought Stress.  

Science.gov (United States)

Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined. PMID:24984087

Phimchan, Paongpetch; Chanthai, Saksit; Bosland, Paul W; Techawongstien, Suchila

2014-07-23

223

Endosulfan effects on muscle malate dehydrogenase of the freshwater catfish Clarias batrachus.  

Science.gov (United States)

The effects of a sublethal concentration of an organochlorine pesticide endosulfan were investigated on crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) in the muscle of the freshwater catfish Clarias batrachus. Endosulfan treatment reduced significantly the activity and the specific activity of cMDH and mMDH but had no effect on total cytoplasmic and mitochondrial protein contents. This indicates a decline in the efficiency of aerobic energy metabolism in endosulfan-treated fish. The polyacrylamide gel electrophoresis showed two distinct isoforms (C1 and C2) of cMDH and only one form of mMDH. The inhibition produced by endosulfan in vivo was of mixed noncompetitive/uncompetitive type for crude as well as purified cMDH and mMDH. However, in vitro inhibition was of simple noncompetitive and mixed competitive/noncompetitive type for purified cMDH and mMDH, respectively. Citrate was found to be an uncompetitive inhibitor of cMDH and mixed noncompetitive/uncompetitve inhibitor of mMDH. The inhibitory patterns were modulated by endosulfan. These results demonstrate inhibitory effects of endosulfan on skeletal muscle MDH of the freshwater catfish Clarias batrachus and inhibition effects are mediated through enzyme/substrate/endosulfan complexing. PMID:14575683

Mishra, Rajnikant; Shukla, S P

2003-11-01

224

Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase  

International Nuclear Information System (INIS)

Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol 32P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ? subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn2+ or Ni2+ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn2+ or Ni2+ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni2+ was a more potent activator of phosphorylated calcineurin compared to Mn2+. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni2+ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca2+

1986-05-01

225

Isolation of streptococcal hyaluronate synthase.  

Science.gov (United States)

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

Prehm, P; Mausolf, A

1986-05-01

226

A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules  

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Full Text Available A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein, has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

Hildgund Schrempf

2010-09-01

227

Important differences in nitric oxide synthase activity and predominant isoform in reproductive tissues from human and rat  

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Full Text Available Abstract For the extrapolation of data obtained from experimental animals to the human situation, it is important to know the similarities and differences between human and animal species. Some important characteristics of nitric oxide synthase (NOS in myometrium and vagina from human and rat were compared. NOS-activity was measured by the formation of 14C-citrulline from 14C-arginine and the expression of NOS isoforms was examined by Western blotting. NOS activity in human uterus and vagina was significantly lower than in the tissues from rat. In contrast to the rat where NOS activity was predominantly found in the cytosolic fractions, NOS activity in particulate and cytosolic fractions from both human myometrium and vagina was similar. Data from Western blots confirmed that eNOS and nNOS isoforms were concentrated in the particulate and cytosolic fractions, respectively. Estrogen treatment of rats resulted in a down regulation of uterine cytosolic NOS activity. A down regulation of NOS in the cytosolic fraction was also seen in the human pregnant myometrium as compared with the nonpregnant myometrium. The vaginal NOS activity was considerably higher than the uterus in both species. In spite of some clear-cut qualitative and other differences between human and rat tissues, there are some interesting similarities. Downregulation in pregnancy of human uterine NOS is probably due to, at least in part, the influence of estrogen and progesterone.

Al-Hijji J

2003-02-01

228

Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.  

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The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level. PMID:24793581

Pi?syk, Sebastian; Perli?ska-Lenart, Urszula; Górka-Nie?, Wioletta; Graczyk, Sebastian; Antosiewicz, Beata; Zembek, Patrycja; Palamarczyk, Gra?yna; Kruszewska, Joanna S

2014-07-10

229

L-Malate content in irradiated onions (Allium Cepa L.) cv. Valenciana sintetica 14  

International Nuclear Information System (INIS)

Results of L-malate evaluation in control and irradiated onions, (v. 'Valenciana sintetica 14') and its correlation with sprouting cumulative values are reported. It was concluded that if on the 150th day of storage, the malate content reaches a maximum value and the sprouting is 1 per cent or less, then it would indicate that the samples have been irradiated. L-malate values are positively correlated to sprouting in control samples, while for irradiated ones correlation was negative. (author)

1987-01-01

230

Another Unusual Type of Citric Acid Cycle Enzyme in Helicobacter pylori: the Malate:Quinone Oxidoreductase  

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The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the elect...

2000-01-01

231

An Arabidopsis callose synthase.  

Science.gov (United States)

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

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

2002-08-01

232

Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes  

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Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ..mu..g/ml) permeabilized rat adipocytes. Incorporation of /sup 3/H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10/sup -9/M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different.

Mooney, R.A.; Wisniewski, J.L.

1986-05-01

233

Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes  

International Nuclear Information System (INIS)

Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ?g/ml) permeabilized rat adipocytes. Incorporation of "3H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10"-"9M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different

1986-05-01

234

Citrate synthase, sarcoplasmic reticular calcium ATPase, and choline acetyltransferase activities of specific pelvic floor muscles of the rabbit.  

Science.gov (United States)

There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic-endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity. PMID:22911511

Spettel, Sara; De, Elise; Elias, Tamer; Schuler, Catherine; Leggett, Robert E; Levin, Robert M

2012-11-01

235

Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The effect of foliar application of salicylic acid (SA) at different concentrations (10?3 M and 10?5 M) was investigated on the production of secondary metabolites (flavonoids), chalcone synthase (CHS) activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231) in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC) analysis showed that application of SA induc...

2012-01-01

236

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.  

Science.gov (United States)

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME. PMID:1291764

Banu, M J; Nellaiappan, K; Dhandayuthapani, S

1992-06-01

237

Steroid Receptor RNA Activator (SRA) Modification by the Human Pseudouridine Synthase 1 (hPus1p): RNA Binding, Activity, and Atomic Model.  

Science.gov (United States)

The most abundant of the modified nucleosides, and once considered as the "fifth" nucleotide in RNA, is pseudouridine, which results from the action of pseudouridine synthases. Recently, the mammalian pseudouridine synthase 1 (hPus1p) has been reported to modulate class I and class II nuclear receptor responses through its ability to modify the Steroid receptor RNA Activator (SRA). These findings highlight a new level of regulation in nuclear receptor (NR)-mediated transcriptional responses. We have characterised the RNA association and activity of the human Pus1p enzyme with its unusual SRA substrate. We validate that the minimal RNA fragment within SRA, named H7, is necessary for both the association and modification by hPus1p. Furthermore, we have determined the crystal structure of the catalytic domain of hPus1p at 2.0 Å resolution, alone and in a complex with several molecules present during crystallisation. This model shows an extended C-terminal helix specifically found in the eukaryotic protein, which may prevent the enzyme from forming a homodimer, both in the crystal lattice and in solution. Our biochemical and structural data help to understand the hPus1p active site architecture, and detail its particular requirements with regard to one of its nuclear substrates, the non-coding RNA SRA. PMID:24722331

Huet, Tiphaine; Miannay, François-Alexandre; Patton, Jeffrey R; Thore, Stéphane

2014-01-01

238

2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.  

Science.gov (United States)

There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

Bitok, J Kipchirchir; Meyers, Caren Freel

2012-10-19

239

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.  

Science.gov (United States)

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625?mM. A hyperosmotic condition (1.2?M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. PMID:21721062

Yutani, Masahiro; Hashimoto, Yukie; Ogita, Akira; Kubo, Isao; Tanaka, Toshio; Fujita, Ken-ichi

2011-11-01

240

The tumor-suppressing activity of the prenyl diphosphate synthase subunit 2 gene in lung cancer cells.  

Science.gov (United States)

The prenyl diphosphate synthase subunit 2 (PDSS2) gene has recently been proposed as a novel tumor suppressor in several types of solid tumors. However, the mechanism of its tumor-suppressing activity is not known. Our previous study found a decreased expression of PDSS2 in clinical samples of non-small-cell lung cancer, and an inverse correlation between PDSS2 levels and stages of tumor differentiation and lymph node metastasis. In this study, we further investigated the tumor-suppressing activity of PDSS2 in lung cancer cells using cellular and molecular tools. The PDSS2 gene has low levels of expression in human lung cancer cell lines. We transfected and overexpressed PDSS2 in the NCI-H1299 lung cancer cell line. The forced overexpression caused massive cell death (?70%) through apoptotic pathways and significantly inhibited colony formation. At the same time, repression of PDSS2 expression by siRNA enhanced the growth of a noncancerous lung epithelial cell line MRC-5. There was an inverse correlation (Pearson's test, r=-0.9373) between PDSS2 expression and gelsolin expression, which is known to inhibit apoptosis and enhance cell invasion and metastasis. The ability of PDSS2 to repress gelsolin might contribute to its tumor-suppressing activity. However, PDSS2 did not influence the sensitivity of the lung cancer cells to chemotherapeutic drugs. Taken together, PDSS2 has tumor-suppressing activity in human lung cancer cells by enhancing apoptosis and inhibiting tumorigenic capacity. PMID:24608273

Chen, Ping; Zhang, Yin; Polireddy, Kishore; Chen, Qi

2014-08-01

 
 
 
 
241

Spectroscopic, thermal and structural studies on manganous malate crystals  

Science.gov (United States)

Prismatic crystals of manganous malate have been prepared by controlled ionic diffusion in hydrosilica gel. The structure was elucidated using single crystal X-ray diffraction. The crystals are orthorhombic with space group Pbca. Vibrations of the functional groups were identified by the FTIR spectrum. Thermogravimetric and differential thermal analyses (TG-DTA) were carried out to explore the thermal decomposition pattern of the material. Structural information derived from FTIR and TG-DTA studies is in conformity with the single crystal XRD data.

Thomas, J.; Lincy, A.; Mahalakshmi, V.; Saban, K. V.

2013-01-01

242

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

Science.gov (United States)

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

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

1994-01-01

243

Exercise Protects against Chronic ?-Adrenergic Remodeling of the Heart by Activation of Endothelial Nitric Oxide Synthase  

Science.gov (United States)

Extensive data have shown that exercise training can provide cardio-protection against pathological cardiac hypertrophy. However, how long the heart can retain cardio-protective phenotype after the cessation of exercise is currently unknown. In this study, we investigated the time course of the loss of cardio-protection after cessation of exercise and the signaling molecules that are responsible for the possible sustained protection. Mice were made to run on a treadmill six times a week for 4 weeks and then rested for a period of 0, 1, 2 and 4 weeks followed by isoproterenol injection for 8 days. Morphological, echocardiographic and hemodynamic changes were measured, gene reactivation was determined by real-time PCR, and the expression and phosphorylation status of several cardio-protective signaling molecules were analyzed by Western-blot. HW/BW, HW/TL and LW/BW decreased significantly in exercise training (ER) mice. The less necrosis and lower fetal gene reactivation induced by isoproterenol injection were also found in ER mice. The echocardiographic and hemodynamic changes induced by ?-adrenergic overload were also attenuated in ER mice. The protective effects can be sustained for at least 2 weeks after the cessation of the training. Western-blot analysis showed that the alterations in the phosphorylation status of endothelial nitric oxide synthase (eNOS) (increase in serine 1177 and decrease in threonine 495) continued for 2 weeks after the cessation of the training whereas increases of the phosphorylation of Akt and mTOR disappeared. Further study showed that L-NG-Nitroarginine methyl ester (L-NAME) treatment abolished the cardio-protective effects of ER. Our findings demonstrate that stimulation of eNOS in mice through exercise training provides acute and sustained cardioprotection against cardiac hypertrophy.

Yang, Liang; Jia, Zhe; Yang, Lei; Zhu, Mengmeng; Zhang, Jincai; Liu, Jie; Wu, Ping; Tian, Wencong; Li, Jing; Qi, Zhi; Tang, Xiangdong

2014-01-01

244

Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.  

Science.gov (United States)

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders. PMID:19826105

Gupta, Vineet; Patwardhan, Gauri A; Zhang, Qian-Jin; Cabot, Myles C; Jazwinski, S Michal; Liu, Yong-Yu

2010-04-01

245

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301 with Thromboxane Synthase Inhibitory Activity in Rats Subjected to Cerebral Ischemia  

Directory of Open Access Journals (Sweden)

Full Text Available ONO-1301 has been developed as a novel long-acting prostacyclin agonist with thromboxane synthase inhibitory activity. In the present study, we investigated the cerebroprotective effect of ONO-1301 on post-ischemic injury induced by cerebral ischemia in rats. ONO-1301 (1 and 10 mg/kg was administrated orally at reperfusion and then twice a day for 42 days. The cell damage induced by cerebral ischemia in the hippocampal CA1 was evaluated using both Nissl staining and proliferating cell nuclear antigen (PCNA staining on the 42 days after cerebral ischemia. Activated astrocytes were evaluated using immunofluorescence staining with GFAP on the 42 days after cerebral ischemia. Spatial learning was assessed using a Morris water maze (MWM task on the 56 days (i.e. after a 14 days washout period. ONO-1301- treated rats (1 and 10 mg/kg significantly improved cell death in the hippocampal CA1, the number of PCNA-positive cells and astrocyte activation. The spatial learning of ONO-1301-treated rats compared with vehicle- treated rats in the MWM task. These results suggest that repeated treatment with oral ONO-1301 could prevent or limit post-ischemic brain damage. In particular, treatment with ONO-1301 within 7 days after ischemia is most effective to improve ischemic damage.

Mai Hazekawa

2011-10-01

246

Thymidylate synthase activity and the cell growth are inhibited by the beta-carboline-benzoquinolizidine alkaloid deoxytubulosine.  

Science.gov (United States)

Employing thymidylate synthase (TS) (5, 10-CH2-H4PteGlu: dUMP C-methyltransferase, EC 2.1.1.45), a key target enzyme in chemotherapy, the biological activity of the beta-carboline-benzoquinolizidine alkaloid deoxytubulosine (DTB) isolated from the Indian medicinal plant Alangium lamarckii has been evaluated and assessed for the first time. The TS employed in the present studies was purified from Lactobacillus leichmannii. The DTB was demonstrated to exhibit potent cytotoxicity and inhibited the cell growth of L. leichmannii, and DTB potently inhibited TS activity (IC50 = 40 microM). The DTB concentrations > 80 microM resulted in a total loss of the TS activity, thus suggesting that the beta-carboline-benzoquinolizidine alkaloid is a promising potential antitumor agent. The DTB binding to TS appears to be irreversible and tight through a possible covalent linkage. Although DTB strongly binds to DNA, it is not known whether DTB binds to RNA associated with TS. Inhibition kinetics showed that TS has a Ki value of 7 x 10(-6) M for DTB and that the inhibition is a simple linear "noncompetitive" type. PMID:9522276

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

1998-01-01

247

Determination of delta-aminolaevulinic acid synthase activity in human bone marrow using high performance liquid chromatography  

International Nuclear Information System (INIS)

A high performance liquid chromatographic (HPLC) method is described for the rapid and specific determination of the activity of the enzyme delta-aminolaevulinic acid synthase (ALA-S) in mitochondria prepared by sonication of human bone marrow cells. After incubation with "1"4C-?-ketoglutarate the "1"4C-delta-aminolaevulinic acid (ALA) formed is converted to a pyrrole derivative, 2-methyl-3-carbethoxy-4-(3-propionic acid) pyrrole. This is isolated by reversed-phase ion-pair chromatography on a Hypersil-SAS column with methanol-water (45:155, v/v) in the presence of 0.005 mol/l l-heptanesulphonic acid (PIC B-7) as the mobile phase. The radioactivity of the isolated pyrrole is determined by scintillation counting. The optimal substrate concentration and pH were 0.17 mmol/l ?-ketoglutarate and pH 7.4, with an optimal period of sonication of 18s. Under these conditions ALA production was proportional to the concentrations of erythroblasts in the initial sample and was linear with time up to 60 min. The addition of pyridoxal phosphate (PLP) did not affect ALA-S activity in normal subjects. The mean ALA-S activity in 10 haematologically normal control subjects was found to be 318.8 pmol.10"-"6 erythroblasts.h"-"1 (S.D. +- 125.8, range 193-444.6). (Auth.)

1981-06-02

248

Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells.  

Science.gov (United States)

Hydrogen sulfide (H(2)S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H(2)S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H(2)S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H(2)S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H(2)S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H(2)S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function. PMID:23148920

Kida, Michiya; Sugiyama, Toru; Yoshimoto, Takanobu; Ogawa, Yoshihiro

2013-01-23

249

Modulation of the Heme Electronic Structure and Cystathionine ?-synthase Activity by Second Coordination Sphere Ligands: The Role of Heme Ligand Switching in Redox Regulation†  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In humans, cystathionine ?-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While...

2009-01-01

250

Equol-Stimulated Mitochondrial Reactive Oxygen Species Activate Endothelial Nitric Oxide Synthase and Redox Signaling in Endothelial Cells: Roles for F-Actin and GPR30  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We reported previously that dietary isoflavones modulate arterial blood pressure in vivo and that the daidzein metabolite equol rapidly activates endothelial NO synthase (eNOS) via Akt and extracellular signal–regulated kinase 1/2– dependent signaling. In this study, we report the first evidence in human endothelial cells that acute stimulation of mitochondrial superoxide generation by equol (100 nmol/L) is required for eNOS activation. Scavengers of superoxide (superoxide dismutase and m...

Rowlands, David J.; Chapple, Sarah; Siow, Richard C. M.; Mann, Giovanni E.

2011-01-01

251

Association between accumulation of allene oxide synthase activity and development of resistance against downy mildew disease of pearl millet.  

Science.gov (United States)

The present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars. PMID:24166513

Hosur Gnanaprakash, Pushpalatha; Jogaiah, Sudisha; Sreedhara, Ashok Prabhu; Nagraj Prashanth, Geetha; Kini, Ramachandra K; Shetty, Shekar Hunthrike

2013-12-01

252

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

Science.gov (United States)

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu catalytic triad [Murray-Rust, J., Leiper, J. M., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P. & McDonald, N. (2001) Nat. Struct. Biol. 8, 679-683]. The presence of a reactive cysteine residue (Cys-249) in the active site of DDAH raised the possibility that DDAH activity might be directly regulated by S-nitrosylation of this residue by NO. In the present study, we demonstrate that recombinant DDAH is reversibly inhibited after incubation with NO donors in vitro. Similarly mammalian DDAH in cytosolic extracts is also reversibly inhibited by NO donors. In cultured endothelial cells, heterologously expressed human DDAH II was S-nitrosylated after cytokine induced expression of the inducible NOS isoforms. The implication of these findings is that under certain conditions when NO generation increases, S-nitrosylation diminishes DDAH activity and this would be expected to lead to accumulation of asymmetric dimethylarginine and inhibition of NOS. This observation may help explain why expression of iNOS often leads to inhibition of activity of constitutively expressed NOS isozymes. We also identify Cys-His-Glu as a nitrosylation motif that is conserved in a family of arginine handling enzymes. PMID:12370443

Leiper, James; Murray-Rust, Judith; McDonald, Neil; Vallance, Patrick

2002-10-15

253

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

DEFF Research Database (Denmark)

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 labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger

2007-01-01

254

High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities.  

DEFF Research Database (Denmark)

Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method,we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC. Udgivelsesdato: null-null

Volke, A; Wegener, Gregers

2006-01-01

255

Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry.  

Science.gov (United States)

Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of the C(5) terpenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, to form geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes-monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to detect the short-chain prenyl diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C(10) enzyme products (geranyl diphosphate and neryl diphosphate) and three C(15) products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could determine their IDS activity in a reproducible fashion. PMID:22266300

Nagel, Raimund; Gershenzon, Jonathan; Schmidt, Axel

2012-03-01

256

Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene.  

Science.gov (United States)

We began characterization of the protein--DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression experiments in parsley protoplasts. Two of the three footprinted sequence 'boxes' reside within the minimal promoter. Replacement of 10 bp within either of these 'boxes' leads to complete loss of light responsiveness. We conclude that these sequences define the necessary cis elements of the minimal CHS promoter's light responsive element. One of the functionally defined 'boxes' is homologous to an element implicated in regulation of genes involved in photosynthesis. These data represent the first example in a plant defense gene of an induced change in protein--DNA contacts necessary for transcriptional activation. Also, our data argue strongly that divergent light induced biosynthetic pathways share common regulatory units. PMID:2566481

Schulze-Lefert, P; Dangl, J L; Becker-André, M; Hahlbrock, K; Schulz, W

1989-03-01

257

Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and extractable sucrose phosphate synthase activity  

Energy Technology Data Exchange (ETDEWEB)

Mild water stress, on the order of {minus}1.0 megapascals xylem water potential, can reduce the rate of photosynthesis and eliminate the inhibition of photosynthesis caused by O{sub 2} in water-stress-sensitive plants such as Phaseolus vulgaris. To investigate the lack of O{sub 2} inhibition of photosynthesis, we measured stromal and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase, and partitioning of newly fixed carbon between starch and sucrose before, during, and after mild water stress. The extractable activity of the fructose bisphosphatases was unaffected by mild water stress. The extractable activity of SPS was inhibited by more than 60% in plants stressed to water potentials of {minus}0.9 megapascals. Water stress caused a decline in the starch/sucrose partitioning ratio indicating that starch synthesis was inhibited more than sucrose synthesis. We conclude that the reduced rate of photosynthesis during water stress is caused by stomatal closure, and that the restriction of CO{sub 2} supply caused by stomatal closure leads to a reduction in the capacity for both starch and sucrose synthesis. This causes the reduced O{sub 2} inhibition and abrupt CO{sub 2} saturation of photosynthesis.

Vassey, T.L.; Sharkey, T.D. (Univ. of Wisconsin, Madison (USA))

1989-04-01

258

Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase  

DEFF Research Database (Denmark)

Background-Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. Methods and Results-Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 9226 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility C, shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). Conclusions-In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Wallerath, Thomas; Deckert, Göran

2002-01-01

259

The ketogenic diet component decanoic acid increases mitochondrial citrate synthase and complex I activity in neuronal cells.  

Science.gov (United States)

The Ketogenic diet (KD) is an effective treatment with regards to treating pharmaco-resistant epilepsy. However, there are difficulties around compliance and tolerability. Consequently, there is a need for refined/simpler formulations that could replicate the efficacy of the KD. One of the proposed hypotheses is that the KD increases cellular mitochondrial content which results in elevation of the seizure threshold. Here, we have focussed on the medium-chain triglyceride form of the diet and the observation that plasma octanoic acid (C8) and decanoic acid (C10) levels are elevated in patients on the medium-chain triglyceride KD. Using a neuronal cell line (SH-SY5Y), we demonstrated that 250-?M C10, but not C8, caused, over a 6-day period, a marked increase in the mitochondrial enzyme, citrate synthase along with complex I activity and catalase activity. Increased mitochondrial number was also indicated by electron microscopy. C10 is a reported peroxisome proliferator activator receptor ? agonist, and the use of a peroxisome proliferator activator receptor ? antagonist was shown to prevent the C10-mediated increase in mitochondrial content and catalase. C10 may mimic the mitochondrial proliferation associated with the KD and raises the possibility that formulations based on this fatty acid could replace a more complex diet. We propose that decanoic acid (C10) results in increased mitochondrial number. Our data suggest that this may occur via the activation of the PPAR? receptor and its target genes involved in mitochondrial biogenesis. This finding could be of significant benefit to epilepsy patients who are currently on a strict ketogenic diet. Evidence that C10 on its own can modulate mitochondrial number raises the possibility that a simplified and less stringent C10-based diet could be developed. PMID:24383952

Hughes, Sean David; Kanabus, Marta; Anderson, Glenn; Hargreaves, Iain P; Rutherford, Tricia; O'Donnell, Maura; Cross, J Helen; Rahman, Shamima; Eaton, Simon; Heales, Simon J R

2014-05-01

260

c-Fos Activates Glucosylceramide Synthase and Glycolipid Synthesis in PC12 Cells*  

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It has been demonstrated that c-Fos has, in addition to its well recognized AP-1 transcription factor activity, the capacity to associate to the endoplasmic reticulum and activate key enzymes involved in the synthesis of phospholipids required for membrane biogenesis during cell growth and neurite formation. Because membrane genesis requires the coordinated supply of all its integral membrane components, the question emerges as to whether c-Fos also activates the synth...

Crespo, Pilar M.; Silvestre, David C.; Gil, Germa?n A.; Maccioni, Hugo J. F.; Daniotti, Jose? L.; Caputto, Beatriz L.

2008-01-01

 
 
 
 
261

Nitric oxide synthase-dependent NADPH-diaphorase activity in the optic lobes of male and female Ceratitis capitata mutants  

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Full Text Available Nitric oxide (NO is acknowledged as a messenger molecule in the nervous system with a pivotal role in the modulation of the chemosensory information. It has been shown to be present in the optic lobes of several insect species. In the present study, we used males and females from four different strains of the medfly Ceratitis capitata (Diptera, Tephritidae: or; or,wp (both orange eyed; w,M360 and w,Heraklion (both white eyed, as models to further clarify the involvement of NO in the mutants’ visual system and differences in its activity and localization in the sexes. Comparison of the localization pattern of NO synthase (NOS, through NADPH-diaphorase (NADPHd staining, in the optic lobes of the four strains, revealed a stronger reaction intensity in the retina and in the neuropile region lamina than in medulla and lobula. Interestingly, the intensity of NADPHd staining differs, at least in some strains, in the optic lobes of the two sexes; all the areas are generally strongly labelled in the males of the or and w,M360 strains, whereas the w,Heraklion and or,wp mutants do not show evident sexdependent NADPHd staining. Taken as a whole, our data point to NO as a likely transmitter candidate in the visual information processes in insects, with a possible correlation among NOS distribution, eye pigmentation and visual function in C. capitata males. Moreover, NO could influence behavioural differences linked to vision in the two sexes.

G Bernocchi

2004-06-01

262

Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor.  

Science.gov (United States)

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H(+)-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H(+) ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody-antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4-340.0 microg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was -0.073+/-0.002 mV/microg ml(-1). The detection limit of nNOS was determined to be 0.2 microg/ml based on five-time measurements (95% confidence level, k=3, n=5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis. PMID:19632105

Koh, Wei Choon Alvin; Choe, Eun Sang; Lee, Dong Kun; Chang, Seung-Cheol; Shim, Yoon-Bo

2009-09-15

263

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

Science.gov (United States)

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

Cox, Timothy M

2010-10-01

264

Evaluation of the prostaglandin F synthase activity of human and bovine aldo-keto reductases: AKR1A1s complement AKR1B1s as potent PGF synthases.  

Science.gov (United States)

AKR1B1 of the polyol pathway was identified as a prostaglandin F2? synthase (PGFS). Using a genomic approach we have identified in the endometrium five bovine and three human AKRs with putative PGFS activity and generated the corresponding recombinant enzymes. The PGFS activity of the recombinant proteins was evaluated using a novel assay based on in situ generation of the precursor of PG biosynthesis PGH2. PGF2? was measured by ELISA and the relative potencies of the different enzymes were compared. We identified AKR1A1 and confirmed AKR1B1 as the most potent PGFS expressing characteristic inhibition patterns in presence of methylglyoxal, ponalrestat and glucose. PMID:23747692

Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Fortier, Michel A

2013-10-01

265

Plasma Interleukin-12 in Malaria-Tolerant Papua New Guineans: Inverse Correlation with Plasmodium falciparum Parasitemia and Peripheral Blood Mononuclear Cell Nitric Oxide Synthase Activity  

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Interleukin-12 (IL-12) has been inversely associated with disease severity in human and murine malaria, and a polymorphism in the IL-12 p40 subunit gene (IL12B) has been associated with susceptibility to human cerebral malaria and reduced nitric oxide (NO) production. To better define the relationships between IL-12, NO, malaria parasitemia, and IL12B polymorphisms during malarial tolerance, plasma IL-12 levels and peripheral blood mononuclear cell NO synthase (NOS) activity were measured in ...

Boutlis, Craig S.; Lagog, Moses; Chaisavaneeyakorn, Sujittra; Misukonis, Mary A.; Bockarie, Moses J.; Mgone, Charles S.; Wang, Zhiqiang; Morahan, Grant; Weinberg, J. Brice; Udhayakumar, Venkatachalam; Anstey, Nicholas M.

2003-01-01

266

Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury  

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Abstract Background Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown.

Okubo Masamichi; Yamanaka Hiroki; Kobayashi Kimiko; Kanda Hirosato; Dai Yi; Noguchi Koichi

2012-01-01

267

Citrus Flavonoids Luteolin, Apigenin, and Quercetin Inhibit Glycogen Synthase Kinase-3? Enzymatic Activity by Lowering the Interaction Energy Within the Binding Cavity  

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Pancreatic cancer studies have shown that inhibition of glycogen synthase kinase-3? (GSK-3?) leads to decreased cancer cell proliferation and survival by abrogating nuclear factor ?B (NF?B) activity. In this investigation, various citrus compounds, including flavonoids, phenolic acids, and limonoids, were individually investigated for their inhibitory effects on GSK-3? by using a luminescence assay. Of the 22 citrus compounds tested, the flavonoids luteolin, apigenin, and quercetin had t...

Johnson, Jodee L.; Rupasinghe, Sanjeewa G.; Stefani, Felicia; Schuler, Mary A.; Mejia, Elvira Gonzalez

2011-01-01

268

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

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

Brayman, Timothy G.; Wilks, John W.

2003-01-01

269

Beneficial effects of antioxidants and l-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress  

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Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by ...

Nigris, Filomena; Lerman, Lilach O.; Ignarro, Sharon Williams; Sica, Giacomo; Lerman, Amir; Palinski, Wulf; Ignarro, Louis J.; Napoli, Claudio

2003-01-01

270

Differential induction of chalcone synthase mRNA activity at the onset of phytoalexin accumulation in compatible and incompatible plant-pathogen interactions  

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Changes in the mRNA activity of chalcone synthase, the first enzyme of phenylpropanoid metabolism specific to flavonoid/isoflavonoid biosynthesis, have been investigated in relation to expression of the phytoalexin defense response in race-cultivar specific interactions between hypocotyls of Phaseolus vulgaris and the partially biotrophic fungus Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there is an early but localized increase ...

Bell, John N.; Dixon, Richard A.; Bailey, John A.; Rowell, Pat M.; Lamb, Chris J.

1984-01-01

271

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions  

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Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2? are the main prostanoids produced in the human endometrium and proper balance in their relative production is important...

Bresson, Eva; Lacroix-pe?pin, Nicolas; Boucher-kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A.

2012-01-01

272

A novel interaction between Glycogen Synthase Kinase-3? (GSK-3?) and the scaffold protein Receptor for Activated C-Kinase 1 (RACK1) regulates the circadian clock  

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Glycogen synthase kinase-3? (GSK-3?) and GSK-3? are intracellular kinases with largely redundant functions. However, the deletion of each GSK-3 isoform in the mouse has distinct consequences, suggesting that these related enzymes also have non-overlapping isoform-specific functions. A yeast two-hybrid screen for GSK-3? interacting partners revealed an interaction with the Receptor for Activated C-Kinase 1 (RACK1). We confirm this interaction in mammalian cells, and provide evidence that R...

Zeidner, Leigh C.; Buescher, Jessica L.; Phiel, Christopher J.

2011-01-01

273

Nitric oxide synthase activity in rat gastric mucosa contributes to mucin synthesis elicited by calcitonin gene-related peptide.  

Science.gov (United States)

The majority of research for the calcitonin gene-related peptide (CGRP) in the stomach has been devoted to the submucosal blood flow, and only slight attention has been paid to its involvement in the gastric epithelial function. In this study, we examined the age-related change in the CGRP-containing nerves and its effects on the mucus metabolism. We compared the immunoreactivity for CGRP in the gastric mucosa of 7-week-old rats (young) to that of 52-week-old animals (middle-aged). The effects of CGRP on the mucin biosynthesis were compared using the stomachs from both young and middle-aged rats. The nitric oxide synthase (NOS) activity was measured in the surface and deep mucosa of the gastric corpus. The density of the CGRP nerve fibers was reduced in both the lamina propria and submucosa of the middle-aged rats compared to the young rats. CGRP stimulated the mucin biosynthesis in the cultured corpus mucosa from the 7-week-old rats, but not from the 52-week-old rats. The total NOS activity of the surface layer in the corpus mucosa was markedly reduced in the middle-aged rats compared to the young rats. These findings demonstrate the age-dependent reduction in the CGRP-induced mucin biosynthesis, as well as in the density of the CGRP fibers in the rat stomach. The decreased NOS activity in the surface layer of the oxyntic mucosa in the aged rats may also be a principal cause for the lack of regulation of the mucin biosynthesis by CGRP. PMID:16847357

Ichikawa, Takafumi; Kusakabe, Tatsumi; Gono, Yukari; Shikama, Nobuaki; Hiruma, Hiromi; Kawakami, Tadashi; Ishihara, Kazuhiko

2006-06-01

274

Observation of an Unusual Electronically Distorted Semiquinone Radical of PCB Metabolites in the Active Site of Prostaglandin H Synthase-2  

Science.gov (United States)

The activation of the metabolites of airborne polychlorinated biphenyls (PCBs) into highly reactive radicals is of fundamental importance. We found that human recombinant prostaglandin H synthase-2 (hPGHS-2) biotransforms dihydroxy-PCBs, such as 4-chlorobiphenyl-2?,5?-hydroquinone (4-CB-2?,5?H2Q), into semiquinone radicals via one-electron oxidation. Using electron paramagnetic resonance (EPR) spectroscopy, we observed the formation of the symmetric quartet spectrum (1:3:3:1 by area) of 4-chlorobiphenyl-2?,5?-semiquinone radical (4-CB-2?,5?-SQ•?) from 4-CB-2?,5?H2Q. This spectrum changed to an asymmetric spectrum with time: the change can be explained as the overlap of two different semiquinone radical species. Hindered rotation of the 4-CB-2?,5?-SQ•? appears not to be a major factor for the change in lineshape because increasing the viscosity of the medium with glycerol produced no significant change in lineshape. Introduction of a fluorine, which increases the steric hindrance for rotation of the dihydroxy-PCB studied, also produced no significant changes. An in silico molecular docking model of 4-CB-2?,5?H2Q in the peroxidase site of hPGHS-2 together with ab initio quantum mechanical studies indicate that the close proximity of a negatively charged carboxylic acid in the peroxidase active site may be responsible for the observed perturbation in the spectrum. This study provides new insights into the formation of semiquinones from PCB metabolites and underscores the potential role of PGHS-2 in the metabolic activation of PCBs.

Wangpradit, Orarat; Moman, Edelmiro; Nolan, Kevin B.; Buettner, Garry R.; Robertson, Larry W.; Luthe, Gregor

2013-01-01

275

Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3  

Science.gov (United States)

In the present study we report that a replication-defective adenovirus construct of GMF cDNA (GMF-V) induced overexpression of GMF protein in neuroblastoma (N18) cells caused cytotoxicity and loss of cell viability. A significant increase in activation of GSK-3? occurred after infection with GMF-V when compared with mock and lacZ controls. Overexpression of GMF also increased caspase-3 activity, an early marker of apoptosis. Depletion of GMF gene by introducing GMF-specific siRNA (GsiRNA) completely blocked both activation of GSK-3? and caspase-3 activation whereas a control scrambled siRNA (CsiRNA) had no effect. A cell-permeable peptide inhibitor of GSK-3?, and lithium completely prevented GMF-dependent activation of caspase-3. These results demonstrate that GSK-3 mediates activation of the death domain caspase by GMF overexpression. We also show that the phosphorylation of GSK-3-dependent site of Tau was a consequence of GMF-overexpression in N18 cells. Taken together our results imply that GMF is involved in the signaling leading to the activation of GSK-3? and caspase-3 in N18 cells and strongly suggest its involvement in neurodegeneration since, GSK-3? is known to hyperphosphorylate tau which is associated with the neurotoxicity of neurofibrillary tangles in Alzheimer's disease.

Zaheer, Asgar; Knight, Scott; Zaheer, Ashna; Ahrens, Marcus; Sahu, Shailendra K.; Yang, Baoli

2008-01-01

276

Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3.  

Science.gov (United States)

In the present study we report that a replication-defective adenovirus construct of GMF cDNA (GMF-V) induced overexpression of GMF protein in neuroblastoma (N18) cells caused cytotoxicity and loss of cell viability. A significant increase in activation of GSK-3beta occurred after infection with GMF-V when compared with mock and lacZ controls. Overexpression of GMF also increased caspase-3 activity, an early marker of apoptosis. Depletion of GMF gene by introducing GMF-specific siRNA (GsiRNA) completely blocked both activation of GSK-3beta and caspase-3 activation whereas a control scrambled siRNA (CsiRNA) had no effect. A cell-permeable peptide inhibitor of GSK-3beta, and lithium completely prevented GMF-dependent activation of caspase-3. These results demonstrate that GSK-3 mediates activation of the death domain caspase by GMF overexpression. We also show that the phosphorylation of GSK-3-dependent site of Tau was a consequence of GMF-overexpression in N18 cells. Taken together our results imply that GMF is involved in the signaling leading to the activation of GSK-3beta and caspase-3 in N18 cells and strongly suggest its involvement in neurodegeneration since GSK-3beta is known to hyperphosphorylate tau which is associated with the neurotoxicity of neurofibrillary tangles in Alzheimer's disease. PMID:18054898

Zaheer, Asgar; Knight, Scott; Zaheer, Ashna; Ahrens, Marcus; Sahu, Shailendra K; Yang, Baoli

2008-01-23

277

In Vivo Activities of Farnesyl Pyrophosphate Synthase Inhibitors against Leishmania donovani and Toxoplasma gondii  

Science.gov (United States)

The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.

Yardley, Vanessa; Khan, Anis A.; Martin, Michael B.; Slifer, Teri R.; Araujo, Fausto G.; Moreno, Silvia N. J.; Docampo, Roberto; Croft, Simon L.; Oldfield, Eric

2002-01-01

278

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.  

Science.gov (United States)

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself. PMID:12354108

Willemoës, Martin; Sigurskjold, Bent W

2002-10-01

279

Mice deficient in cystathionine beta synthase display increased Dyrk1A and SAHH activities in brain.  

Science.gov (United States)

Hyperhomocysteinemia is associated with brain disease. However, biological actions linking hyperhomocysteinemia to neuronal abnormalities are not well understood. We recently found a relationship between Dyrk1A protein expression, a serine/threonine kinase that might be responsible for cognitive functions in Down's syndrome, and hepatic S-adenosylhomocysteine hydrolase (SAHH) activity, which plays a key role in S-adenosylmethionine-dependent methylation reactions. Considering the role of methylation and Dyrk1A in cognitive functions, the aim of this study was to investigate the relationship between Dyrk1A and SAHH activity in brain of hyperhomocysteinemic mice. We found an increase in Dyrk1A protein expression and activity in brain of hyperhomocysteinemic mice, concomitant with an increased SAHH activity. The effect of overexpression of protein Dyrk1A on SAHH activity was confirmed in brain of Dyrk1A transgenic mice, and additionally we found a positive correlation between Dyrk1A and SAHH activity. These observations suggest a potential effect of Dyrk1A on brain phenotypes linked to hyperhomocysteinemia. PMID:22700376

Planque, Chris; Dairou, Julien; Noll, Christophe; Bui, Linh-Chi; Ripoll, Clémentine; Guedj, Fayçal; Delabar, Jean-Maurice; Janel, Nathalie

2013-05-01

280

Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3  

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In the present study we report that a replication-defective adenovirus construct of GMF cDNA (GMF-V) induced overexpression of GMF protein in neuroblastoma (N18) cells caused cytotoxicity and loss of cell viability. A significant increase in activation of GSK-3? occurred after infection with GMF-V when compared with mock and lacZ controls. Overexpression of GMF also increased caspase-3 activity, an early marker of apoptosis. Depletion of GMF gene by introducing GMF-specific siRNA (GsiRNA) co...

Zaheer, Asgar; Knight, Scott; Zaheer, Ashna; Ahrens, Marcus; Sahu, Shailendra K.; Yang, Baoli

2008-01-01

 
 
 
 
281

Pengaruh Pengasapan (Thermal Fogging Insektisida Piretroid (Malation 95% Terhadap Nyamuk Aedes aegypti dan Culex quinquefasciatus di Pemukiman  

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Full Text Available The evaluation of piretroid insecticide (active ingredient Malation 95% was con-ducted in Sub district Tengarang, Semarang Segency, Central Java Province. The insecti-cide was applied using thermal fogging method for dosages of 125, 250, 375, 500 and 625 ml/ha (diluted in diesel to 10 litters. The evaluation of the efficacy was conducted against two mosquito species, Aedes aegypti (the main dengue haemorrhagic fever and Culex quinquefasciatus (the urban lymphatic fil-ariasis vector. Result of the evaluation was revealed that dosages of 500 and 625 ml/ha were effective against both tested mosquito species indoor and outdoor.

Hasan Boesri

2009-12-01

282

Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.  

Science.gov (United States)

Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 ?M) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (?13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ?1.9 fold while its inactive analog 4?PDD was ineffective. Calcium and PMA activated the enzyme to ?3 folds. Interestingly, 1,2-DG6 (2.5 ?M) and PS (1 ?M) with calcium activated the enzyme activity to ?7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (?90%) and calphostin-C (?40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. PMID:22947512

Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

2012-11-01

283

A sesquiterpene, dehydrocostus lactone, inhibits the expression of inducible nitric oxide synthase and TNF-alpha in LPS-activated macrophages.  

Science.gov (United States)

Nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) are the major mediators produced in activated macrophages which contribute to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (dehydrocostus lactone) isolated from the medicinal plant, Saussurea lappa, inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW 264.7 cells by suppressing inducible nitric oxide synthase enzyme expression. This compound also decreased the TNF-alpha level in LPS-activated systems in vitro and in vivo. Thus, dehydrocostus lactone may be a possible candidate for the development of new drugs to treat endotoxemia accompanied by the overproduction of NO and TNF-alpha. PMID:10193198

Lee, H J; Kim, N Y; Jang, M K; Son, H J; Kim, K M; Sohn, D H; Lee, S H; Ryu, J H

1999-03-01

284

Phenylalanine ammonia-lyase, flavanone 3?-hydroxylase and flavonol synthase enzyme activity by a new in vitro assay method in berry fruits.  

Science.gov (United States)

An HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3?-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method considered kinetic studies to establish the incubation time to perform the assay. The method here described proved to be an interesting approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods. The enzyme activity method developed was applied to strawberry, raspberry, blackberry, redcurrant and blackcurrant fruits. PMID:24491710

Flores, Gema; De la Peña Moreno, Fernando; Blanch, Gracia Patricia; Del Castillo, Maria Luisa Ruiz

2014-06-15

285

Changes in Carbohydrate Content and the Activities of Acid Invertase, Sucrose Synthase and Sucrose Phosphate Synthase in Asparagus Spears During Storage  

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We held asparagus (Asparagus officinalis L.) spears at 25?C for up to 5 days after harvest and examined changes in soluble carbohydrates and the activities of enzymes concerned with carbohydrate breakdown in both top and bottom portions of the spears. The acid invertase in soluble fraction showed a higher activity than that in cell wall bound fraction and the top portion of the spear showed a significantly higher soluble acid invertase activity than the bottom portion. But the activi...

Pankaj Kumar Bhowmik; Toshiyuki Matsui; Fabio Gimena Enriquez; Shameem Alam, A. K. M.; Kazuhide Kawada

2001-01-01

286

Inhibition of p38 mitogen-activated protein kinase enhances c-Jun N-terminal kinase activity: Implication in inducible nitric oxide synthase expression  

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Full Text Available Abstract Background Nitric oxide (NO is an inflammatory mediator, which acts as a cytotoxic agent and modulates immune responses and inflammation. p38 mitogen-activated protein kinase (MAPK signal transduction pathway is activated by chemical and physical stress and regulates immune responses. Previous studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli. The aim of the present study was to investigate the mechanisms involved in the regulation of inducible NO synthesis by p38 MAPK pathway. Results p38 MAPK inhibitors SB203580 and SB220025 stimulated lipopolysaccharide (LPS-induced inducible nitric oxide synthase (iNOS expression and NO production in J774.2 murine macrophages. Increased iNOS mRNA expression was associated with reduced degradation of iNOS mRNA. Treatment with SB220025 increased also LPS-induced c-Jun N-terminal kinase (JNK activity. Interestingly, JNK inhibitor SP600125 reversed the effect of SB220025 on LPS-induced iNOS mRNA expression and NO production. Conclusion The results suggest that inhibition of p38 MAPK by SB220025 results in increased JNK activity, which leads to stabilisation of iNOS mRNA, to enhanced iNOS expression and to increased NO production.

Kankaanranta Hannu

2006-02-01

287

Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase  

Energy Technology Data Exchange (ETDEWEB)

X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

2010-03-08

288

Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture  

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Full Text Available Abstract Background Platelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture. Methods Fetal membranes were collected from Caesarean sections at term. RNA was extracted from membranes and subjected to a qualitative RT-PCR to assess the baseline expression of iNOS. Discs of fetal membranes were cultured for 24 hours in the presence of platelet-activating factor at a dose range of 0.1 nanomolar – 1 micomolar or 1 microgram/ml lipopolysaccharide. Nitric oxide production was measured via nitrite ions in the culture medium and mRNA for iNOS was detected by RT-PCR. Results Culturing the membrane discs in medium containing serum induced nitric oxide synthase expression and platelet-activating factor significantly stimulated the production of nitric oxide under these conditions. When cultured without serum inducible nitric oxide synthase expression was induced by lipopolysaccharide, but not by platelet-activating factor. Conclusion Platelet-activating factor may have a role in the initiation of labour, at term or preterm, via the increased local production of nitric oxide as an inflammatory mediator. In this model of intrauterine infection, lipopolysaccharide was found to induce iNOS expression by fetal membranes, and this mechanism could be involved in preterm labour.

Seyffarth Gunter

2004-06-01

289

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase  

International Nuclear Information System (INIS)

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-?-stimulated monocytes to endothelial cells and suppressed the TNF-? induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-?-induced nuclear factor-?B activation, which was attenuated by pretreatment with NG-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ? Puerarin induced the phosphorylation of eNOS and the production of NO. ? Puerarin activated eNOS through ER-dependent PI3-kinase and Ca2+-dependent AMPK. ? Puerarin-induced NO was involved in the inhibition of NF-kB activation. ? Puerarin may help for prevention of vascular dysfunction and diabetes.

2011-11-15

290

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase  

Energy Technology Data Exchange (ETDEWEB)

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2011-11-15

291

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

Science.gov (United States)

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

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

2009-10-01

292

Overexpression of a resveratrol synthase gene (PcRS) from Polygonum cuspidatum in transgenic Arabidopsis causes the accumulation of trans-piceid with antifungal activity.  

Science.gov (United States)

Although resveratrol-forming stilbene synthase (STS) genes have been well characterized in many plant species, there are only a few descriptions about STS genes from Polygonum cuspidatum Sieb. et Zucc, an important medicinal crop in Asian countries. To evaluate the biological functions of a Polygonum cuspidatum resveratrol synthase gene (PcRS), the PcRS gene was expressed in Arabidopsis under the control of Cauliflower mosaic virus (CaMV) 35S promoter. Integration and expression of transgene in the plant genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Transgenic plants accumulated a new compound in both the leaves and seeds, which was identified as trans-piceid by high-pressure liquid chromatography (HPLC) and electrospray mass spectrometry (HPLC-ESI-MS). Overexpression of PcRS in transgenic Arabidopsis caused restriction of Colletotrichum higginsianum colonization by inhibition of spore production, resulting in enhanced resistance against C. higginsianum. So, the PcRS gene could be deployed in other crop plants to significantly enhance resistance to fungal pathogens and improve the nutritional quality. In addition, altered seed coat pigmentation and significant reduction in anthocyanin levels were observed in transgenic Arabidopsis, while the expression of endogenous chalcone synthase (CHS) gene was not down-regulated. These results suggest that additional STS activities cause a lack of precursors for CHS which leads to the disturbance of the subsequent flavonoid biosynthesis steps in Arabidopsis. PMID:21717185

Liu, Zhongyu; Zhuang, Chuxiong; Sheng, Shujing; Shao, Li; Zhao, Wei; Zhao, Shujin

2011-11-01

293

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

Science.gov (United States)

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

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

2004-03-11

294

Nitric oxide synthase isoforms and NF-kappaB activity in normal and osteoarthritic human chondrocytes: regulation by inducible nitric oxide  

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To elucidate the role of endogenous inducible nitric oxide (NO) on the regulation of NF-kappaB activity in human chondrocytes, we evaluated (i) the pattern of expression of the neuronal (nNOS) and inducible (iNOS) NO synthase isoforms and the basal NF-kappaB activity in normal and osteoarthritic (OA) human chondrocytes, (ii) the role of cytokines and growth factors in modulating the protein levels of the two NOS isoforms, and (iii) the effect of inhibiting endogenous inducible NO production o...

Rosa, Sc; Judas, F.; Lopes, Mc; Mendes, Af

2008-01-01

295

Potent activity of 5-fluoro-2'-deoxyuridine and related compounds against thymidine kinase-deficient (TK-) herpes simplex virus: targeted at thymidylate synthase.  

Science.gov (United States)

5-Fluorouracil, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorocytidine (FCyd), 5-fluoro-2'-deoxycytidine (FdCyd), 5-trifluoro-2'-deoxythymidine (F3dThd), and the 5'-monophosphates and 3',5'-cyclic monophosphates thereof were found to inhibit thymidine kinase-deficient (TK-) mutant strains of herpes simplex virus (HSV) at a much lower concentration than the wild-type (TK+) HSV strains. Other 5-substituted 2'-deoxyuridines that have previously been recognized as potent thymidylate synthase inhibitors behaved in a similar fashion. The activity of FdUrd, FdCyd, F3dThd, and their 3',5'-cyclic monophosphates against TK-HSV was readily reversed by 2'-deoxythymidine (dThd) but not by 2'-deoxyuridine (dUrd). These compounds also inhibited the incorporation of [6-3H]dUrd into DNA at a concentration which was up to 5 orders of magnitude lower than the concentration at which the incorporation of [methyl-3H] dThd was inhibited. Thus, while not being a target for the well established anti-HSV compounds in TK+HSV-infected cells, thymidylate synthase appears to be an important target in TK-HSV-infected cells. In addition to dTMP synthase, TK-HSV-infected cells appear to reveal other therapeutically exploitable targets such as OMP decarboxylase (towards pyrazofurin), CTP synthase (towards carbodine and its cyclopentenyl analogue), dihydrofolate reductase (towards methotrexate), and S-adenosylhomocysteine hydrolase (towards neplanocins). PMID:3039343

De Clercq, E; Bères, J; Bentrude, W G

1987-08-01

296

Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.  

Science.gov (United States)

Treatment of mouse astrocyte cultures with combined interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha induced expression of inducible nitric-oxide synthase (iNOS), resulting in sustained release of large amounts of nitric oxide, whereas TNF-alpha and IL-1alpha individually were unable to induce iNOS expression in astrocytes. The role of MAPK cascades and of NF-kappaB activation in the early intracellular signal transduction involved in iNOS transcription in TNF-alpha/IL-1alpha-stimulated astrocytes was investigated. TNF-alpha and IL-1alpha activated all p42/44(MAPK), p38(MAPK), and p54(JNK) pathways as determined by immunoprecipitation kinase assays using specific antibodies and substrates. The p38(MAPK) pathway is specifically involved in TNF-alpha/IL-1alpha-induced iNOS expression, since iNOS protein and nitric oxide release in the presence of a specific inhibitor of p38(MAPK), 4-(4-fluorophenyl)-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (FHPI), were dramatically diminished. In contrast, PD98059, a specific inhibitor of MEK1 had no effect on iNOS expression. p38(MAPK) did not couple NF-kappaB to iNOS transcription, but NF-kappaB had a clear role in iNOS transcription regulation. Northern blot analysis showed that the p38(MAPK) pathway controlled iNOS expression at the transcriptional level, since iNOS mRNA was reduced in the presence of FHPI in TNF-alpha/IL-1alpha-stimulated astrocytes. iNOS expression was investigated with TNF receptor (TNFR)-1- and TNFR-2-deficient mice. The TNF-alpha activity in TNF-alpha/IL-1alpha-stimulated astrocytes was exclusively mediated through TNFR-1, most likely because TNFR-2-mediated signals in astrocytes did not connect to the p38(MAPK) pathway. These data suggest that TNF-alpha/IL-1alpha-induced iNOS expression depends on a yet undetermined second pathway in addition to p38(MAPK). PMID:9353295

Da Silva, J; Pierrat, B; Mary, J L; Lesslauer, W

1997-11-01

297

Activation of AMP-activated protein kinase and phosphorylation of glycogen synthase kinase3 ? mediate ursolic acid induced apoptosis in HepG2 liver cancer cells.  

Science.gov (United States)

Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3?) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3? at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3? inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3? phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3? phosphorylation as a potent chemopreventive agent. PMID:23325562

Son, Hyun-Soo; Kwon, Hee Young; Sohn, Eun Jung; Lee, Jang-Hoon; Woo, Hong-Jung; Yun, Miyong; Kim, Sung-Hoon; Kim, Young-Chul

2013-11-01

298

AMP-activated Protein Kinase (AMPK) Activating Agents Cause Dephosphorylation of Akt and Glycogen Synthase Kinase-3  

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AMP-activated protein kinase (AMPK) is a key cellular sensor of reduced energy supply that is activated by increases in the cellular ratio of AMP/ATP. Phenformin and 5-aminoimida-zole-4-carboxamide riboside (AICAR) are two drugs widely used to activate AMPK experimentally. In both differentiated hippocampal neurons and neuroblastoma SH-SY5Y cells we found that these two agents not only activated AMPK, but conversely greatly reduced the activating Ser/Thr phosphorylation of Akt. This blockade ...

King, Taj D.; Song, Ling; Jope, Richard S.

2006-01-01

299

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

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

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

2009-01-01

300

Human platelet nitric oxide synthase activity: an optimized method Atividade da óxido nítrico sintase em plaquetas humanas: um método otimizado  

Directory of Open Access Journals (Sweden)

Full Text Available We investigated the kinetic analysis of human platelet Nitric Oxide Synthase (NOS activity by the rate of conversion of [³H] arginine to [³H]-citrulline in unstimulated fresh platelets. NOS activity was present in the membrane fraction and cytosol, and was Ca2+- and calmodulin dependent which is a characteristic of endothelial NOS. NOS activity was also dependent of NADPH since the omission of this cofactor induced an important decrease (85,2% in the enzyme activity. The kinetic varied with protein and arginine concentration but optimum concentrations were found up to 60 minutes, and up to 80 µg of protein at 120 nM of arginine and 0.5 µCi of ³H-arginine. NOS activity in the absence of FAD (flavin adenine dinucleotide, FMN (flavin mononucleotide and BH4 (tetrahydrobiopterin was only 2.8% of the activity measured in the presence of these three cofactors. The enzyme activity was completely inhibited by L-NAME (1 mM (98.1 % and EGTA (5 mM (98.8 %. Trifluoperazine (TFP caused 73.2% inhibition of the enzyme activity at 200 µM and 83.8 % at 500 µM. Under basal conditions, NOS Km for L-arginine was 0.84 ± 0.08 µM and mean Vmax values were 0.122 ± 0.025 pmol.mg-1.min-1. Mean human NOS platelet activity was 0.020 ± 0.010 pmol.mg-1.min-1. Results indicate that the eNOS in human platelet can be evaluated by conversion of [³H]-arginine to [³H]citrulline in an optimized method, which provide reproducible and accurate results with good sensitivity to clinical experiments involving neurological and psychiatric diseases.A análise cinética da atividade da óxido nítrico sintase (NOS plaquetária foi avaliada pela conversão de [³H]-arginina em [³H]-citrulina em plaquetas humanas frescas não estimuladas. A atividade da NOS foi detectada na fração citosólica e na membrana, além de ser dependente de Ca2+-calmodulina, que é uma característica da NOS endotelial (eNOS. A omissão de NADPH levou à diminuição da atividade da NOS dependente da dose causando redução de 85,2% da atividade enzimática. A cinética variou de acordo com as concentrações de proteína e de arginina, sendo que as melhores leituras foram obtidas com 80 µg de proteína, 120 nM de arginina em 0,5 µCi de ³H arginina, em 60 minutos de incubação. A atividade da NOS na ausência de FAD (flavina adenina dinucleotídeo, FMN (flavina mononucleotídeo e BH4 (tetrahidrobiopterina foi de apenas 2,8% da atividade medida na presença destes três cofatores. A atividade da enzima foi completamente inibida pelo L-NAME (1 mM; 98,1 %, EGTA (5 mM; 98,8 % e adição de trifluoperazina (TFP, nas concentrações de 200 µM e 500 µM, inibiu a atividade da enzima em 73,2% e 83,8 %, respectivamente. Em condições basais, o Km da NOS para Larginina foi de 0,84 ± 0,08 µM e o valor de Vmax foi de 0,122 ± 0,025 pmol.mg-1.min-1. A atividade média da NOS plaquetária humana foi de 0,020 ± 0,010 pmol.mg-1.min-1. Os resultados indicam que a eNOS em plaquetas humanas pode ser avaliada pelo método da conversão de [³H]-arginina em [³H]-citrulina, que em condições otimizadas, fornece resultados reprodutíveis e precisos com ótima sensibilidade para experimentos clínicos envolvendo doenças neurológicas e psiquiátricas.

Elisa Mitiko Kawamato

2002-09-01

 
 
 
 
301

Human platelet nitric oxide synthase activity: an optimized method / Atividade da óxido nítrico sintase em plaquetas humanas: um método otimizado  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese A análise cinética da atividade da óxido nítrico sintase (NOS) plaquetária foi avaliada pela conversão de [³H]-arginina em [³H]-citrulina em plaquetas humanas frescas não estimuladas. A atividade da NOS foi detectada na fração citosólica e na membrana, além de ser dependente de Ca2+-calmodulina, que [...] é uma característica da NOS endotelial (eNOS). A omissão de NADPH levou à diminuição da atividade da NOS dependente da dose causando redução de 85,2% da atividade enzimática. A cinética variou de acordo com as concentrações de proteína e de arginina, sendo que as melhores leituras foram obtidas com 80 µg de proteína, 120 nM de arginina em 0,5 µCi de ³H arginina, em 60 minutos de incubação. A atividade da NOS na ausência de FAD (flavina adenina dinucleotídeo), FMN (flavina mononucleotídeo) e BH4 (tetrahidrobiopterina) foi de apenas 2,8% da atividade medida na presença destes três cofatores. A atividade da enzima foi completamente inibida pelo L-NAME (1 mM; 98,1 %), EGTA (5 mM; 98,8 %) e adição de trifluoperazina (TFP), nas concentrações de 200 µM e 500 µM, inibiu a atividade da enzima em 73,2% e 83,8 %, respectivamente. Em condições basais, o Km da NOS para Larginina foi de 0,84 ± 0,08 µM e o valor de Vmax foi de 0,122 ± 0,025 pmol.mg-1.min-1. A atividade média da NOS plaquetária humana foi de 0,020 ± 0,010 pmol.mg-1.min-1. Os resultados indicam que a eNOS em plaquetas humanas pode ser avaliada pelo método da conversão de [³H]-arginina em [³H]-citrulina, que em condições otimizadas, fornece resultados reprodutíveis e precisos com ótima sensibilidade para experimentos clínicos envolvendo doenças neurológicas e psiquiátricas. Abstract in english We investigated the kinetic analysis of human platelet Nitric Oxide Synthase (NOS) activity by the rate of conversion of [³H] arginine to [³H]-citrulline in unstimulated fresh platelets. NOS activity was present in the membrane fraction and cytosol, and was Ca2+- and calmodulin dependent which is a [...] characteristic of endothelial NOS. NOS activity was also dependent of NADPH since the omission of this cofactor induced an important decrease (85,2%) in the enzyme activity. The kinetic varied with protein and arginine concentration but optimum concentrations were found up to 60 minutes, and up to 80 µg of protein at 120 nM of arginine and 0.5 µCi of ³H-arginine. NOS activity in the absence of FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide) and BH4 (tetrahydrobiopterin) was only 2.8% of the activity measured in the presence of these three cofactors. The enzyme activity was completely inhibited by L-NAME (1 mM) (98.1 %) and EGTA (5 mM) (98.8 %). Trifluoperazine (TFP) caused 73.2% inhibition of the enzyme activity at 200 µM and 83.8 % at 500 µM. Under basal conditions, NOS Km for L-arginine was 0.84 ± 0.08 µM and mean Vmax values were 0.122 ± 0.025 pmol.mg-1.min-1. Mean human NOS platelet activity was 0.020 ± 0.010 pmol.mg-1.min-1. Results indicate that the eNOS in human platelet can be evaluated by conversion of [³H]-arginine to [³H]citrulline in an optimized method, which provide reproducible and accurate results with good sensitivity to clinical experiments involving neurological and psychiatric diseases.

Elisa Mitiko, Kawamato; Isaias, Glezer; Carolina Demarchi, Munhoz; Cristiane, Bernardes; Cristoforo, Scavone; Tania, Marcourakis.

302

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex  

International Nuclear Information System (INIS)

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

2010-05-07

303

Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis.  

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Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N ter...

Charnock, C.; Refseth, U. H.; Sireva?g, R.

1992-01-01

304

Processes of malate catabolism during the anaerobic metabolism of grape berries  

International Nuclear Information System (INIS)

In order to precise malate fate during the anaerobic metabolism of grape, malate-3-14C was injected into Carignan berries kept in darkness at 350C under carbon dioxide atmosphere. The injection of labelled malate was effected in presence or not of non-labelled oxalate which inhibits malic enzyme (EC I.I.I.40). The analyses of the samples fixed after 3 and 7 days anaerobiosis concerned the titration of various substrates, organic acids, amino-acids and glycolysis products, and the measuring of the NADP+-malic enzyme (EC I.I.I.40) and malate dehydrogenase (EC I.I.I.40). Radioactivity is mainly observed in ethanol, amino-butyrate the non-separated group glycerate-shikimate and succinate. Malic enzyme acts in the first sequence of a process leading from malate to ethanol. Alanin synthesis seems to be stimulated in presence of oxalate. The results obtained and some hypotheses presented in the literature induce to suggest a utilization scheme for malate in the anaerobic metabolism of grape

1976-01-01

305

Exclusive selectivity of multidentate ligands independent on the oxidation state of cobalt: influence of steric hindrance on dioxygen binding and phenoxazinone synthase activity.  

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The present report describes the syntheses, characterizations, crystal structures and study of the phenoxazinone synthase activity of two peroxo-bridged dicobalt(III) complexes, [Co2(L(1))2(?-O2)](ClO4)4·2CH3CN (1) and [Co2(L(2))2(?-O2)](ClO4)4 (2), and three mononuclear cobalt(II) complexes, [Co(L(3))(CH3CN)](ClO4)2 (3), [Co(L(4))(H2O)](ClO4)2 (4) and [Co(L(5))(H2O)](ClO4)2 (5), derived from the pentadentate ligands L(1)-L(5), which are the 1?:?2 condensation products of triamines and 2-acetylpyridine or 2-pyridinecarboxaldehyde (6-methyl-2-pyridinecarboxaldehyde). X-ray crystallography reveals exclusive selectivity of the acyclic Schiff dibasic form of the ligands over the heterocyclic analogues, and this selectivity is found to be insensitive to the oxidation state of cobalt. Other first row transition metals have been characterized in either form of the ligands in their complexes but it is specific for cobalt established in the present study. The pronounced effect of the methyl substitutions is observed from their crystal structures; substitution at imine-C does not have any significant influence on the peroxo-bridging but substitution at sixth position of pyridyl ring prevents the formation of peroxo-bridging, and both the steric and electronic factors play vital roles on such chemical diversity. All the complexes show the phenoxazinone synthase mimicking activity and the comparative catalytic activity has been explored. Although electrochemical behaviors of all the complexes are very similar, their relative catalytic activity mimicking the function of phenoxazinone synthase arises from the electronic and steric factors of the methyl substitution. PMID:24699794

Panja, Anangamohan

2014-06-01

306

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.  

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Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding. PMID:12752439

Simard, Dave; Hewitt, Kerry A; Lunn, Faylene; Iyengar, Akshai; Bearne, Stephen L

2003-05-01

307

Characterization of site-specific mutations in a short-chain-length/medium-chain-length polyhydroxyalkanoate synthase: in vivo and in vitro studies of enzymatic activity and substrate specificity.  

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Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu; Sudesh, Kumar; Numata, Keiji

2013-06-01

308

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions.  

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Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2? are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2?/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2? production in response to IL-1? in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2? synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1? is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2? production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1? particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2? acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2? production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies. PMID:22654757

Bresson, Eva; Lacroix-Pépin, Nicolas; Boucher-Kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A

2012-01-01

309

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions  

Science.gov (United States)

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2? are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2?/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2? production in response to IL-1? in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2? synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1? is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2? production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1? particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2? acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2? production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

Bresson, Eva; Lacroix-Pepin, Nicolas; Boucher-Kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A.

2012-01-01

310

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force.  

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H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation. PMID:17904517

Feniouk, Boris A; Rebecchi, Alberto; Giovannini, Donatella; Anefors, Sofie; Mulkidjanian, Armen Y; Junge, Wolfgang; Turina, Paola; Melandri, B Andrea

2007-11-01

311

Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation?  

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Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa tra...

2008-01-01

312

A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules  

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A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin...

Yury Herasimenka; Marta Kotasinska; Stefan Walter; Hildgund Schrempf

2010-01-01

313

The malate-aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast.  

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Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Deltaaat1Delta double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles. PMID:18381895

Easlon, Erin; Tsang, Felicia; Skinner, Craig; Wang, Chen; Lin, Su-Ju

2008-04-01

314

[The changes of the activity of NO-synthases and oxidative processes under conditions of 5-HT receptors activation in the stomach and large intestine in streptozocin-induced diabetes mellitus].  

Science.gov (United States)

We investigated the activity of NO-synthases, lipoperoxidation processes, antioxidant defense enzymes in the muscular layers of stomach and large intestine. The L-arginine concentration in blood plasma was also monitored under conditions of 2-weeks activation of 5-HT4 receptors by mosaprid in streptozocin-induced diabetes mellitus. We showed that the onset of diabetes mellitus is accompanied by a 2.4-2.8-fold increase in the activity of inducible NO-synthase and a 23-40% increase in the SOD activity. The nitric oxide content and TBA products in the muscular layers of stomach and large intestine were increased, whereas the motor-evacuational function of the stomach and large intestine decreased. Activation of 5-HT4 receptors by mosaprid under conditions of diabetes mellitus decreased the activity of inducible NO-synthase, the lipoperoxidation processes, nitrite anion content and TBA products in muscular layers of stomach and large intestine by 23%. At the same time, we observed an increase in the motor-evacuational function of stomach and large intestine without affecting the blood sugar level. PMID:23233943

Skliarov, O Ia; Detsyk, O I

2012-01-01

315

Estradiol Regulation of Lipocalin-Type Prostaglandin D Synthase Promoter Activity: Evidence for Direct and Indirect Mechanisms  

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In the CNS, lipocalin-type prostaglandin D synthase (L-PGDS) is predominantly a non-neuronal enzyme responsible for the production of PGD2, an endogenous sleep promoting substance. We have previously demonstrated that estradiol differentially regulates L-PGDS transcript levels in the rodent brain. In hypothalamic nuclei, estradiol increases L-PGDS transcript expression, whereas in the ventrolateral preoptic area L-PGDS gene expression is reduced after estradiol treatment. In the present study...

Devidze, Nino; Fujimori, Ko; Urade, Yoshihiro; Pfaff, Donald W.; Mong, Jessica A.

2010-01-01

316

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling  

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Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that...

Lu Frances Fangjia; Su Ping; Liu Fang; Daskalakis Zafiris J

2012-01-01

317

Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath  

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Background: The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled br...

2004-01-01

318

Farnesyl diphosphate synthase assay.  

Science.gov (United States)

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

Arró, Montserrat; Manzano, David; Ferrer, Albert

2014-01-01

319

Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury  

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Full Text Available Abstract Background Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown. Methods Using the rat spared nerve injury (SNI model, we investigated the expression of PAF synthases (LPCAT1 and 2 and PAF receptor (PAFr mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR and double-labeling analysis of in situ hybridization histochemistry (ISHH with immunohistochemistry (IHC were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086. Results RT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia. Conclusions Our data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development of neuropathic pain.

Okubo Masamichi

2012-02-01

320

Manipulation of Rumen Ecology by Yeast and Malate in Dairy Heifer  

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Full Text Available Four, one-year old of dairy heifers were randomly assigned according to a 2x2 Factorial arrangement in a 4x4 Latin square design to study supplementation of malate level at 500 vs 1,000 g with yeast at 1,000 vs 2,000 g in concentrate. The treatments were as follows: T1 = supplementation of malate at 500 g + yeast at 1,000 g; T2 = supplementation of malate at 500 g + yeast at 2,000 g; T3 = supplementation of malate at 1,000 g + yeast at 1,000 g; T4 = supplementation of malate at 1,000 g + yeast at 2,000 g in concentrate, respectively. The cows were offered the treatment concentrate at 1 %BW and ruzi grass was fed ad libitum. The results have revealed that rumen fermentation and blood metabolites were similar for all treatments. However, the concentration of volatile fatty acid was significantly different especially the concentration of propionic acid was slightly higher in heifer receiving T4 than T3, T2 and T1 (24.4, 22.9, 22.4 and 19.7%, respectively. The populations of protozoa and fungal zoospores were significantly different as affected by malate level and yeast. In conclusion, the combined use of concentrate containing high level of cassava chip at 70% DM with malate at 1,000 g and yeast at 2,000 g in concentrate with ruzi grass as a roughage could improved rumen ecology in dairy heifers.

Sittisak Khampa

2009-01-01

 
 
 
 
321

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles  

Science.gov (United States)

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

Miyagawa, Ryu; Tano, Keiko; Mizuno, Rie; Nakamura, Yo; Ijiri, Kenichi; Rakwal, Randeep; Shibato, Junko; Masuo, Yoshinori; Mayeda, Akila; Hirose, Tetsuro; Akimitsu, Nobuyoshi

2012-01-01

322

Transfer of label from aspartate to malate by the cell-free extract of Sedum mexicanum leaves  

International Nuclear Information System (INIS)

The cell-free extract from the leaves of Sedum mexicanum, a typical CAM plant, formed 14C-malate from 14C-aspartate in the presence of NAD. No reduction of NAD was observed during the reaction. Analysis of this reaction revealed that the transfer of label from 14C-aspartate to malate took place by the action of malate dehydrogenase and aspartate aminotransferase, and the reaction was reversible in the model experiment with commercial enzymes. The pitfalls in assessing the data on dark 14CO2 fixation in CAM are discussed with reference to the transfer of label between malate and aspartate without actual synthesis. (Kaihara, S.)

1979-01-01

323

Mild reductions in mitochondrial citrate synthase activity result in a compromised nitrate assimilation and reduced leaf pigmentation but have no effect on photosynthetic performance or growth.  

Science.gov (United States)

Transgenic tomato (Solanum lycopersicum) plants, expressing a fragment of the mitochondrial citrate synthase gene in the antisense orientation and exhibiting mild reductions in the total cellular activity of this enzyme, displayed essentially no visible phenotypic alteration from the wild type. A more detailed physiological characterization, however, revealed that although these plants were characterized by relatively few changes in photosynthetic parameters they displayed a decreased relative flux through the tricarboxylic acid cycle and an increased rate of respiration. Furthermore, biochemical analyses revealed that the transformants exhibited considerably altered metabolism, being characterized by slight decreases in the levels of organic acids of the tricarboxylic acid cycle, photosynthetic pigments, and in a single line in protein content but increases in the levels of nitrate, several amino acids, and starch. We additionally determined the maximal catalytic activities of a wide range of enzymes of primary metabolism, performed targeted quantitative PCR analysis on all three isoforms of citrate synthase, and conducted a broader transcript profiling using the TOM1 microarray. Results from these studies confirmed that if the lines were somewhat impaired in nitrate assimilation, they were not severely affected by this, suggesting the presence of strategies by which metabolism is reprogrammed to compensate for this deficiency. The results are discussed in the context of carbon-nitrogen interaction and interorganellar coordination of metabolism. PMID:18359839

Sienkiewicz-Porzucek, Agata; Nunes-Nesi, Adriano; Sulpice, Ronan; Lisec, Jan; Centeno, Danilo C; Carillo, Petronia; Leisse, Andrea; Urbanczyk-Wochniak, Ewa; Fernie, Alisdair R

2008-05-01

324

TCA cycle activity in Staphylococcus aureus is essential for iron-regulated synthesis of staphyloferrin A, but not staphyloferrin B: the benefit of a second citrate synthase.  

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Staphylococcus aureus elaborates two citrate-containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron-restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron-regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S.?aureus in iron-restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB-deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron-restricted media, and is significantly more impaired for virulence than either of the single-deletion mutants. We propose that SB is the more important of the two siderophores for S.?aureus insofar as it is synthesized, and supports iron-restricted growth, without need of TCA cycle activity. PMID:24666349

Sheldon, Jessica R; Marolda, Cristina L; Heinrichs, David E

2014-05-01

325

The efficacy of topical ivermectin versus malation 0.5% lotion for the treatment of scabies.  

Science.gov (United States)

Objective: There are different medications for the treatment of scabies but the treatment of choice is still controversial. This study aimed at comparing the efficacy of topical ivermectin versus malation 0.5% lotion for the treatment of scabies. Methods: In total, 340 patients with scabies were enrolled, and randomized into two groups: the first group received 1% ivermectin applied topically to the affected skin and the second group received topical malation 0.5% lotion and were told to apply this twice with 1 week interval. Treatment was evaluated at intervals of 2 and 4 weeks, and if there was treatment failure at the 2-week follow-up, treatment was repeated. Results: Two application of topical ivermectin provided a cure rate of 67.6% at the 2-week follow-up, which increased to 85.2% at the 4-week follow-up after repeating the treatment. Treatment with two applications of malation 0.5% lotion was effective in 44.1% of patients at the 2-week follow-up, which increased to 67.6% at the 4-week follow-up after this treatment was repeated. Conclusion:Two application of ivermectin was as effective as single applications of malation 0.5% lotion at the 2-week follow-up. After repeating the treatment, ivermectin was superior to malation 0.5% lotion at the 4-week follow up. PMID:23472617

Goldust, Mohamad; Rezaee, Elham

2013-05-01

326

Arabidopsis peroxisomal malate dehydrogenase functions in beta-oxidation but not in the glyoxylate cycle.  

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The aim was to determine the function of peroxisomal NAD(+)-malate dehydrogenase (PMDH) in fatty acid beta-oxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in beta-oxidation, even though microarray analysis shows that beta-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in beta-oxidation in mutant seedlings, [(14)C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid beta-oxidation and does not participate directly in the glyoxylate cycle. PMID:17376163

Pracharoenwattana, Itsara; Cornah, Johanna E; Smith, Steven M

2007-05-01

327

Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax.  

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Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors. PMID:18003612

Lunn, Faylene A; MacDonnell, Jennifer E; Bearne, Stephen L

2008-01-25

328

Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.  

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We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received...

Golino, P.; Rosolowsky, M.; Yao, S. K.; Mcnatt, J.; Clerck, F.; Buja, L. M.; Willerson, J. T.

1990-01-01

329

Nitric Oxide synthases and atrial fibrillation  

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

CynthiaAnnCarnes

2012-04-01

330

Post-translational Modification Regulates Prostaglandin D2 Synthase Apoptotic Activity: Characterization by Site-directed Mutagenesis  

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Lipocalin-type prostaglandin D2 synthase (L-PGDS) is a highly glycosylated protein found in several body fluids. Elevated L-PGDS levels have been observed in the serum of patients with renal impairment, diabetes mellitus, and hypertension. Recently, we demonstrated the ability of L-PGDS to induce apoptosis in a variety of cell types including epithelial cells, neuronal cells, and vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the effect several site-directed mu...

Ragolia, Louis; Hall, Christopher E.; Palaia, Thomas

2007-01-01

331

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

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

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

2002-01-01

332

Bacterial ?-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis  

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Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. We now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolating the wild-type hemA gene encoding ?-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizob...

Guerinot, Mary Lou; Chelm, Barry K.

1986-01-01

333

Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity  

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Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1? , IL-6 and TNF?-induced NO levels in RAW 264.7 macrop...

Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K.; Gupta, Sanjay

2010-01-01

334

Analysis of the Polymerization Initiation and Activity of Pasteurella multocida Heparosan Synthase PmHS2, an Enzyme with Glycosyltransferase and UDP-sugar Hydrolase Activity*  

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Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUA?1–4GlcNAc?1-)n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated...

Chavaroche, Anais A. E.; Den Broek, Lambertus A. M.; Springer, Jan; Boeriu, Carmen; Eggink, Gerrit

2011-01-01

335

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity  

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Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUA?1-4GlcNAc?1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated t...

Chavaroche, A. A. E.; Broek, L. A. M.; Springer, J.; Boeriu, C.; Eggink, G.

2011-01-01

336

Increase in flavan-3-ols by silencing flavonol synthase mRNA affects the transcript expression and activity levels of antioxidant enzymes in tobacco.  

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Flavonoids are plant secondary metabolites widespread throughout the plant kingdom involved in many physiological and biochemical functions. Amongst the flavonoids, flavan-3-ols (catechin and epicatechin) are known for their direct free radical scavenging activity in vitro, but studies on their antioxidant potential and interaction with antioxidant enzymes in vivo are lacking. Here, the flavonoid pathway was engineered by silencing a gene encoding flavonol synthase (FLS) in tobacco to direct the flow of metabolites towards production of flavan-3-ols. FLS silencing reduced flavonol content 17-53%, while it increased catechin and epicatechin content 51-93% and 18-27%, respectively. The silenced lines showed a significant increase in expression of genes for dihydroflavonol reductase and anthocyanidin synthase, a downstream gene towards epicatechin production, with no significant change in expression of other genes of the flavonoid pathway. Effects of accumulation of flavan-3-ols in FLS silenced lines on transcript level and activities of antioxidant enzymes were studied. Transcripts of the antioxidant enzymes glutathione reductase (GR), ascorbate peroxidase (APx), and catalase (CAT) increased, while glutathione-S-transferase (GST), decreased in FLS silenced lines. Enhanced activity of all the antioxidant enzymes was observed in silenced tobacco lines. To validate the affect of flavan-3-ols on the antioxidant system, in vitro experiments were conducted with tobacco seedlings exposed to two concentrations of catechin (10? and 50??m) for 2?days. In vitro exposed seedlings produced similar levels of transcripts and activity of antioxidant enzymes as FLS silenced seedlings. Results suggest that flavan-3-ols (catechin) might be increasing activity of GR, Apx and CAT by elevating their mRNAs levels. Since these enzymes are involved in scavenging of reactive oxygen species, this strategy would help in tailoring crops for enhanced catechin production as well as making them tolerant to oxidative stresses. PMID:22324650

Mahajan, M; Joshi, R; Gulati, A; Yadav, S K

2012-02-10

337

Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.  

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The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their ?1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the ?1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts. PMID:23661084

Li, Yanhong; Yu, Hai; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Qu, Jingyao; Hie, Liana; Chen, Xi

2014-02-01

338

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

Science.gov (United States)

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

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

2011-07-01

339

Fimasartan, anti-hypertension drug, suppressed inducible nitric oxide synthase expressions via nuclear factor-kappa B and activator protein-1 inactivation.  

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Since inhibition of angiotensin II type 1 (AT1) receptor reduces chronic inflammation associated with hypertension, we evaluated the anti-inflammatory potential and the underlying mechanism of fimasartan, a Korean Food and Drug Administration approved anti-hypertension drug, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Fimasartan suppressed the expressions of inducible nitric oxide synthase (iNOS) by down-regulating its transcription, and subsequently inhibited the productions of nitric oxide (NO). In addition, fimasartan attenuated LPS-induced transcriptional and DNA-binding activities of nuclear factor-kappa B (NF-?B) and activator protein-1 (AP-1). These reductions were accompanied by parallel reductions in the nuclear translocation of NF-?B and AP-1. Taken together, our data suggest that fimasartan down-regulates the expression of the iNOS in macrophages via NF-?B and AP-1 inactivation. PMID:23449332

Ryu, Suran; Shin, Ji-Sun; Cho, Young-Wuk; Kim, Hyoung Kook; Paik, Soo Heui; Lee, Joo Han; Chi, Yong Ha; Kim, Ji Han; Kim, Je Hak; Lee, Kyung-Tae

2013-01-01

340

Effects of polyporus polysaccharide on activity and mRNA expression of inducible nitric oxide synthase in peritoneal macrophages of mice  

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Full Text Available Objective: To study the mechanisms of the antitumor and immunoregulation functions of polyporus polysaccharide (PPS. Methods: The production of nitric oxide (NO, the activity and mRNA expression of inducible nitric oxide synthase (iNOS in peritoneal macrophages of mice administered with different dose of PPS were observed by Griess reaction, fluorimetry assay and RT-PCR, respectively. Results: PPS could elevate the iNOS activity with dose-dependence and stimulate the iNOS mRNA expression of peritoneal macrophages in mice. Conclusion: The regulation of PPS on the production of NO in peritoneal macrophages of mice may occur at transcriptional level of iNOS. This indicates that the mechanism of PPS's antitumor and immunoregulation functions may be related to increasing NO output of macrophages through stimulating iNOS's denovo synthesis.

HUANG Di-Nan

2004-09-01

 
 
 
 
341

The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue.  

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Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis. PMID:19074144

Veyron-Churlet, Romain; Molle, Virginie; Taylor, Rebecca C; Brown, Alistair K; Besra, Gurdyal S; Zanella-Cléon, Isabelle; Fütterer, Klaus; Kremer, Laurent

2009-03-01

342

Is stored malate the quantitatively most important substrate utilised by respiration and ethanolic fermentation in grape berry pericarp during ripening?  

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A widely held view is that in grape pericarp glycolysis is inhibited during ripening, and that stored malate rather than sugars become the major substrate for respiration. In this study we determined what contribution stored malate could make to the substrate requirements of respiration and ethanolic fermentation in the pericarp of Cabernet Sauvignon berries during ripening. At a number of time points through development the amount of malate in the pericarp was measured. The change in malate content between each time point was then calculated, having first allowed for dilution arising from expansion of the fruit. The amount of CO2 that was released by the berry in the interval between each pair of time points was measured. It was found that the contribution that stored malate could make to the substrate requirements of respiration and ethanolic fermentation of grape pericarp was dependent on the stage of ripening. At the beginning of ripening stored malate could provide a greater proportion of substrate than later in ripening, and during the latter its contribution was relatively low. Therefore, stored malate was not the quantitatively most important substrate utilised by respiration and ethanolic fermentation in the pericarp of grape berries during most of ripening. It is likely that sugars provide the bulk of the deficit in substrate. Further, the increase in the respiratory quotient during most of ripening does not arise from the use of malate as main respiratory substrate. PMID:24463535

Famiani, Franco; Farinelli, Daniela; Palliotti, Alberto; Moscatello, Stefano; Battistelli, Alberto; Walker, Robert P

2014-03-01

343

The prostaglandin F synthase activity of the human aldose reductase AKR1B1 brings new lenses to look at pathologic conditions.  

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Full Text Available Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs, lead us to the discovery that AKR1B5 and later AKR1B1 were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2? are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2 and PGF2? may constitute a functional dyad with physiological relevance at least as important as the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2? production in response to IL-1? in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231 also known as ALDR1 or ALR2 is a functional PGF2? synthase in different models of living cells and tissues. Using human endometrial cells, prostate and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1? is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2? production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1? particularly around the multiple stress response region (MSRR containing two putative antioxidant response elements (ARE adjacent to TonE and AP1.We also show that AKR1B1 is able to regulate PGE2 production through PGF2? acting on its FP receptor and that aldose reductase inhibitors (ARIs like alrestatin, statil (ponalrestat and EBPC exhibit distinct and characteristic inhibition of PGF2? production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

MichelAFortier

2012-05-01

344

Mitochondria from the left heart ventricles of both normotensive and spontaneously hypertensive rats oxidize externally added NADH mostly via a novel malate/oxaloacetate shuttle as reconstructed in vitro.  

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A substantial increase in NADH production, arising from accelerated glycolysis, occurs in cardiac hypertrophy and this raises the question of how the NADH is oxidised. We have addressed this problem by reconstructing appropriate mitochondrial shuttles in vitro, using mitochondria from the left ventricles of both normotensive and spontaneously hypertensive rats at 5 and 24 weeks of age as model systems for left ventricle hypertrophy and hypertrophy/hypertension respectively. We found that most NADH oxidation occurs via a novel malate/oxaloacetate shuttle, the activity of which increases with time and with the progression of hypertrophy and development of hypertension as judged by statistical ANOVA analysis. In contrast, alpha-glycerol-phosphate and the malate/aspartate shuttles were shown to make only a minor contribution to NADH oxidation in a manner essentially independent of age and progression of hypertrophy/hypertension. The rate of malate transport in exchange with oxaloacetate proved to limit the rate of NADH oxidation via this malate/oxaloacetate shuttle. PMID:16786170

Atlante, Anna; Seccia, Teresa M; De Bari, Lidia; Marra, Ersilia; Passarella, Salvatore

2006-07-01

345

Mechanistic and bioinformatic investigation of a conserved active site helix in ?-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.  

Science.gov (United States)

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily. PMID:24720347

Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

2014-05-13

346

Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties.  

Science.gov (United States)

The effect of foliar application of salicylic acid (SA) at different concentrations (10-3 M and 10-5 M) was investigated on the production of secondary metabolites (flavonoids), chalcone synthase (CHS) activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231) in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC) analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin) decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS) enzyme activity (involving flavonoid synthesis) and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10-5 M SA treatment. As the SA concentration was decreased from 10-3 M to 10-5 M, the free radical scavenging power (FRAP) increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 ?g mL-1, the DPPH antioxidant activity recorded the highest value of 58.30%-72.90% with the 10-5 M SA treatment followed by the 10-3 M SA (52.14%-63.66%) treatment. The lowest value was recorded in the untreated control plants (42.5%-46.7%). These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10-5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of flavonoids in ginger can be increased by foliar application of SA in a controlled environment and that the anticancer activity in young ginger extracts could be improved. PMID:23203096

Ghasemzadeh, Ali; Jaafar, Hawa Z E; Karimi, Ehsan

2012-01-01

347

Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells.  

Science.gov (United States)

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes. PMID:7654209

Grivell, A R; Korpelainen, E I; Williams, C J; Berry, M N

1995-09-01

348

Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes  

DEFF Research Database (Denmark)

AIMS/HYPOTHESIS: In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved insulin signalling to glycogen synthesis remains to be clarified. METHODS: Whole-body glucose metabolism was studied in 12 patients with type 2 diabetes, and 10 lean and 10 obese non-diabetic controls by means of indirect calorimetry and tracers during a euglycaemic-hyperinsulinaemic clamp. The diabetic patients underwent a second isoglycaemic-hyperinsulinaemic clamp maintaining fasting hyperglycaemia. Muscle biopsies from m. vastus lateralis were obtained before and after the clamp for examination of GS and relevant insulin signalling components. RESULTS: During euglycaemia, insulin-stimulated glucose disposal, glucose oxidation and non-oxidative glucose metabolism were reduced in the diabetic group compared with both control groups (p¿

Vind, B F; Birk, Jesper Bratz

2012-01-01

349

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RAR?/RXR? heterodimer to a novel retinoic acid response element in the promoter  

International Nuclear Information System (INIS)

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RAR?/RXR? heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5 bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RAR?/RXR? directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RAR?/RXR?) in the induction of hiNOS by RA

2007-04-06

350

Biochemistry: Acetohydroxyacid Synthase  

Directory of Open Access Journals (Sweden)

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

Pham Ngoc Chien

2010-02-01

351

Biochemistry: Acetohydroxyacid Synthase  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Pham Ngoc Chien

2010-01-01

352

Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.  

LENUS (Irish Health Repository)

Cystathionine ?-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 ?L of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

Krijt, Jakub

2011-02-01

353

Citrate Synthase Mutants of Sinorhizobium meliloti Are Ineffective and Have Altered Cell Surface Polysaccharides  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruva...

Mortimer, Michael W.; Mcdermott, Timothy R.; York, Gregory M.; Walker, Graham C.; Kahn, Michael L.

1999-01-01

354

Homochiral Cu(II) and Ni(II) malates with tunable structural features  

Science.gov (United States)

Four new homochiral metal-organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H2O (1), [Cu(mal)(bpe)]·2H2O (2), [Ni(mal)(bpy)]·1.3CH3OH (3) and [Ni(mal)(bpe)]·4H2O (4) (mal=S-malate, bpy=4,4?-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1-3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral {Ni(mal)} motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %.

Zavakhina, Marina S.; Samsonenko, Denis G.; Virovets, Alexander V.; Dybtsev, Danil N.; Fedin, Vladimir P.

2014-02-01

355

The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading  

DEFF Research Database (Denmark)

The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the alpha2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in alpha1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-beta and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that alpha2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken.

Jørgensen, Sebastian B; Nielsen, Jakob N.

2004-01-01

356

Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity.  

Science.gov (United States)

Transformed cells are highly glycolytic and overexpress hexokinase II (HXK II). HXK II is capable of binding to the mitochondria through an interaction with the voltage-dependent anion channel (VDAC), an abundant outer mitochondrial membrane protein. The binding of HXK II to mitochondria has been shown to protect against loss of cell viability. Akt activation inhibits apoptosis partly by promoting the binding of HXK II to the mitochondria, but the mechanism through which Akt accomplishes this has not been characterized. The present report shows that Akt mediates the binding of HXK II to the mitochondria by negatively regulating the activity of glycogen synthase kinase 3beta (GSK3beta). On inhibition of Akt, GSK3beta is activated and phosphorylates VDAC. HXK II is unable to bind VDAC phosphorylated by GSK3beta and dissociates from the mitochondria. Inhibition of Akt potentiates chemotherapy-induced cytotoxicity, an effect that is dependent on GSK3beta activation and its attendant ability to disrupt the binding of HXK II to the mitochondria. Moreover, agents that can force the detachment of HXK II from mitochondria in the absence of Akt inhibition or GSK3beta activation promoted a synergistic increase in cell killing when used in conjunction with chemotherapeutic drugs. Such findings indicate that interference with the binding of HXK II to mitochondria may be a practicable modality by which to potentiate the efficacy of conventional chemotherapeutic agents. PMID:16288047

Pastorino, John G; Hoek, Jan B; Shulga, Nataly

2005-11-15

357

Role of constitutive nitric oxide synthase in regulation of Helicobacter pylori-induced gastric mucosal cyclooxygenase-2 ac-tivation through S-nitrosylation: mechanism of ghrelin action  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Gastric mucosal inflammatory responses to H. pylori lipopolysaccharide (LPS), are characterized by the excessive NO and prostaglandin (PGE2) generation due to the disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) systems. Here, we report that the LPS-induced enhancement in gastric mucosal inducible (i) iNOS) activity and up-regulation in PGE2 production was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS activation. The sti...

Slomiany, Bronislaw L.; Amalia Slomiany

2011-01-01

358

Unusual features of a recombinant apple alpha-farnesene synthase.  

Science.gov (United States)

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity. PMID:17140613

Green, Sol; Friel, Ellen N; Matich, Adam; Beuning, Lesley L; Cooney, Janine M; Rowan, Daryl D; MacRae, Elspeth

2007-01-01

359

Functional prostacyclin synthase promoter polymorphisms. Impact in pulmonary arterial hypertension.  

Science.gov (United States)

Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH-relevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations. Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes' transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH. PMID:24605778

Stearman, Robert S; Cornelius, Amber R; Lu, Xiao; Conklin, David S; Del Rosario, Mark J; Lowe, Anita M; Elos, Mihret T; Fettig, Lynsey M; Wong, Randall E; Hara, Naoko; Cogan, Joy D; Phillips, John A; Taylor, Matthew R; Graham, Brian B; Tuder, Rubin M; Loyd, James E; Geraci, Mark W

2014-05-01

360

A new pathway for salvaging the coenzyme B12 precursor cobinamide in archaea requires cobinamide-phosphate synthase (CbiB) enzyme activity.  

Science.gov (United States)

The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5'-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica. A cbiP mutant strain of the archaeon Halobacterium sp. strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon. A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide. The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid. The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway. The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea. PMID:14645280

Woodson, Jesse D; Zayas, Carmen L; Escalante-Semerena, Jorge C

2003-12-01

 
 
 
 
361

Methylmercury Alters the Activities of Hsp90 Client Proteins, Prostaglandin E Synthase/p23 (PGES/23) and nNOS  

Science.gov (United States)

Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2? and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity.

Caito, Samuel; Zeng, Heng; Aschner, Judy L.; Aschner, Michael

2014-01-01

362

Manipulation of Rumen Ecology by Malate and Cassava Hay in High-Quality Feed Block in Dairy Steers  

Directory of Open Access Journals (Sweden)

Full Text Available Four, dairy steers were randomly assigned according to a 2x2 Factorial arrangement in a 4x4 Latin square design to study supplementation of malate level at 500 and 1,000 g and cassava hay in high-quality feed block. The treatments were as follows: T1 = supplementation of high-quality feed block without cassava hay + malate at 500 g; T2 = supplementation of high-quality feed block without cassava hay + malate at 1,000 g; T3 = supplementation of high-quality feed block with cassava hay + malate at 500 g; T4 = supplementation of high-quality feed block with cassava hay + malate at 1,000 g, respectively. The cows were offered the treatment concentrate at 1.0% BW and ruzi grass was fed ad libitum. The results have revealed that populations of protozoa and fungal zoospores were significantly different as affected by malate level and cassava hay supplementation. However, rumen fermentation and blood metabolites were similar for all treatments. In conclusion, the combined use of cassava hay and malate at 1,000 g in high-quality feed block with concentrates containing high levels of cassava chip at 65% DM could highest improved rumen ecology in dairy steers.

Sittisak Khampa

2009-01-01

363

Supplementation of Malate and Yeast in Concentrate Containing High Cassava Chip on Rumen Ecology in Dairy Steers  

Directory of Open Access Journals (Sweden)

Full Text Available Four, one-year old of dairy steers were randomly assigned according to a 2x2 Factorial arrangement in a 4x4 Latin square design to study supplementation of malate level at 500 vs 1,000 g with yeast (Saccharomyces cerevisiae at 1,000 vs 2,000 g in concentrate containing high levels of cassava chip. The treatments were as follows: T1 = supplementation of malate at 500 g with yeast at 1,000 g; T2 = supplementation of malate at 500 g with yeast at 2,000 g; T3 = supplementation of malate at 1,000 g with yeast at 1,000 g; T4 = supplementation of malate at 1,000 g with yeast at 2,000 g in concentrate, respectively. The animals were offered the treatment concentrate at 1% BW and ruzi grass was fed ad libitum. The results have revealed that rumen fermentation and blood metabolites were similar for all treatments. The populations of protozoa and fungal zoospores were significantly different as affected by malate level and yeast. In conclusion, the combined use of concentrate containing high level of cassava chip at 70%DM with malate at 1,000 g and yeast at 2,000 g in concentrate with ruzi grass as a roughage could improved rumen ecology in dairy steers.

Sittisak Khampa

2009-01-01

364

Evidence for the mechanism of hydroxylation by 4-hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase from intermediate partitioning in active site variants.  

Science.gov (United States)

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS. PMID:21815644

Shah, Dhara D; Conrad, John A; Heinz, Brian; Brownlee, June M; Moran, Graham R

2011-09-01

365

Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells  

International Nuclear Information System (INIS)

Highlights: ? As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. ? RhoA activation increases PGE2 levels and its metabolic enzyme mPGES-1. ? RhoA-activated NF-?B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E2 (PGE2), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE2 levels and gene expression of the rate-limiting PGE2 producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1?-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1?-mediated phosphorylated nuclear factor-?B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE2 production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

2011-09-30

366

Inhibition of endothelial nitric oxide synthase activity and suppression of endothelium-dependent vasorelaxation by 1,2-naphthoquinone, a component of diesel exhaust particles  

Energy Technology Data Exchange (ETDEWEB)

Diesel exhaust particles contain redox-active quinones, such as 9,10-phenanthraquinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which act as potent electron acceptors, thereby altering electron transfer on proteins. We have previously found that 9,10-PQ inhibits constitutive nitric oxide synthase (NOS) activity, by shunting electrons away from NADPH on the cytochrome P450 reductase domain of NOS, and thus suppresses acetylcholine (Ach)-induced vasorelaxation in the aortic ring. However, the effect of 1,2-NQ on endothelial NOS (eNOS) activity is still poorly understood. With the membrane fraction of cultured bovine aortic endothelial cells, we found that 1,2-NQ was a potent inhibitor of eNOS with an IC{sub 50} value of 1.4 {mu}M, whereas trans-1,2-dihydroxy-1,2-dihydronaphthalene (1,2-DDN), a redox-negative naphthalene analog of 1,2-NQ, did not show such an inhibitory action. Although 1,2-DDN (5 {mu}M) did not affect Ach-mediated vasorelaxation, 1,2-NQ caused a significant suppression of Ach-induced endothelium-dependent vasorelaxation in the aortic ring. However, 1,2-NQ did not affect sodium nitroprusside-induced endothelium-independent vasorelaxation. These results suggest that 1,2-NQ is an environmental quinone that inhibits eNOS activity, thereby disrupting NO-dependent vascular tone. (orig.)

Sun, Yang; Taguchi, Keiko; Sumi, Daigo [University of Tsukuba, Department of Environmental Medicine, Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Sciences, Ibaraki (Japan); Yamano, Shigeru [Fukuoka University, Faculty of Pharmaceutical Sciences, Fukuoka (Japan); Kumagai, Yoshito [University of Tsukuba, Department of Environmental Medicine, Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Sciences, Ibaraki (Japan); Southern California Particle Center and Supersite, Los Angeles, CA (United States)

2006-05-15

367

Analysis of cis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea.  

Science.gov (United States)

Cis-regulatory elements involved in the activation of the plant defense-related gene encoding chalcone synthase 1 (PsChs1) in pea (Pisum sativum L.) were examined by transient transfection, gel mobility shift assay and in vitro DNase I-footprinting analysis. Transient transfection assay revealed that a 61 bp DNA fragment spanning from -242 to -182 of PsChs1 was required for the maximal promoter activity and possibly involved in the enhancement of elicitor-mediated activation. Nuclear isolate from elicitor-treated pea epicotyl tissues contained some factor(s) that specifically bound to this DNA fragment to form a complex with low mobility (LMC, low mobility complex) in gel mobility shift assay. DNase I-footprinting analysis of LMC revealed that among three protected regions detected in a 61 bp DNA fragment, two regions contained identical AT-rich sequence, TAAAATACT. Site directed mutation in either or both identical sequences, TAAAATACT to TGGAATACT, resulted in the reduction or loss in the ability to form LMC. Detailed analysis of 61 bp DNA fragment demonstrated that the region from -242 to -226 containing promoter-distal TAAAATACT motif was imperative for the maximal elicitor-mediated activation of PsChs1. PMID:8790282

Seki, H; Ichinose, Y; Kato, H; Shiraishi, T; Yamada, T

1996-06-01

368

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures.  

Science.gov (United States)

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons. PMID:19187438

d'Anglemont de Tassigny, Xavier; Campagne, Céline; Steculorum, Sophie; Prevot, Vincent

2009-04-01

369

Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer's disease.  

Science.gov (United States)

Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias. PMID:23801246

Lin, Ai-Ling; Zheng, Wei; Halloran, Jonathan J; Burbank, Raquel R; Hussong, Stacy A; Hart, Matthew J; Javors, Martin; Shih, Yen-Yu Ian; Muir, Eric; Solano Fonseca, Rene; Strong, Randy; Richardson, Arlan G; Lechleiter, James D; Fox, Peter T; Galvan, Veronica

2013-09-01

370

Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)  

Energy Technology Data Exchange (ETDEWEB)

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

2010-02-15

371

Glycogen Synthase Kinase (GSK) 3? Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells  

Science.gov (United States)

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3? phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3? selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3??/? mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3? directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3?-mediated phosphorylation of NM1 is required for pol I transcription activation.

Sarshad, Aishe A.; Corcoran, Martin; Al-Muzzaini, Bader; Borgonovo-Brandter, Laura; Von Euler, Anne; Lamont, Douglas; Visa, Neus; Percipalle, Piergiorgio

2014-01-01

372

Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).  

Energy Technology Data Exchange (ETDEWEB)

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the U.S. Department of Energy’s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

Wilkins, Michael J.; Callister, Stephen J.; Miletto, Marzia; Williams, Kenneth H.; Nicora, Carrie D.; Lovely, Derek R.; Long, Philip E.; Lipton, Mary S.

2011-01-01

373

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

Directory of Open Access Journals (Sweden)

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

Cho Yu

2010-08-01

374

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp. Atividade da sintetase da glutamina e sintase do glutamato em relação a fixação de nitrogênio em Lotus spp.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of no...

2000-01-01

375

Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NHâ??-terminal (sites 2 + 2a) phosphorylation  

DEFF Research Database (Denmark)

Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr³⁰⁸ on Akt (p-Akt-Thr³⁰⁸), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr³⁰⁸ (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser⁴⁷³ or IRS-1-PI3K activity. Furthermore, p-Akt-Thr³⁰⁸ and Akt2 activity were negatively associated with NHâ??-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P <0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NHâ??-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NHâ??-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NHâ??-terminal dephosphorylation may be the site for "fine-tuning" insulin-mediated GS activation in humans.