WorldWideScience

Sample records for citrate synthase activity

  1. Lactate Dehydrogenase and Citrate Synthase activity in cardiac and skeletal muscle of lowland and highland tinamous

    OpenAIRE

    Aira, Naomi

    2013-01-01

    Tinamous (Tinamidae) have the smallest heart in relation to body mass compared to any other flying bird today (Bishop 1997). This means that heart size is likely to restrict aerobic metabolism. Tinamous inhabit areas from sea level to 4800 m a.s.l., which means that the high altitude living species, Nothoprocta ornata (NO), is exposed to hypoxia. In this study the activity of the two metabolic enzymes Lactate Dehydrogenase (LDH) and Citrate Synthase (CS) was measured and the ratio between the...

  2. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    OpenAIRE

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-01-01

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites.

  3. Improved L-lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity.

    Science.gov (United States)

    van Ooyen, Jan; Noack, Stephan; Bott, Michael; Reth, Alexander; Eggeling, Lothar

    2012-08-01

    We here developed a series of Corynebacterium glutamicum strains with gradual decreased specific citrate synthase (CS) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on L-lysine formation and growth. We achieved an intended gradual L-lysine yield increase and recognized and overcame further new limitations in the L-lysine biosynthesis pathway to result in a strain with the highest yield reported so far when assayed under comparable conditions. As a non-intended outcome, a detailed flux analysis revealed an almost constant flux through CS at 10% remaining CS activity, whereas the metabolome data revealed an increase in the oxaloacetate and acetyl-CoA concentrations. Hence reduced CS activity is apparently efficiently buffered by increased concentrations of CS substrates, implying a certain robustness of the central metabolism in response of the imposed gene expressions. PMID:22392073

  4. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu [Purdue University, 175 South University Street, West Lafayette, IN 47907-2063 (United States)

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  5. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    International Nuclear Information System (INIS)

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS

  6. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Science.gov (United States)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-01-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  7. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    Science.gov (United States)

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  8. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Andersen, Nynne Bjerre; Dela, Flemming

    2014-01-01

    changes in CS activity is often assumed. However, this relationship and absolute values of CS and maximal oxygen uptake (V.O2max) has never been assessed across different studies. A systematic PubMed search on literature published from 1983 to 2013 was performed. The search profile included: citrate...... CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P < 0.001) between the relative change in V.O2max and the relative change in CS activity. All reported absolute values of CS and V.O2max did not correlate (r =- 0.07, n = 148, P = 0.......4). Training induced changes in whole body oxidative capacity is matched by changes in muscle CS activity in a nearly 1:1 relationship. Absolute values of CS across different studies cannot be compared unless a standardized analytical method is used by all laboratories...

  9. Citrate-release-mediated aluminum resistance is coupled to the inducible expression of mitochondrial citrate synthase gene in Paraserianthes falcataria.

    Science.gov (United States)

    Osawa, Hiroki; Kojima, Katsumi

    2006-05-01

    Aluminum (Al) resistance in some leguminous plants is achieved by enhanced citrate release from roots. Enhancement requires several hours for complete activation and is postulated to involve Al-responsive genes or components. We examined the mechanism of Al-induced citrate release by studying the relationship between citrate release and expression of the mitochondrial citrate synthase (mCS) gene in three leguminous trees. Root elongation in Leucaena leucocephala (Lam.) de Wit was arrested within 24 h by 30 microM Al, whereas root elongation in Paraserianthes falcataria (L.) Neilson and Acacia mangium Willd. was inhibited mangium maintained enhanced release and accumulation of citrate for at least 28 days in response to Al treatment. Aluminum increased the accumulation of mCS transcripts in P. falcataria roots, but not in L. leucocephala roots, and thus up-regulation decreased following removal of Al. Lanthanum did not alter the expression level of mCS. Aluminum increased mCS activity concomitantly with enhanced mCS gene expression in P. falcataria, whereas it did not affect mCS activity in L. leucocephala. Aluminum content in root apices of P. falcataria was increased by cycloheximide, supporting the idea that de novo synthesis of proteins is a prerequisite for Al resistance. Our findings suggest that Al-inducible expression of mCS coupled with enhanced citrate release mediates Al resistance in P. falcataria. PMID:16452070

  10. 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)

    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

    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.

  11. Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Mogensen, Martin; Petersen, Ingrid;

    2005-01-01

    In myotubes established from patients with type 2 diabetes (T2D), lipid oxidation and insulin-mediated glucose oxidation are reduced, whereas in myotubes from obese non-diabetic subjects, exposure to palmitate impairs insulin-mediated glucose oxidation. To determine the underlying mechanisms of...... obese subjects and T2D patients. Basal CS activity was lower (14%) in diabetic myotubes compared with myotubes from lean controls (P=0.03). Incubation with insulin (1 microM) for 4 h increased the CS activity (26-33%) in myotubes from both lean (P=0.02) and obese controls (P<0.001), but not from...

  12. Enhancement of Aminoacylase Activity by Sodium Citrate

    Institute of Scientific and Technical Information of China (English)

    于范利; 曹志方; 李森; 周海梦

    2001-01-01

    Kidney and other tissues of animals and humans have a high concentration of citrate which is an important intermediate substance in the citrate cycle. Citrate may play an important physiological role in metabolism. In this paper, we studied the interaction of the sodium salt of citrate with aminoacylase which is an important enzyme in metabolism and found sodium citrate can enhance the activity of aminoacylase. The maximum enzyme activity induced by sodium citrate increased approximately 3 folds over the enzyme activity without sodium citrate. The initial reaction rates (Ⅴ) for different concentrations of sodium citrate were obtained, showing that sodium citrate is a non-competitive activator. The result of the ANS binding fluorescence measurements for aminoacylase indicated that increasing sodium citrate concentrations markedly increased the ANS binding fluorescence with a blue shift of the emission spectra peak. This suggests the formation of more hydrophobic regions. Aggregates formed quickly when aminoacylase was incubated with sodium citrate (0.3 mol/L) and guanidinium chloride (0- 3. 5 mol/L). Aminoacylase lost enzyme activity in the guanidinium chloride more quickly in the presence of sodium citrate than in the absence of sodium citrate. The intrinsic fluorescence emission intensity decreased more quickly and the red shift of the emission spectra peak was larger than that without sodium citrate.

  13. Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse Inhibition of brain citrate synthase activity in an animal model of sepsis

    Directory of Open Access Journals (Sweden)

    Giselli Scaini

    2011-06-01

    fisiopatologia desta doença.OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS. Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

  14. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

    OpenAIRE

    Landschütze, V; Willmitzer, L; Müller-Röber, B

    1995-01-01

    The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistin...

  15. Mitochondrial citrate synthase crystals: novel finding in Sengers syndrome caused by acylglycerol kinase (AGK) mutations.

    Science.gov (United States)

    Siriwardena, Komudi; Mackay, Nevena; Levandovskiy, Valeriy; Blaser, Susan; Raiman, Julian; Kantor, Paul F; Ackerley, Cameron; Robinson, Brian H; Schulze, Andreas; Cameron, Jessie M

    2013-01-01

    We report on two families with Sengers syndrome and mutations in the acylglycerol kinase gene (AGK). In the first family, two brothers presented with vascular strokes, lactic acidosis, cardiomyopathy and cataracts, abnormal muscle cell histopathology and mitochondrial function. One proband had very abnormal mitochondria with citrate synthase crystals visible in electron micrographs, associated with markedly high citrate synthase activity. Exome sequencing was used to identify mutations in the AGK gene in the index patient. Targeted sequencing confirmed the same homozygous mutation (c.3G>A, p.M1I) in the brother. The second family had four affected members, of which we examined two. They also presented with similar clinical symptoms, but no strokes. Postmortem heart and skeletal muscle tissues showed low complex I, III and IV activities in the heart, but normal in the muscle. Skin fibroblasts showed elevated lactate/pyruvate ratios and low complex I+III activity. Targeted sequencing led to identification of a homozygous c.979A>T, p.K327* mutation. AGK is located in the mitochondria and phosphorylates monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid. Disruption of these signaling molecules affects the mitochondria's response to superoxide radicals, resulting in oxidative damage to mitochondrial DNA, lipids and proteins, and stimulation of cellular detoxification pathways. High levels of manganese superoxide dismutase protein were detected in all four affected individuals, consistent with increased free radical damage. Phosphatidic acid is also involved in the synthesis of phospholipids and its loss will result in changes to the lipid composition of the inner mitochondrial membrane. These effects manifest as cataract formation in the eye, respiratory chain dysfunction and cardiac hypertrophy in heart tissue. These two pedigrees confirm that mutation of AGK is responsible for the severe neonatal presentation of Sengers syndrome. The

  16. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    OpenAIRE

    Rozycki, Bartosz; Cieplak, Marek

    2015-01-01

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermo...

  17. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    Energy Technology Data Exchange (ETDEWEB)

    Różycki, Bartosz, E-mail: rozycki@ifpan.edu.pl; Cieplak, Marek [Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668 Warsaw (Poland)

    2014-12-21

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme.

  18. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    Science.gov (United States)

    RóŻycki, Bartosz; Cieplak, Marek

    2014-12-01

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme.

  19. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    International Nuclear Information System (INIS)

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme

  20. Molecular Cloning and Characterization of Citrate Synthase Gene in Rice( Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shan-shan; MING Feng; LU Qun; GUO Bin; SHEN Da-leng

    2005-01-01

    The full-length OsCS encoding citrate synthase was isolated from rice (Oryza sativa L. subsp. japonica). OsCS is 1477-bp long and encodes a 474 amino acid polypeptide. Its putative protein sequence is highly identical to Daucus carota, Nicotiana tabacum,Beta vulgaris subsp., Arabidopsis thaliana, and Citrus junos (>70%). The deduced amino-terminal sequence of OsCS showes characteristics of mitochondrial targeting signal. Southern blot analysis using ORF of the OsCS as the probe indicated that this gene exists in multiple copies in rice genome. The band with predicated size of 82 kD was detected by Western blot after being induced by 0.4 mmol/L IPTG.

  1. Evaluation of the role of mitochondrial citrate synthase, mitochondrial and cytosolic isoforms of isocitrate dehydrogenase in tomato leaf metabolism

    OpenAIRE

    Sienkiewicz-Porzucek, Agata

    2010-01-01

    Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosoli...

  2. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant.

    Science.gov (United States)

    Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

    2016-01-01

    'Hong Anliu' (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of 'Anliu' (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between 'HAL' and 'AL'. Compared with 'AL', the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in 'HAL'. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H(+)-ATPase, vacuolar H(+)-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in 'HAL'. These results implied that 'HAL' has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with 'AL'. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in 'HAL'. PMID:27385485

  3. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

    Science.gov (United States)

    Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

    2016-01-01

    ‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’. PMID:27385485

  4. Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

    Science.gov (United States)

    Kulla, H; Gottschalk, G

    1977-12-01

    Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation. PMID:924971

  5. Unique animal prenyltransferase with monoterpene synthase activity

    Science.gov (United States)

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

    2009-06-01

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

  6. Aluminum-activated citrate and malate transporters encoded by distinct Al tolerance genes function independently in Arabidopsis

    Science.gov (United States)

    Aluminum (Al) -activated malate and citrate exudation from roots plays an important role in conferring Al tolerance to many plant species. Here, we report on the identification and characterization of AtMATE, the gene encoding an Al-activated root citrate efflux transporter that functions in Arabid...

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  9. Activities and regulation of peptidoglycan synthases

    NARCIS (Netherlands)

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

    2015-01-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have b

  10. Effect of citrate on Aspergillus niger phytase adsorption and catalytic activity in soil

    Science.gov (United States)

    Mezeli, Malika; Menezes-Blackburn, Daniel; Zhang, Hao; Giles, Courtney; George, Timothy; Shand, Charlie; Lumsdon, David; Cooper, Patricia; Wendler, Renate; Brown, Lawrie; Stutter, Marc; Blackwell, Martin; Darch, Tegan; Wearing, Catherine; Haygarth, Philip

    2015-04-01

    Current developments in cropping systems that promote mobilisation of phytate in agricultural soils, by exploiting plant-root exudation of phytase and organic acids, offer potential for developments in sustainable phosphorus use. However, phytase adsorption to soil particles and phytate complexion has been shown to inhibit phytate dephosphorylation, thereby inhibiting plant P uptake, increasing the risk of this pool contributing to diffuse pollution and reducing the potential benefits of biotechnologies and management strategies aimed to utilise this abundant reserve of 'legacy' phosphorus. Citrate has been seen to increase phytase catalytic efficiency towards complexed forms of phytate, but the mechanisms by which citrate promotes phytase remains poorly understood. In this study, we evaluated phytase (from Aspergillus niger) inactivation, and change in catalytic properties upon addition to soil and the effect citrate had on adsorption of phytase and hydrolysis towards free, precipitated and adsorbed phytate. A Langmuir model was fitted to phytase adsorption isotherms showing a maximum adsorption of 0.23 nKat g-1 (19 mg protein g-1) and affinity constant of 435 nKat gˉ1 (8.5 mg protein g-1 ), demonstrating that phytase from A.niger showed a relatively low affinity for our test soil (Tayport). Phytases were partially inhibited upon adsorption and the specific activity was of 40.44 nKat mgˉ1 protein for the free enzyme and 25.35 nKat mgˉ1 protein when immobilised. The kinetics of adsorption detailed that most of the adsorption occurred within the first 20 min upon addition to soil. Citrate had no effect on the rate or total amount of phytase adsorption or loss of activity, within the studied citrate concentrations (0-4mM). Free phytases in soil solution and phytase immobilised on soil particles showed optimum activity (>80%) at pH 4.5-5.5. Immobilised phytase showed greater loss of activity at pH levels over 5.5 and lower activities at the secondary peak at pH 2

  11. Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding

    Directory of Open Access Journals (Sweden)

    Victor S. Blancato

    2016-02-01

    Full Text Available The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC, indicated that CitO has a high affinity for citrate (KD= 1.2±0.2 µM, while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.

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

    OpenAIRE

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

    2015-01-01

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

  13. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance

    Science.gov (United States)

    Aluminum (Al) activated root malate and citrate exudation plays an important role in Al tolerance in many plant species. Here, we report on the identification and characterization of AtMATE, a homolog of the recently discovered sorghum and barley Al tolerance genes, here shown to encode an Al-activ...

  14. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance

    Science.gov (United States)

    Aluminum (Al) activated root malate and citrate exudation play an important role in Al tolerance in many plant species. AtALMT1, an Al-activated malate transporter, is a major contributor to Arabidopsis Al tolerance. Here, we demonstrate that a second, unrelated gene, AtMATE, encodes an Arabidopsi...

  15. Citrate confers less filter-induced complement activation and neutrophil degranulation than heparin when used for anticoagulation during continuous venovenous haemofiltration in critically ill patients

    OpenAIRE

    Schilder, Louise; Nurmohamed, Shaikh Azam; Wee, Piet; Paauw, Nanne; Girbes, Armand; Beishuizen, Albertus; Beelen, Robert; Groeneveld, Johan

    2014-01-01

    textabstractBackground: During continuous venovenous haemofiltration (CVVH), regional anticoagulation with citrate may be superior to heparin in terms of biocompatibility, since heparin as opposed to citrate may activate complement (reflected by circulating C5a) and induce neutrophil degranulation in the filter and myeloperoxidase (MPO) release from endothelium. Methods. No anticoagulation (n = 13), unfractionated heparin (n = 8) and trisodium citrate (n = 17) regimens during CVVH were compar...

  16. Citrate confers less filter-induced complement activation and neutrophil degranulation than heparin when used for anticoagulation during continuous venovenous haemofiltration in critically ill patients

    OpenAIRE

    Schilder, Louise; Nurmohamed, S Azam; ter Wee, Pieter M.; Paauw, Nanne J.; Girbes, Armand RJ; Beishuizen, Albertus; Beelen, Robert HJ; Groeneveld, AB Johan

    2014-01-01

    Background During continuous venovenous haemofiltration (CVVH), regional anticoagulation with citrate may be superior to heparin in terms of biocompatibility, since heparin as opposed to citrate may activate complement (reflected by circulating C5a) and induce neutrophil degranulation in the filter and myeloperoxidase (MPO) release from endothelium. Methods No anticoagulation (n = 13), unfractionated heparin (n = 8) and trisodium citrate (n = 17) regimens during CVVH were compared. Blood samp...

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Thromboelastography results on citrated whole blood from clinically healthy cats depend on modes of activation

    Directory of Open Access Journals (Sweden)

    Wiinberg Bo

    2010-06-01

    Full Text Available Abstract Background During the last decade, thromboelastography (TEG has gained increasing acceptance as a diagnostic test in veterinary medicine for evaluation of haemostasis in dogs, however the use of TEG in cats has to date only been described in one previous study and a few abstracts. The objective of the present study was to evaluate and compare three different TEG assays in healthy cats, in order to establish which assay may be best suited for TEG analyses in cats. Methods 90 TEG analyses were performed on citrated whole blood samples from 15 clinically healthy cats using assays without activator (native or with human recombinant tissue factor (TF or kaolin as activators. Results for reaction time (R, clotting time (K, angle (α, maximum amplitude (MA and clot lysis (LY30; LY60 were recorded. Results Coefficients of variation (CVs were highest in the native assay and comparable in TF and kaolin activated assays. Significant differences were observed between native and kaolin assays for all measured parameters, between kaolin and TF for all measured parameters except LY60 and between native and TF assays for R and K. Conclusion The results indicate that TEG is a reproducible method for evaluation of haemostasis in clinically healthy cats. However, the three assays cannot be used interchangeably and the kaolin- and TF activated assays have the lowest analytical variation indicating that using an activator may be superior for performing TEG in cats.

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

    OpenAIRE

    Kida, Y; Esposito-Del Puente, A; Bogardus, C; Mott, D M

    1990-01-01

    Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatas...

  20. Redox properties and activity of iron-citrate complexes: evidence for redox cycling.

    Science.gov (United States)

    Adam, Fatima I; Bounds, Patricia L; Kissner, Reinhard; Koppenol, Willem H

    2015-04-20

    Iron in iron overload disease is present as non-transferrin-bound iron, consisting of iron, citrate, and albumin. We investigated the redox properties of iron citrate by electrochemistry, by the kinetics of its reaction with ascorbate, by ESR, and by analyzing the products of reactions of ascorbate with iron citrate complexes in the presence of H2O2 with 4-hydroxybenzoic acid as a reporter molecule for hydroxylation. We report -0.03 V +0.01 V for the (Fe(3+)-cit/Fe(2+)-cit) couple. The first step in the reaction of iron citrate with ascorbate is the rapid formation of mixed complexes of iron with citrate and ascorbate, followed by slow reduction to Fe(2+)-citrate with k = ca. 3 M(-1) s(-1). The ascorbyl radical is formed by iron citrate oxidation of Hasc(-) with k = ca. 0.02 M(-1) s(-1); the majority of the ascorbyl radical formed is sequestered by complexation with iron and remains EPR silent. The hydroxylation of 4-hydroxybenzoic acid driven by the Fenton reduction of iron citrate by ascorbate in the presence of H2O2 proceeds in three phases: the first phase, which is independent of the presence of O2, is revealed as a nonzero intercept that reflects the rapid reaction of accumulated Fe(2+) with H2O2; the intermediate oxygen-dependent phase fits a first-order accumulation of product with k = 5 M(-1) s(-1) under aerobic and k = 13 M(-1) s(-1) under anaerobic conditions; the slope of the final linear phase is ca. k = 5 × 10(-2) M(-1) s(-1) under both aerobic and anaerobic conditions. Product yields under aerobic conditions are greater than predicted from the initial concentration of iron, but they are less than predicted for continuous redox cycling in the presence of excess ascorbate. The ongoing formation of hydroxylated product supports slow redox cycling by iron citrate. Thus, when H2O2 is available, iron-citrate complexes may contribute to pathophysiological manifestations of iron overload diseases. PMID:25654270

  1. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content...... expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review will...... discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  2. Aluminum resistance in common bean (Phaseolus vulgaris) involves induction and maintenance of citrate exudation from root apices.

    Science.gov (United States)

    Rangel, Andrés Felipe; Rao, Idupulapati Madhusudana; Braun, Hans-Peter; Horst, Walter Johannes

    2010-02-01

    Two common bean (Phaseolus vulgaris L.) genotypes differing in aluminum (Al) resistance, Quimbaya (Al-resistant) and VAX-1 (Al-sensitive) were grown in hydroponics for up to 25 h with or without Al, and several parameters related to the exudation of organic acids anions from the root apex were investigated. Al treatment enhanced the exudation of citrate from the root tips of both genotypes. However, its dynamic offers the most consistent relationship between Al-induced inhibition of root elongation and Al accumulation in and exclusion from the root apices. Initially, in both genotypes the short-term (4 h) Al-injury period was characterized by the absence of citrate efflux independent of the citrate content of the root apices, and reduction of cytosolic turnover of citrate conferred by a reduced Nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase (EC 1.1.1.42) activity. Transient recovery from initial Al stress (4-12 h) was found to be dependent mainly on the capacity to utilize internal citrate pools (Al-resistant genotype Quimbaya) or enhanced citrate synthesis [increased activities of NAD-malate dehydrogenase (EC 1.1.1.37) and ATP-phosphofructokinase (EC 2.7.1.11) in Al-sensitive VAX-1]. Sustained recovery from Al stress through citrate exudation in genotype Quimbaya after 24 h Al treatment relied on restoring the internal citrate pool and the constitutive high activity of citrate synthase (CS) (EC 4.1.3.7) fuelled by high phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity. In the Al-sensitive genotype VAX-1 the citrate exudation and thus Al exclusion and root elongation could not be maintained coinciding with an exhaustion of the internal citrate pool and decreased CS activity. PMID:20053183

  3. Mosapride citrate, a 5-HT₄ receptor agonist, increased the plasma active and total glucagon-like peptide-1 levels in non-diabetic men.

    Science.gov (United States)

    Aoki, Kazutaka; Kamiyama, Hiroshi; Masuda, Kiyomi; Togashi, Yu; Terauchi, Yasuo

    2013-01-01

    Mosapride citrate, a selective agonist of the 5-hydroxytryptaine (5-HT)₄ receptor, is typically used to treat heartburn, nausea, and vomiting associated with chronic gastritis or to prepare for a barium enema X-ray examination. Mosapride citrate reportedly improves insulin sensitivity in patients with type 2 diabetes. As mosapride citrate activates the motility of the gastrointestinal tract, we hypothesized that mosapride citrate affects incretin secretion. We examined the effect of the administration of mosapride citrate on the plasma glucose, serum insulin, plasma glucagon, and plasma incretin levels before breakfast and at 60, 120, and 180 min after breakfast in men with normal glucose tolerance (NGT) or impaired glucose tolerance (IGT) to exclude gastropathy. Mosapride citrate was administered according to two different intake schedules (C: control (no drug), M: mosapride citrate 20 mg) in each of the subject groups. The area under the curve (AUC) of the plasma glucose levels was smaller in the M group than in the C group. The time profiles for the serum insulin levels at 60 and 120 min after treatment with mosapride citrate tended to be higher, although the difference was not statistically significant. The AUCs of the plasma active and total glucagon-like peptide-1 (GLP-1) levels were significantly larger in the M group than in the C group. No significant difference in the AUC of the plasma glucose-dependent insulinotropic polypeptide (GIP) level was observed between the two groups. Our results suggest that mosapride citrate may have an antidiabetic effect by increasing GLP-1 secretion. PMID:23257734

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

    Science.gov (United States)

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

    2008-05-01

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

  5. Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants.

    Science.gov (United States)

    Montgomery, Heather J; Bartlett, Ryan; Perdicakis, Basil; Jervis, Eric; Squier, Thomas C; Guillemette, J Guy

    2003-07-01

    Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite. PMID:12820885

  6. Modulatory Effect of Citrate Reduced Gold and Biosynthesized Silver Nanoparticles on α-Amylase Activity

    OpenAIRE

    Kantrao Saware; Ravindra Mahadappa Aurade; Kamala Jayanthi, P. D.; Venkataraman Abbaraju

    2015-01-01

    Amylase is one of the important digestive enzymes involved in hydrolysis of starch. In this paper, we describe a novel approach to study the interaction of amylase enzyme with gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) and checked its catalytic function. AuNPs are synthesized using citrate reduction method and AgNPs were synthesized using biological route employing Ficus benghalensis and Ficus religiosa leaf extract as a reducing and stabilizing agent to reduce silver nitrate...

  7. Regulatory Citrate Lyase Mutants of Salmonella typhimurium

    OpenAIRE

    Kulla, Hans G.

    1983-01-01

    Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from Salmonella typhimurium. The only class of mutants found had a mode of covalent regulation that strongly resembled the Escherichia coli system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present.

  8. Regulatory citrate lyase mutants of Salmonella typhimurium.

    Science.gov (United States)

    Kulla, H G

    1983-01-01

    Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from Salmonella typhimurium. The only class of mutants found had a mode of covalent regulation that strongly resembled the Escherichia coli system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present. PMID:6336740

  9. A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies.

    OpenAIRE

    Combet, S.; Balligand, Jean-Luc; Lameire, N.; Goffin, Eric; Devuyst, Olivier

    2000-01-01

    A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies. BACKGROUND: Nitric oxide (NO) is synthesized by NO synthase (NOS) isoforms that are expressed in the peritoneum. Thus far, NOS activity in the peritoneum has been assessed by nonspecific methods. We describe the application of a specific method for determination of NOS activity in rat and human peritoneal biopsies. METHODS: The L-citrulline assay is based on the stoechiometric production of N...

  10. 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 125I-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

  11. Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

    Directory of Open Access Journals (Sweden)

    Alistair K Brown

    Full Text Available BACKGROUND: There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin. METHODS AND FINDINGS: We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml and against Mycobacterium tuberculosis (MIC = 12 microg/ml. Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH. SIGNIFICANCE: Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

  12. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    Science.gov (United States)

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg. PMID:23467831

  13. 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)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

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

    OpenAIRE

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

    2008-01-01

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this s...

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

    Science.gov (United States)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

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

  16. Glycogen synthase activation in human skeletal muscle: effects of diet and exercise.

    Science.gov (United States)

    Kochan, R G; Lamb, D R; Lutz, S A; Perrill, C V; Reimann, E M; Schlender, K K

    1979-06-01

    We investigated the role of glycogen synthase in supranormal resynthesis (supercompensation) of skeletal muscle glycogen after exhaustive exercise. Six healthy men exercised 60 min by cycling with one leg at 75% VO2max, recovered 3 days on a low-carbohydrate diet, exercised again, and recovered 4 days on high-carbohydrate diet. Glycogen and glycogen synthase activities at several glucose-6-phosphate (G6P) concentrations were measured in biopsy samples of m. vastus lateralis. Dietary alterations alone did not affect glycogen, whereas exercise depleted glycogen stores. After the second exercise bout, glycogen returned to normal within 24 h and reached supercompensated levels by 48 h of recovery. Glycogen synthase activation state strikingly increased after exercise in exercised muscle and remained somewhat elevated for the first 48 h of recovery in both muscles. We suggest that 1) forms of glycogen synthase intermediate to I (G6P-independent) and D (G6P-dependent) forms are present in vivo, and 2) glycogen supercompensation can in part be explained by the formation of intermediate forms of glycogen synthase that exhibit relatively low activity ratios, but an increased sensitivity to activation by G6P. PMID:109015

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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

  18. Effect of aging on expression of nitric oxide synthase I and activity of nitric oxide synthase in rat penis

    Institute of Scientific and Technical Information of China (English)

    Jun-PingSHI; Yong-MeiZHAO; Yu-TongSONG

    2003-01-01

    Aim: To investigate the effect of aging on the expression of nitric oxide synthase I (NOS I) and the activity of NOS in rat penis. Methods: Sixty male rats from 3 age groups (adult, old and senescent) were investigated.The expression of NOS I protein and mRNA in rat penis were detected by Western blot and RT-PCR respectively and the NOS activity, with ultraviolet spectrophotometry. Results: In the old and senescent group, NOS I protein expression was significantly decreased as compared with the adult. NOS I mRNA expression was well correlated with the protein expression. NOS activity was not statistically different between the adult and old groups, but it was significantly reduced in the senescent compared with the adult group (P<0.01). Conclusion: The aging-induced decreases in NOS I expression and NOS activity may be one of the main mechanisms leading to erectile dysfunctionin the senescent rats. ( Asian J Androl 2003 Jun; 5: 117-120)

  19. Nitrate Protects Cucumber Plants Against Fusarium oxysporum by Regulating Citrate Exudation.

    Science.gov (United States)

    Wang, Min; Sun, Yuming; Gu, Zechen; Wang, Ruirui; Sun, Guomei; Zhu, Chen; Guo, Shiwei; Shen, Qirong

    2016-09-01

    Fusarium wilt causes severe yield losses in cash crops. Nitrogen plays a critical role in the management of plant disease; however, the regulating mechanism is poorly understood. Using biochemical, physiological, bioinformatic and transcriptome approaches, we analyzed how nitrogen forms regulate the interactions between cucumber plants and Fusarium oxysporum f. sp. cucumerinum (FOC). Nitrate significantly suppressed Fusarium wilt compared with ammonium in both pot and hydroponic experiments. Fewer FOC colonized the roots and stems under nitrate compared with ammonium supply. Cucumber grown with nitrate accumulated less fusaric acid (FA) after FOC infection and exhibited increased tolerance to chemical FA by decreasing FA absorption and transportation in shoots. A lower citrate concentration was observed in nitrate-grown cucumbers, which was associated with lower MATE (multidrug and toxin compound extrusion) family gene and citrate synthase (CS) gene expression, as well as lower CS activity. Citrate enhanced FOC spore germination and infection, and increased disease incidence and the FOC population in ammonium-treated plants. Our study provides evidence that nitrate protects cucumber plants against F. oxysporum by decreasing root citrate exudation and FOC infection. Citrate exudation is essential for regulating disease development of Fusarium wilt in cucumber plants. PMID:27481896

  20. Investigation of the effect of kaolin and tissue factor-activated citrated whole blood, on clot forming variables, as evaluated by thromboelastograph

    DEFF Research Database (Denmark)

    Johansson, Per Ingemar; Bochsen, L.; Andersen, S.;

    2008-01-01

    laboratory-based analysis, however, requires validation of the activators employed and the effect of storage of the WB sample in citrate before analysis. STUDY DESIGN AND METHODS: The effect of kaolin, tissue factor (TF) 1:17,000, or TF 1:42,500 on TEG clotting time (R), Angle (velocity of clot formation......), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time. RESULTS: Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer...... rested for 0 and 30 minutes were evaluated with kaolin as the activator. CONCLUSION: The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in...

  1. Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Lim, Xin Ying; Pickford, Russell; Don, Anthony S

    2016-01-01

    Sphingolipids are one the major lipid families in eukaryotes, incorporating a diverse array of structural and signaling lipids such as sphingomyelin and gangliosides. The core lipid component for all complex sphingolipids is ceramide, a diacyl lipid consisting of a variable length fatty acid linked through an amide bond to a long chain base such as sphingosine or dihydrosphingosine. This reaction is catalyzed by a family of six ceramide synthases (CERS1-6), each of which preferentially catalyzes the synthesis of ceramides with different fatty acid chain lengths. Ceramides are themselves potent cellular and physiological signaling molecules heavily implicated in diabetes and neurodegenerative diseases, making it important for researchers to have access to sensitive and accurate assays for ceramide synthase activity. This chapter describes methods for assaying ceramide synthase activity in cell or tissue lysates, or in cultured cells (in situ), using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the readout. LC-MS/MS is a very sensitive and accurate means for assaying ceramide synthase reaction products. PMID:26552671

  2. Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase▿†

    Science.gov (United States)

    Baumgart, Meike; Mustafi, Nurije; Krug, Andreas; Bott, Michael

    2011-01-01

    The aconitase gene acn of Corynebacterium glutamicum is regulated by four transcriptional regulators, indicating that the synthesis of this enzyme is carefully controlled. To understand the causes for this elaborate regulation, the properties of the Δacn-1 deletion mutant were analyzed in detail. The mutant was glutamate auxotrophic in glucose minimal medium, showed a strong growth defect, and secreted large amounts of acetate. None of these phenotypes could be complemented by plasmid-encoded aconitase, suggesting the presence of a secondary mutation. In fact, a point mutation within the gltA gene encoding citrate synthase was identified that caused the instability of the protein and an almost complete lack of its enzymatic activity. Subsequently, 27 further, independent Δacn clones were isolated, and 15 of them were found to contain distinct mutations in gltA, causing the loss of citrate synthase activity. A similar result was observed for mutants lacking the isocitrate dehydrogenase gene icd. In this case, 8 of 24 Δicd clones contained additional mutations in gltA. Indirect evidence was obtained that elevated intracellular citrate concentrations could be the cause of this selection pressure. Accordingly, the careful control of aconitase synthesis might have evolved due to the necessity to avoid inhibitory cytoplasmic citrate levels on the one hand and to prevent the excessive synthesis of an oxygen-sensitive protein requiring both iron and sulfur on the other hand. PMID:21984793

  3. Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene[S

    OpenAIRE

    Burns, Tara Ann; Subathra, Marimuthu; signorelli, Paola; Choi, Young; Yang, Xiaofeng; Wang, Yong; Villani, Maristella; Bhalla, Kapil; Zhou, Daohong; Luberto, Chiara

    2013-01-01

    Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the ...

  4. "Dopamine-first" mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

    OpenAIRE

    Lichman, B. R.; Gershater, M. C.; Lamming, E. D.; Pesnot, T.; Sula, A.; Keep, N.H.; Hailes, H. C.; Ward, J. M.

    2015-01-01

    Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet-Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two propo...

  5. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  6. S-Glutathionylation Enhances Human Cystathionine β-Synthase Activity Under Oxidative Stress Conditions

    OpenAIRE

    Niu, Wei-Ning; Yadav, Pramod Kumar; Adamec, Jiri; Banerjee, Ruma

    2015-01-01

    Aims: Cystathionine β-synthase (CBS) catalyzes the first and rate-limiting step in the two-step trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of three major enzymes responsible for the biogenesis of H2S, a signaling molecule. We have previously demonstrated that CBS is activated in cells challenged by oxidative stress, but the underlying molecular mechanism of this regulation has remained unclear. Results: Here, we demonstrate that S-glutathionylation of CBS...

  7. Histochemical study of the nitric oxide synthase activity in experimental trichinellosis.

    Science.gov (United States)

    Hadaś, E; Gustowska, L; Boczoń, K; Janczewska, D

    1999-01-01

    Nitric oxide plays a critical role in a variety of biological activities. It has been nicknamed a "killer" and "mediator" due to its toxic and signalling properties. Apart from its regular physiological function, nitric oxide indirectly participates in infectious diseases. Our report seems to be the first presentation of the nitric oxide synthase participation in the host biochemical defence mechanisms and in morphological transformation of muscle cells in trichinellosis. PMID:16883715

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

    Science.gov (United States)

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

    2006-07-01

    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

  9. Increased nitric oxide synthase activity and Hsp90 association in skeletal muscle following chronic exercise

    OpenAIRE

    Harris, M. Brennan; Mitchell, Brett M.; Sood, Sarika G.; Webb, R. Clinton; Venema, Richard C.

    2008-01-01

    Exercise training results in dynamic changes in skeletal muscle blood flow and metabolism. Nitric oxide (NO) influences blood flow, oxidative stress, and glucose metabolism. Hsp90 interacts directly with nitric oxide synthases (NOS), increasing NOS activity and altering the balance of superoxide versus NO production. In addition, Hsp90 expression increases in various tissues following exercise. Therefore, we tested the hypothesis that exercise training increases Hsp90 expression as well as Hs...

  10. 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.

  11. Hyperhomocysteinaemia in rats is associated with erectile dysfunction by impairing endothelial nitric oxide synthase activity.

    Science.gov (United States)

    Jiang, Weijun; Xiong, Lei; Bin Yang; Li, Weiwei; Zhang, Jing; Zhou, Qing; Wu, Qiuyue; Li, Tianfu; Zhang, Cui; Zhang, Mingchao; Xia, Xinyi

    2016-01-01

    To investigate the effect of hyperhomocysteinaemia (HHCy) on penile erectile function in a rat model, a methionine-rich diet was used in which erectile function, the reproductive system, and nitric oxide synthase were characterized. The intracavernous pressure, apomorphine experiments, measurement of oxidative stress, hematoxylin and eosin staining, immunohistochemistry analysis, reverse transcription-polymerase chain reactions and measurement of endothelial nitric oxide synthase activity were utilized. Our results showed that erections in the middle-dose, high-dose, and interference (INF) groups were significantly lower than the control (P < 0.05). INF group, being fed with vitamins B and folic acid, demonstrated markedly improved penile erections compared with the middle-dose group (P < 0.05). HHCy-induced eNOS and phospho-eNOS protein expression was reduced and the antioxidant effect was markedly impaired. The data of the present data provide evidence that HHCy is a vascular risk factor for erectile dysfunction by impairing cavernosa endothelial nitric oxide synthase activity. Intake of vitamins B can alleviate this abnormality. PMID:27221552

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

    International Nuclear Information System (INIS)

    Here we demonstrate that human keratinocytes possess a Ca2+/ calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[3H]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/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) 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- released by UVB-irradiated human keratinocytes in skin erythema and inflammation. (Author)

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

    Science.gov (United States)

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

    2013-04-01

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

  14. Citrat og nyresten

    DEFF Research Database (Denmark)

    Osther, P J

    1993-01-01

    Citrate is an important naturally occurring inhibitor of calcium stone formation in urine. Urinary citrate excretion was examined in 43 consecutive patients with recurrent idiopathic calcium nephrolithiasis and in 50 normal controls by a specific enzymatic technique. Hypocitraturia (<1.6 mmol/24h...

  15. Physicochemical action of potassium-magnesium citrate in nephrolithiasis

    Science.gov (United States)

    Pak, C. Y.; Koenig, K.; Khan, R.; Haynes, S.; Padalino, P.

    1992-01-01

    Effect of potassium-magnesium citrate on urinary biochemistry and crystallization of stone-forming salts was compared with that of potassium citrate at same dose of potassium in five normal subjects and five patients with calcium nephrolithiasis. Compared to the placebo phase, urinary pH rose significantly from 6.06 +/- 0.27 to 6.48 +/- 0.36 (mean +/- SD, p less than 0.0167) during treatment with potassium citrate (50 mEq/day for 7 days) and to 6.68 +/- 0.31 during therapy with potassium-magnesium citrate (containing 49 mEq K, 24.5 mEq Mg, and 73.5 mEq citrate per day). Urinary pH was significantly higher during potassium-magnesium citrate than during potassium citrate therapy. Thus, the amount of undissociated uric acid declined from 118 +/- 61 mg/day during the placebo phase to 68 +/- 54 mg/day during potassium citrate treatment and, more prominently, to 41 +/- 46 mg/day during potassium-magnesium citrate therapy. Urinary magnesium rose significantly from 102 +/- 25 to 146 +/- 37 mg/day during potassium-magnesium citrate therapy but not during potassium citrate therapy. Urinary citrate rose more prominently during potassium-magnesium citrate therapy (to 1027 +/- 478 mg/day from 638 +/- 252 mg/day) than during potassium citrate treatment (to 932 +/- 297 mg/day). Consequently, urinary saturation (activity product) of calcium oxalate declined significantly (from 1.49 x 10(-8) to 1.03 x 10(-8) M2) during potassium-magnesium citrate therapy and marginally (to 1.14 x 10(-8) M2) during potassium citrate therapy.(ABSTRACT TRUNCATED AT 250 WORDS).

  16. Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

    Science.gov (United States)

    Hwang, Hye Jin; Steidemann, Michelle; Dunivin, Taylor K; Rizzo, Mike; LaPres, John J

    2016-09-01

    2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments. PMID:27284569

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

    International Nuclear Information System (INIS)

    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 NaB3H4 or Ado[14C]Met. Peptide sequencing of both 3H- and 14C-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 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-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 [14C]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

  18. Assembly of the eukaryotic PLP-synthase complex from plasmodium and activation of the Pdx1 enzyme

    OpenAIRE

    Guedez, Gabriela; Hipp, Katharina; Windeisen, Volker; Derrer, Bianca; Gengenbacher, Martin; Boettcher, Bettina; Sinning, Irmgard; Kappes, Barbara; Tews, Ivo

    2012-01-01

    Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B6 (pyridoxal 5-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alter...

  19. [Activity of 5-aminolevulinate synthase in rat liver during degradation of cytochrome P-450 caused by administration of cadmium chloride].

    Science.gov (United States)

    Kaliman, P A; Inshina, N N

    2003-01-01

    The 5-aminolevulinate synthase, tryptophan-2,3-dioxygenase activities and cytochrome P-450 content in the rat liver was studied in different terms after CdCl2 administration and after administration of metal salt against a background of 2-hours action of alpha-tocopherol. The lowering of activity of 5-aminolevulinate synthase in 2 h with the consequent increase of the enzyme activity in 6 h and 24 h was detected. The holoenzyme activity and heme saturation of tryptophan-2,3-dioxygenase increased 6 h after CdCl2 administration. The holoenzyme activity and the total activity of tryptophan-2,3-dioxygenase rised in 24 h. The level of cytochrome P-450 lowered. Preliminary administration of alpha-tocopherol prevented changes of studied parameters 24 h after CdCl2 administration. The relationship between decrease of cytochrome P-450 level and 5-aminolevulinate synthase activation are discussed. PMID:14577179

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

    Directory of Open Access Journals (Sweden)

    Thi Le Nhung Nguyen-Deroche

    2012-01-01

    Full Text Available Zinc-supplementation (20 μM 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 Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

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

    Science.gov (United States)

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

    2016-06-01

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

  2. Substrate structure-activity relationships guide rational engineering of modular polyketide synthase ketoreductases.

    Science.gov (United States)

    Bailey, Constance B; Pasman, Marjolein E; Keatinge-Clay, Adrian T

    2016-01-14

    Modular polyketide synthase ketoreductases can set two chiral centers through a single reduction. To probe the basis of stereocontrol, a structure-activity relationship study was performed with three α-methyl, β-ketothioester substrates and four ketoreductases. Since interactions with the β-ketoacyl moiety were found to be most critical, residues implicated in contacting this moiety were mutated. Two mutations were sufficient to completely reverse the stereoselectivity of the model ketoreductase EryKR1, converting it from an enzyme that generates (2S,3R)-products into one that yields (2S,3S)-products. PMID:26568113

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

    OpenAIRE

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

    2014-01-01

    The bacterial signaling molecule cyclic-di-GMP stimulates the synthesis of bacterial cellulose, frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of the cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the cyclic-di-GMP-activated BcsA–B complex. The structures reveal that cyclic-di-GMP releases an auto-inhibited state of the enzyme by breaking a salt bridge which otherwise tethers a conserve...

  4. Trichothecenes induce accumulation of glucosylceramide in neural cells by interfering with lactosylceramide synthase activity

    International Nuclear Information System (INIS)

    Trichothecenes are sesquiterpenoid metabolites produced by several fungal strains that impair human and animal health. Since sphingolipids were connected with fungal toxicity the aim of the present study was to test the influence of fungal metabolites on sphingolipid metabolism in neural cells. The crude extract of fungal strain Spicellum roseum induced accumulation of glucosylceramide (GlcCer), and simultaneous reduction of the formation of lactosylceramide (LacCer) and complex gangliosides in primary cultured neurons. Following a bioassay-guided fractionation of the respective fungal extract we could demonstrate that the two isolated trichothecene derivatives, 8-deoxy-trichothecin (8-dT) and trichodermol (Td-ol) were responsible for this effect. Thus, incubation of primary cultured neurons as well as of neuroblastoma B104 cells for 24 h with 30 μM of either of the two fungal metabolites resulted in uncoupling of sphingolipid biosynthesis at the level of LacCer. For the observed reduction of LacCer synthase activity by about 90% cell integrity was crucial in both cell types. In neuroblastoma cells the amount of LacCer synthase mRNA was reduced in the presence of trichothecenes, whereas in primary cultured neurons this was not the case, suggesting a post-transcriptional mechanism of action in the latter cell type. The data also show that the compounds did not interfere with the translocation of GlcCer in neuroblastoma cells. Collectively, our results demonstrate that trichodermol and 8-deoxy-trichothecin inhibit LacCer synthase activity in a cell-type-specific manner

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-12

    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.

  6. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet; O'Neil-Johnson, Mark; Starks, Courtney; Hamberger, Björn; Bohlmann, Jörg

    2014-09-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual βα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids. PMID:24990389

  7. Gastrointestinal citrate absorption in nephrolithiasis

    Science.gov (United States)

    Fegan, J.; Khan, R.; Poindexter, J.; Pak, C. Y.

    1992-01-01

    Gastrointestinal absorption of citrate was measured in stone patients with idiopathic hypocitraturia to determine if citrate malabsorption could account for low urinary citrate. Citrate absorption was measured directly from recovery of orally administered potassium citrate (40 mEq.) in the intestinal lavage fluid, using an intestinal washout technique. In 7 stone patients citrate absorption, serum citrate levels, peak citrate concentration in serum and area under the curve were not significantly different from those of 7 normal subjects. Citrate absorption was rapid and efficient in both groups, with 96 to 98% absorbed within 3 hours. The absorption of citrate was less efficient from a tablet preparation of potassium citrate than from a liquid preparation, probably due to a delayed release of citrate from wax matrix. However, citrate absorption from solid potassium citrate was still high at 91%, compared to 98% for a liquid preparation. Thus, hypocitraturia is unlikely to be due to an impaired gastrointestinal absorption of citrate in stone patients without overt bowel disease.

  8. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  9. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Soriano, Erika V.; Zhang, Yang; Colabroy, Keri L.; Sanders, Jennie M.; Settembre, Ethan C.; Dorrestein, Pieter C.; Begley, Tadhg P.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2013-09-01

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ▶), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.

  10. Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice

    OpenAIRE

    Grandati, M; Verrecchia, C; Revaud, M L; Allix, M.; Boulu, R. G.; Plotkine, M.

    1997-01-01

    The temporal changes in constitutive NO-synthase (cNOS) and in calcium-independent NO-synthase activities were studied in mice subjected to 2 h of transient focal cerebral ischaemia. The changes in brain nitrites/nitrates (NOx) content were also studied.NOS activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Brain NOx contents were investigated by the Griess colourimetric method.cNOS activity in the infarcted cortical area was significantly reduced after 6 h ...

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

    Science.gov (United States)

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

    2016-03-01

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

  12. Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

    Science.gov (United States)

    Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

    2012-01-01

    Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

  13. Phase I study using desferrioxamine and iron sorbitol citrate in an attempt to modulate the iron status of tumor cells to enhance doxorubicin activity.

    Science.gov (United States)

    Voest, E E; Neijt, J P; Keunen, J E; Dekker, A W; van Asbeck, B S; Nortier, J W; Ros, F E; Marx, J J

    1993-01-01

    A novel approach to enhance the activity of doxorubicin is to increase the availability of cellular "chelatable" iron to participate in doxorubicin-mediated free-radical generation. To achieve this, we designed a regimen consisting of desferrioxamine (DFO, 50 mg/kg daily given as an i.v. infusion over 72 h) to increase cellular iron uptake. Thereafter, the combination of iron sorbitol citrate (ISC) and doxorubicin (as a single agent or as part of the CHOP regimen) was given. In a phase I study we investigated the toxicity of this regimen in nine patients with refractory malignant disease. Severe but reversible ocular toxicity (i.e., acute maculopathy) was observed in two patients. As these patients were the only ones who were pretreated with cisplatin, we caution against the use of DFO in cisplatin-pretreated patients. Severe phlebitis was encountered in five of nine patients. A partial remission was observed in two of four patients with refractory Non-Hodgkin's lymphoma who were treated with DFO, ISC, and doxorubicin as part of the CHOP regimen. We conclude that pretreatment with DFO and iron sorbitol citrate may be of benefit in the treatment of malignancies with doxorubicin-containing regimens, but ocular toxicity and severe phlebitis limits the use of DFO in this approach. The attachment of DFO to biocompatible polymers may be a method of overcoming the observed toxicity and warrants further study. PMID:8431969

  14. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization

    NARCIS (Netherlands)

    Dong, L.; Miettinen, K.; Verstappen, F.W.A.; Voster, A.; Jongsma, M.A.; Memelink, J.; Krol, van der S.; Bouwmeester, H.J.

    2013-01-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellu

  15. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

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

  16. Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase.

    Science.gov (United States)

    Fischer, Markus; Haase, Ilka; Kis, Klaus; Meining, Winfried; Ladenstein, Rudolf; Cushman, Mark; Schramek, Nicholas; Huber, Robert; Bacher, Adelbert

    2003-02-21

    6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role. PMID:12581640

  17. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  18. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

    OpenAIRE

    Buller, Andrew R.; Brinkmann-Chen, Sabine; Romney, David K.; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H

    2015-01-01

    Many enzymes perform desirable biochemical transformations, but are not suitable to use as biocatalysts outside of the cell. In particular, enzymes from heteromeric complexes typically have decreased activity when removed from their protein partners. We used directed evolution to restore the catalytic efficiency of the tryptophan synthase β-subunit (TrpB), which synthesizes l...

  19. Oligomerization of rice granule-bound starch synthase 1 modulates its activity regulation.

    Science.gov (United States)

    Liu, De-Rui; Huang, Wei-Xue; Cai, Xiu-Ling

    2013-09-01

    Granule-bound starch synthase 1 (GBSS1) is responsible for amylose synthesis in cereals, and this enzyme is regulated at the transcriptional and post-transcriptional levels. In this study, we show that GBSS1 from Oryza sativa L. (OsGBSS1) can form oligomers in rice endosperm, and oligomerized OsGBSS1 exhibits much higher specific enzymatic activity than the monomer. A monomer-oligomer transition equilibrium for OsGBSS1 occurs in the endosperm during development. Redox potential is a key factor affecting the oligomer percentage as well as the enzymatic activity of OsGBSS1. Adenosine diphosphate glucose, the direct donor of glucose, also impacts OsGBSS1 oligomerization in a concentration-dependent manner. OsGBSS1 oligomerization is influenced by phosphorylation status, which was strongly enhanced by Mitogen-activated protein kinase (MAPK) and ATP treatment and was sharply weakened by protein phosphatase (PPase) treatment. The activity of OsGBSS1 affects the ratio of amylose to amylopectin and therefore the eating quality of rice. Understanding the regulation of OsGBSS1 activity may lead to the improvement of rice eating quality. PMID:23849121

  20. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  1. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

    Science.gov (United States)

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

  2. Ferric Citrate Controls Phosphorus and Delivers Iron in Patients on Dialysis

    OpenAIRE

    Lewis, Julia B.; Sika, Mohammed; Koury, Mark J.; Chuang, Peale; Schulman, Gerald; Smith, Mark T.; Whittier, Frederick C.; Linfert, Douglas R.; Galphin, Claude M.; Athreya, Balaji P.; Nossuli, A. Kaldun Kaldun; Chang, Ingrid J.; Blumenthal, Samuel S.; Manley, John; Zeig, Steven

    2014-01-01

    Patients on dialysis require phosphorus binders to prevent hyperphosphatemia and are iron deficient. We studied ferric citrate as a phosphorus binder and iron source. In this sequential, randomized trial, 441 subjects on dialysis were randomized to ferric citrate or active control in a 52-week active control period followed by a 4-week placebo control period, in which subjects on ferric citrate who completed the active control period were rerandomized to ferric citrate or placebo. The primary...

  3. Sodium bicarbonate and sodium citrate: ergogenic aids?

    Science.gov (United States)

    Requena, Bernardo; Zabala, Mikel; Padial, Paulino; Feriche, Belén

    2005-02-01

    Numerous studies have used exogenous administration of sodium bicarbonate (NaHCO(3)) and sodium citrate (Na-citrate) in an attempt to enhance human performance. After ingestion of NaHCO(3) and Na-citrate, two observations have been made: (a) There was great individual variability in the ergogenic benefit reached, which can be attributed to the level of physical conditioning of the subjects and to their tolerance of the buffer substance; and (b) the subjects who had ingested NaHCO(3) and Na-citrate show higher levels of pH, bicarbonate, and lactate ions concentrations in their exercising blood than do the subjects who had ingested the placebo. A majority of the studies have suggested that the ingestion of both substances provides an ergogenic effect due to the establishment and maintenance of an elevated pH level during exercise. However, the exact mechanism by which the ergogenic effects occur has not been demonstrated conclusively. Sodium bicarbonate and Na-citrate seem to be effective in activities with a sufficient duration to generate a difference in the hydrogen ion gradient, characterized by a very high intensity and involving large muscular groups. However, in activities of equally high intensity, but with longer duration, the results obtained have been conflicting and inconclusive. PMID:15705037

  4. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

    Institute of Scientific and Technical Information of China (English)

    Xin Zhang; Kun Yan Zhu

    2013-01-01

    Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control.However,our understanding of biochemical properties of insect CHSs has been very limited.We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito,Anopheles gambiae.Our study,which represents the first time to use a nonradioactive method to assay CHS activity in an insect species,determined the optimal conditions for measuring the enzyme activity,including pH,temperature,and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine (UDPGlcNAc) and Mg++.The optimal pH was about 6.5-7.0,and the highest activity was detected at temperatures between 37℃ and 44℃.Dithithreitol is required to prevent melanization of the enzyme extract.CHS activity was enhanced at low concentration of GlcNAc,but inhibited at high concentrations.Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet.Our study revealed only slight in vitro inhibition ofA.gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5μmol/L) examined.There was no in vitro inhibition by polyoxin D at any concentration examined.Furthermore,we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined.Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A.gambiae.

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

    Science.gov (United States)

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

    2002-06-01

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

  6. Structural basis for the extended substrate spectrum of AmpC BER and structure-guided discovery of the inhibition activity of citrate against the class C β-lactamases AmpC BER and CMY-10.

    Science.gov (United States)

    Na, Jung Hyun; Cha, Sun Shin

    2016-08-01

    AmpC BER is an extended substrate spectrum class C β-lactamase with a two-amino-acid insertion in the R2 loop compared with AmpC EC2. The crystal structures of AmpC BER (S64A mutant) and AmpC EC2 were determined. Structural comparison of the two proteins revealed that the insertion increases the conformational flexibility of the R2 loop. Two citrate molecules originating from the crystallization solution were observed in the active site of the S64A mutant. One citrate molecule makes extensive interactions with active-site residues that are highly conserved among class C β-lactamases, whereas the other one is weakly bound. Based on this structural observation, it is demonstrated that citrate, a primary metabolite that is widely used as a food additive, is a competitive inhibitor of two class C β-lactamases (AmpC BER and CMY-10). Consequently, the data indicate enhancement of the flexibility of the R2 loop as an operative strategy for molecular evolution of extended-spectrum class C β-lactamases, and also suggest that the citrate scaffold is recognized by the active sites of class C β-lactamases. PMID:27487828

  7. Antioxidant and nitric oxide synthase activation properties of water soluble polysaccharides from Pleurotus florida

    Directory of Open Access Journals (Sweden)

    Subarna Saha

    2013-01-01

    Full Text Available Context: Cellular damage caused by reactive oxygen species has been implicated in several diseases, and, at the same time, nitric oxide is recognized as an important messenger molecule for several pathophysiological conditions. Hence, a novel antioxidant and nitric oxide synthase (NOS activator from natural sources have significant importance in human health. Aims: The present study was conducted to evaluate the free radical-scavenging activity and NOS activation properties of water-soluble crude polysaccharide (Floridan from Pleurotus florida. Materials and Methods: Crude polysaccharide was precipitated from hot water extract of P. florida, and their physicochemical parameters were determined. Then, α and β glucan were estimated using mushroom and yeast β glucan assay kit and Fourier transform infrared spectroscopy (FT-IR. Floridan was analyzed for their free radical scavenging activity in different test systems, namely hydroxyl and superoxide radical scavenging activity, ferrous ion chelating ability, determination of reducing power and inhibition of lipid peroxidation. Floridan was also tested for NOS activation using oxyhaemoglobin method. Statistical Analysis: The results were statistically analyzed using the Student′s t-test. Results: Results showed that Floridan was rich in water-soluble β glucan with very low amount of protein and phenols. The EC 50 for hydroxyl and superoxide radical-scavenging activity were 140 and 320 μg/ml, respectively, 450 μg/ml for chelating ability, 300 μg/ml for inhibition of lipid peroxidation and 2 mg/ml for reducing power. Floridan also increased nitric oxide production significantly. Conclusions: The present results revealed that this mushroom polysaccharide may be utilized as a promising dietary supplement to combat several killer diseases.

  8. Sleep-active neuronal nitric oxide synthase-positive cells of the cerebral cortex: a local regulator of sleep?

    OpenAIRE

    Wisor, Jonathan P.; Gerashchenko, Dmitry; Kilduff, Thomas S.

    2011-01-01

    Our recent report demonstrated that a small subset of GABAergic interneurons in the cerebral cortex of rodents expresses Fos protein, a marker for neuronal activity, during slow wave sleep (Gerashchenko et al., 2008). The population of sleep-active neurons consists of strongly immunohistochemically-stained cells for the enzyme neuronal nitric oxide synthase. By virtue of their widespread localization within the cerebral cortex and their widespread projections to other cortical cell types, cor...

  9. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    OpenAIRE

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-01-01

    Catechol oxidases and tyrosinases belong to the family of polyphenol oxidases (PPOs). In contrast to tyrosinases, catechol oxidases were so far defined to lack hydroxylase activity toward monophenols. Aurone synthase (AUS1) is a plant catechol oxidase that specializes in the conversion of chalcones to aurones (flower pigments). We evidence for the first time, to our knowledge, hydroxylase activity for a catechol oxidase (AUS1) toward its natural monophenolic substrate (chalcone). The presente...

  10. Mechanism of influence of phosphorylation on serine 124 on a decrease of catalytic activity of human thymidylate synthase

    OpenAIRE

    Jarmuła, Adam; Frączyk, Tomasz; Cieplak, Piotr; Rode, Wojciech

    2010-01-01

    Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine r...

  11. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  12. 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 [32P]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 [32P]P/sub i/, 32P 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

  13. Vibrational study of tamoxifen citrate polymorphism

    Science.gov (United States)

    Gamberini, M. C.; Baraldi, C.; Tinti, A.; Palazzoli, F.; Ferioli, V.

    2007-09-01

    The trans isomer of ( Z)-2-[ p-(1,2-diphenyl-butenyl)phenoxy]- N, N-dimethyletylamine (tamoxifen) is well known for its endocrine activity as an antiestrogenic agent. Its citrate salt, a widely used pharmaceutical agent, appears in three main polymorphic forms, two of which are well known (I and II) and another form not yet well evidenced. A vibrational study has been conducted for identifying the two known polymorphic forms of tamoxifen citrate (I and II) and for characterising the other form (form III) examined in this study. Other techniques for the characterization of the different polymorphs, such as XRDP, have been used.

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    1995-10-01

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

  16. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

    Science.gov (United States)

    Pinkosky, Stephen L; Filippov, Sergey; Srivastava, Rai Ajit K; Hanselman, Jeffrey C; Bradshaw, Cheryl D; Hurley, Timothy R; Cramer, Clay T; Spahr, Mark A; Brant, Ashley F; Houghton, Jacob L; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

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

    Directory of Open Access Journals (Sweden)

    Fairman Robert

    2004-11-01

    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

  18. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  19. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Directory of Open Access Journals (Sweden)

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  20. Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms.

    Science.gov (United States)

    Foresi, Noelia; Correa-Aragunde, Natalia; Santolini, Jerome; Lamattina, Lorenzo

    2016-01-01

    Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results. PMID:27094418

  1. Rational Improvement of Simvastatin Synthase Solubility in Escherichia coli Leads to Higher Whole-cell Biocatalytic Activity

    OpenAIRE

    Xie, Xinkai; Pashkov, Inna; Gao, Xue; Guerrero, Jennifer L.; Yeates, Todd O; Tang, Yi

    2009-01-01

    Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility o...

  2. Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

    OpenAIRE

    Yan Zhong-Qun; Li Ying-Hua; Brauner Annelie; Tullus Kjell

    2002-01-01

    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 ...

  3. 99mTc-Citrate versus 67Ga-citrate for the scintigraphic visualization of inflammatory lesions

    International Nuclear Information System (INIS)

    Citric acid was labeled with 99mTc with an efficiency of > 99%. The biodistribution of 99mTc-citrate was studied in mice with turpentine-induced abscesses in comparison to 67Ga-citrate. The max. abscess/muscle concentration ratios were 4.61 ± 1.92 (3 h) for 99mTc-citrate and 4.76 ± 2.04 (4 h) for 67Ga-citrate. Arthritis was induced in 10 rabbits by intra-articular injection of ovalbumin Scintigrams obtained 4 days later and at 3 h post-injection of 99mTc-citrate showed increased activity involving the synovium. The max. arthritic/contralateral knee ratio was 3.19 ± 1.29 (3 h) and 6.47 ± 3.71 (24 h) for 99mTc- and 67Ga-citrate, respectively. The blood clearance curve of 99mTc-citrate in rabbits was biexponential with a fast and a slow component, compared to mono-exponential clearance of 67Ga-citrate. In 10 patients with rheumatoid arthritis whole-body scintigrams and spot images of involved joints indicated localization of the tracer in inflamed tissues. The mean target-to-soft tissue ratios were 3.04 ± 0.81 and 4.95 ± 2.56 for 99mTc-citrate and 99mTc-MDP, respectively. Renal clearance of radioactivity was evident from the scintigrams. Our results demonstrated that 99mTc-citrate is effective as a radiopharmaceutical for the visualization of inflammatory lesions and may be preferred to 67Ga-citrate due to the ideal physical characteristics of the radionuclide, easy preparation, low cost, early accumulation and the preference for the renal route of excretion. (Author)

  4. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase.

    Science.gov (United States)

    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng

    2016-01-01

    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved

  5. Sleep active cortical neurons expressing neuronal nitric oxide synthase are active after both acute sleep deprivation and chronic sleep restriction.

    Science.gov (United States)

    Zielinski, M R; Kim, Y; Karpova, S A; Winston, S; McCarley, R W; Strecker, R E; Gerashchenko, D

    2013-09-01

    Non-rapid eye movement (NREM) sleep electroencephalographic (EEG) delta power (~0.5-4 Hz), also known as slow wave activity (SWA), is typically enhanced after acute sleep deprivation (SD) but not after chronic sleep restriction (CSR). Recently, sleep-active cortical neurons expressing neuronal nitric oxide synthase (nNOS) were identified and associated with enhanced SWA after short acute bouts of SD (i.e., 6h). However, the relationship between cortical nNOS neuronal activity and SWA during CSR is unknown. We compared the activity of cortical neurons expressing nNOS (via c-Fos and nNOS immuno-reactivity, respectively) and sleep in rats in three conditions: (1) after 18-h of acute SD; (2) after five consecutive days of sleep restriction (SR) (18-h SD per day with 6h ad libitum sleep opportunity per day); (3) and time-of-day matched ad libitum sleep controls. Cortical nNOS neuronal activity was enhanced during sleep after both 18-h SD and 5 days of SR treatments compared to control treatments. SWA and NREM sleep delta energy (the product of NREM sleep duration and SWA) were positively correlated with enhanced cortical nNOS neuronal activity after 18-h SD but not 5days of SR. That neurons expressing nNOS were active after longer amounts of acute SD (18h vs. 6h reported in the literature) and were correlated with SWA further suggest that these cells might regulate SWA. However, since these neurons were active after CSR when SWA was not enhanced, these findings suggest that mechanisms downstream of their activation are altered during CSR. PMID:23685166

  6. NITRIC OXIDE SYNTHASE ACTIVITY AND PEROXYNITRITE CONTENT IN CELLS OF RAT’S MUCOUS COAT OF STOMACH UNDER EXPERIMENTAL STRESS-INDUCED ULCER

    OpenAIRE

    I. S. Maksymovych; M. V. Mylenko; O. V. Drobinska; L.I. Ostapchenko

    2009-01-01

    Nitric oxide synthase activity in gastric mucosal cells as well as peroxynitrite generation in experimental stress-induced gastric ulcer formation in rats were studied. There was made the conclusion that nitric oxide synthase activity is growing during the stress. It causes increasing of peroxynitrite’ production and ulcer formation. It was established that destructive gastric mucosal damages depend on stress’ duration. It was determined that nitric oxide is involved in complex pathogenetic m...

  7. Cryptococcus neoformans Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins

    OpenAIRE

    Maligie, Marybeth A.; Selitrennikoff, Claude P.

    2005-01-01

    (1,3)β-d-Glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-β-d-glucan 3-β-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-β-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)β-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM M...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...... 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...... 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...

  9. Iridoid synthase activity is common among the plant progesterone 5β-reductase family.

    Science.gov (United States)

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2015-01-01

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterization of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2, and CrP5βR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5βR proteins and has evolved early during evolution. PMID:25578278

  10. Interventional effect of magnesium sulfate on nitric oxide synthase activity after acute craniocerebral injury

    Institute of Scientific and Technical Information of China (English)

    Ximin Yang; Jiangong Zhu; Zongchun Tang

    2007-01-01

    BACKGROUND: Abnormal changes in magnesium ion are closely related to cerebral injury. At present,some evidence indicates that magnesium reagent can improve nerve function and prognosis of patients with cerebral injury.OBJECTIVE: To observe the effect of magnesium sulfate on changes in nitric oxide synthase (NOS)activity in brain tissue of rats with acute craniocerebral injury.DESIGN: Completely randomized grouping design and randomly controlled study.SETTING: Laboratory of Neurosurgery, the Third Hospital of Chinese PLA.MATERIALS: Fifty-four male SD rats of clean grade and weighing 220 - 250 g were randomly divided into normal control group (n =6), cerebral injury group (n =24) and magnesium sulfate group (n =24). Especially,rats in cerebral injury group and magnesium sulfate group were equally divided into four subgroups and observed at 0.5, 2, 6 and 24 hours after model establishment. A solution of 125 g/L of magnesium sulfate was provided by the Seventh Pharmaceutical Factory of Wuxi and the NOS assay kit by Nanjing Jiancheng Bioengineering Institute.METHODS: The experiment was carried out in the Institute of Neurosurgery, the Third Hospital of Chinese PLA from August 2000 to August 2002. ① Rats in the cerebral injury group and magnesium sulfate group were anesthetized to establish cerebral injury models based on modified Feeney technique; magnesium sulfate group were intraperitoneally injected 600 mg/kg magnesium sulfate (125 g/L), but rats in the normal control group remained untreated. ② At 0.5, 2, 6 and 24 hours after cerebral injury, rats in cerebral injury group and magnesium sulfate group were decapitated and brains were dissected. Cerebral cortex of rats in cerebral injury group was selected for NOS assay; in addition, at 0.5 hour after cerebral injury, a portion of the parietal lobe was selected from the brains of rats in the normal control group. Brain samples were homogenized, the homogenated centrifuged and the supernatants were used to measure

  11. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. PMID:26592143

  12. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

    Science.gov (United States)

    Lanone, Sophie; Manivet, Philippe; Callebert, Jacques; Launay, Jean-Marie; Payen, Didier; Aubier, Michel; Boczkowski, Jorge; Mebazaa, Alexandre

    2002-01-01

    Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity. PMID:12097137

  13. Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis.

    Science.gov (United States)

    Polen, Tino; Schluesener, Daniela; Poetsch, Ansgar; Bott, Michael; Wendisch, Volker F

    2007-08-01

    Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate. PMID:17559405

  14. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase.

    Directory of Open Access Journals (Sweden)

    Yixiu Zhao

    Full Text Available Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO and the activity of endothelial nitric oxide synthase (eNOS in human aortic endothelial cells (HAECs were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation

  15. Adenomatous polyposis coli (APC) regulates multiple signaling pathways by enhancing glycogen synthase kinase-3 (GSK-3) activity.

    Science.gov (United States)

    Valvezan, Alexander J; Zhang, Fang; Diehl, J Alan; Klein, Peter S

    2012-02-01

    Glycogen synthase kinase-3 (GSK-3) is essential for many signaling pathways and cellular processes. As Adenomatous Polyposis Coli (APC) functions in many of the same processes, we investigated a role for APC in the regulation of GSK-3-dependent signaling. We find that APC directly enhances GSK-3 activity. Furthermore, knockdown of APC mimics inhibition of GSK-3 by reducing phosphorylation of glycogen synthase and by activating mTOR, revealing novel roles for APC in the regulation of these enzymes. Wnt signaling inhibits GSK-3 through an unknown mechanism, and this results in both stabilization of β-catenin and activation of mTOR. We therefore hypothesized that Wnts may regulate GSK-3 by disrupting the interaction between APC and the Axin-GSK-3 complex. We find that Wnts rapidly induce APC dissociation from Axin, correlating with β-catenin stabilization. Furthermore, Axin interaction with the Wnt co-receptor LRP6 causes APC dissociation from Axin. We propose that APC regulates multiple signaling pathways by enhancing GSK-3 activity, and that Wnts induce APC dissociation from Axin to reduce GSK-3 activity and activate downstream signaling. APC regulation of GSK-3 also provides a novel mechanism for Wnt regulation of multiple downstream effectors, including β-catenin and mTOR. PMID:22184111

  16. Role of Ga-67 citrate imaging in pancreatitis

    Energy Technology Data Exchange (ETDEWEB)

    Aburano, T.; Yokoyama, K.; Hisada, K.; Kakuma, K.; Ichiyanagi, K.

    1988-11-01

    Two patients with pancreatitis in whom an area of predominant uptake of Ga-67 citrate was demonstrated involving the entire pancreas are presented. Ultrasound and x-ray CT did not reveal any morphologic abnormalities in the pancreas, whereas Ga-67 citrate imaging indicated the presence of active inflammatory change. Ga-67 citrate imaging may be useful in confirming the diagnosis of acute pancreatitis or acute exacerbation of chronic pancreatitis based on clinical and laboratory data, especially when ultrasound and/or x-ray CT cannot reveal any morphologic abnormalities in the pancreas.

  17. Ferric citrate controls phosphorus and delivers iron in patients on dialysis.

    Science.gov (United States)

    Lewis, Julia B; Sika, Mohammed; Koury, Mark J; Chuang, Peale; Schulman, Gerald; Smith, Mark T; Whittier, Frederick C; Linfert, Douglas R; Galphin, Claude M; Athreya, Balaji P; Nossuli, A Kaldun Kaldun; Chang, Ingrid J; Blumenthal, Samuel S; Manley, John; Zeig, Steven; Kant, Kotagal S; Olivero, Juan Jose; Greene, Tom; Dwyer, Jamie P

    2015-02-01

    Patients on dialysis require phosphorus binders to prevent hyperphosphatemia and are iron deficient. We studied ferric citrate as a phosphorus binder and iron source. In this sequential, randomized trial, 441 subjects on dialysis were randomized to ferric citrate or active control in a 52-week active control period followed by a 4-week placebo control period, in which subjects on ferric citrate who completed the active control period were rerandomized to ferric citrate or placebo. The primary analysis compared the mean change in phosphorus between ferric citrate and placebo during the placebo control period. A sequential gatekeeping strategy controlled study-wise type 1 error for serum ferritin, transferrin saturation, and intravenous iron and erythropoietin-stimulating agent usage as prespecified secondary outcomes in the active control period. Ferric citrate controlled phosphorus compared with placebo, with a mean treatment difference of -2.2±0.2 mg/dl (mean±SEM) (P<0.001). Active control period phosphorus was similar between ferric citrate and active control, with comparable safety profiles. Subjects on ferric citrate achieved higher mean iron parameters (ferritin=899±488 ng/ml [mean±SD]; transferrin saturation=39%±17%) versus subjects on active control (ferritin=628±367 ng/ml [mean±SD]; transferrin saturation=30%±12%; P<0.001 for both). Subjects on ferric citrate received less intravenous elemental iron (median=12.95 mg/wk ferric citrate; 26.88 mg/wk active control; P<0.001) and less erythropoietin-stimulating agent (median epoetin-equivalent units per week: 5306 units/wk ferric citrate; 6951 units/wk active control; P=0.04). Hemoglobin levels were statistically higher on ferric citrate. Thus, ferric citrate is an efficacious and safe phosphate binder that increases iron stores and reduces intravenous iron and erythropoietin-stimulating agent use while maintaining hemoglobin. PMID:25060056

  18. Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

    OpenAIRE

    Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, Wayne F; Koppisch, Andrew T.; Freel Meyers, Caren L.

    2013-01-01

    The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhi...

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

    OpenAIRE

    Fischer, Marc; Meyer, Sophie; Claudel, Patricia; Steyer, Damien; Bergdoll, Marc; Hugueney, Philippe

    2013-01-01

    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 structura...

  20. Production of technical-grade sodium citrate from glycerol-containing biodiesel waste by Yarrowia lipolytica.

    Science.gov (United States)

    Kamzolova, Svetlana V; Vinokurova, Natalia G; Lunina, Julia N; Zelenkova, Nina F; Morgunov, Igor G

    2015-10-01

    The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144 h, then citrate formation decreased presumably due to inhibition of enzymes involved in this process. In contrast, when the method of repeated batch cultivation was used, the formation of citrate continued for more than 500 h. In this case, the final concentration of citrate in the culture liquid reached 79-82 g/L. Trisodium citrate was isolated from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7-8. This simple and economic isolation procedure gave the yield of crude preparation containing trisodium citrate 5.5-hydrate up to 82-86%. PMID:26141285

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  2. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation.

    Science.gov (United States)

    Buller, Andrew R; Brinkmann-Chen, Sabine; Romney, David K; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H

    2015-11-24

    Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. PMID:26553994

  3. Citrate-Stabilized Gold Nanorods

    OpenAIRE

    Mehtala, Jonathan G; Zemlyanov, Dmitry Y.; Max, Joann P.; Kadasala, Naveen; Zhao, Shou; Wei, Alexander

    2014-01-01

    Stable aqueous dispersions of citrate-stabilized gold nanorods (cit-GNRs) have been prepared in scalable fashion by surfactant exchange from cetyltrimethylammonium bromide (CTAB)-stabilized GNRs, using polystyrenesulfonate (PSS) as a detergent. The surfactant exchange process was monitored by infrared spectroscopy, surface-enhanced Raman scattering (SERS), and X-ray photoelectron spectroscopy (XPS). The latter established the quantitative displacement of CTAB (by PSS) and of PSS (by citrate)....

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

    Science.gov (United States)

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

    1997-12-01

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

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

    OpenAIRE

    UEMURA, Satoshi; Yoshida, Sayaka; Shishido, Fumi; Inokuchi, Jin-ichi

    2009-01-01

    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...

  6. Loss of ceramide synthase 2 activity, necessary for myelin biosynthesis, precedes tau pathology in the cortical pathogenesis of Alzheimer's disease.

    Science.gov (United States)

    Couttas, Timothy A; Kain, Nupur; Suchowerska, Alexandra K; Quek, Lake-Ee; Turner, Nigel; Fath, Thomas; Garner, Brett; Don, Anthony S

    2016-07-01

    The anatomical progression of neurofibrillary tangle pathology throughout Alzheimer's disease (AD) pathogenesis runs inverse to the pattern of developmental myelination, with the disease preferentially affecting thinly myelinated regions. Myelin is comprised 80% of lipids, and the prototypical myelin lipids, galactosylceramide, and sulfatide are critical for neurological function. We observed severe depletion of galactosylceramide and sulfatide in AD brain tissue, which can be traced metabolically to the loss of their biosynthetic precursor, very long chain ceramide. The synthesis of very long chain ceramides is catalyzed by ceramide synthase 2 (CERS2). We demonstrate a significant reduction in CERS2 activity as early as Braak stage I/II in temporal cortex, and Braak stage III/IV in hippocampus and frontal cortex, indicating that loss of myelin-specific ceramide synthase activity precedes neurofibrillary tangle pathology in cortical regions. These findings open a new vista on AD pathogenesis by demonstrating a defect in myelin lipid biosynthesis at the preclinical stages of the disease. We posit that, over time, this defect contributes significantly to myelin deterioration, synaptic dysfunction, and neurological decline. PMID:27255818

  7. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    International Nuclear Information System (INIS)

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na6[TeW6O24] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  8. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Energy Technology Data Exchange (ETDEWEB)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  9. Anaphylaxis related to fentanyl citrate

    Directory of Open Access Journals (Sweden)

    Gaurav Singh Tomar

    2012-01-01

    Full Text Available Anaphylaxis is a fulminant, unexpected, immunoglobulin E-mediated allergic reaction that can be triggered by multiple agents. Common causative agents include neuromuscular blocking drugs, latex, antibiotics, colloids, hypnotics, and opioids. Fentanyl citrate, however, is an extremely unusual cause of anaphylaxis. Pulmonary edema, although uncommon in anaphylaxis, can be a prominent feature, as was in one of the patient. An adverse drug reaction is a noxious or unintended reaction to a drug that is administered in standard doses by the proper route for the purpose of prophylaxis, diagnosis, or treatment. Reactions are classified into two major subtypes: type A, which are dose dependent and predictable; and type B, which are not dose dependent and unpredictable. Unpredictable reactions include immune (allergic or no immune drug hypersensitivity reactions and are related to genetic susceptibilities or undefined mechanisms (formally called idiosyncratic and intolerance reactions. A drug allergy is always associated with an immune mechanism for which evidence of drug-specific antibodies or activated T lymphocytes can be shown. In the last few years, many novel drugs have entered clinical practice (i.e., biologic agents generating novel patterns of drug hypersensitivity reactions. As old drugs continue to be used, new clinical and biologic techniques enable improvement in the diagnosis of these reactions.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Science.gov (United States)

    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

    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, Pendothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (Pdysfunction of aging and may therefore be a therapeutic target.

  12. Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues.

    Science.gov (United States)

    Xie, Xinqiang; Garg, Ashish; Keatinge-Clay, Adrian T; Khosla, Chaitan; Cane, David E

    2016-03-01

    The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-2) and 0.05 equiv of NADP(+) in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography-tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues. PMID:26863427

  13. 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 α 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+

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

    Science.gov (United States)

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

    1997-01-01

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

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

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E;

    2006-01-01

    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...... 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...

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

    Science.gov (United States)

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

    2016-08-15

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

  17. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    Science.gov (United States)

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  18. Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

    Institute of Scientific and Technical Information of China (English)

    Shengqiang Chen; Xuegang Luo; Quan Yang; Weiwen Sun; Kaiyi Cao; Xi Chen; Yueling Huang; Lijun Dai; Yonghong Yi

    2011-01-01

    In the present study, Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome. The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts, indicating a learning and memory disorder. After treatment with 30, 60, 90, 120, or 200 mg/kg lithium chloride, the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated, in particular, the 200 mg/kg lithium chloride treatment had the most significant effect. Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta, an inactive form of glycogen synthase kinase 3 beta, in the cerebral cortex and hippocampus of the Fmr1 KO mice. These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice, possibly by inhibiting glycogen synthase kinase 3 beta activity.

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

    Directory of Open Access Journals (Sweden)

    Hildgund Schrempf

    2010-09-01

    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.

  20. Metabotropic glutamate receptor 5, but not 1, modulates NMDA receptor-mediated activation of neuronal nitric oxide synthase.

    Science.gov (United States)

    Llansola, Marta; Felipo, Vicente

    2010-03-01

    In cerebellar neurons in culture, activation of group I metabotropic glutamate receptors (mGluRs) prevents glutamate and NMDA-induced neuronal death, indicating that it interferes with the excitotoxic mechanisms leading to death. However, it is not known which step of these mechanisms is affected by mGluRs. The aims of this work were to assess: (a) whether activation of group I mGluRs (mGluR1 or mGluR5) impairs NMDA-induced activation of the glutamate-nitric oxide-cGMP pathway; (b) which mGluR (1 or 5) is responsible for this impairment and (c) whether impairment of the pathway occurs at the level of activation of soluble guanylate cyclase by nitric oxide or of activation of neuronal nitric oxide synthase (nNOS) by NMDA. It is shown that activation of mGluR1 enhances the function of the glutamate-nitric oxide-cGMP pathway by increasing activation of soluble guanylate cyclase by nitric oxide. In contrast, mGluR5 activation inhibits the glutamate-nitric oxide-cGMP pathway by reducing NMDA-induced activation of nNOS. This is due to reduced NMDA-induced increase in cAMP, reduced activation of Akt by cAMP and of nNOS by Akt. The impairment of activation of the glutamate-NO-cGMP pathway by activation of mGluR5 would contribute to its neuroprotective effect against excitotoxicity in cerebellar neurons in culture. PMID:20043967

  1. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2+2a) phosphorylation

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Birk, Jesper Bratz; Richter, Erik; Ribel-Madsen, Rasmus; Pehmøller, Christian; Hansen, Bo Falck; Beck-Nielsen, Henning; Hirshman, Michael F; Goodyear, Laurie J; Vaag, Allan; Poulsen, Pernille; Wojtaszewski, Jørgen

    2013-01-01

    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. We previously demonstrated that phosphorylation of Threonine-308 on Akt (pAkt-...

  2. Transcriptional activation of the parsley chalcone synthase promoter in heterologous pea and yeast systems.

    Science.gov (United States)

    Kalbin; Strid; Frohnmeyer

    1999-11-01

    Introduction by electroporation of different parsley (Petroselinum crispum) CHS-promoter/beta-glucuronidase(GUS)-reporter constructs into pea (Pisum sativum L.) protoplasts leads to a high constitutive GUS-expression and to the loss of the light-inducibility seen in the homologous parsley protoplast system. These results indicate that Unit 1 of the parsley CHS-promoter is only partly responsible for the GUS-expression detected. Instead, additional cis-elements, which are located downstream within 100 bp from the transcriptional start site, mediate the de-repression in pea protoplasts. In contrast, in yeast (Saccharomyces cerevisiae) cells, the GUS expression from the heterologous CHS/GUS construct is controlled by elements between Unit 1 and -100 bp. In both pea and yeast cells, transcription factors different from those regulating UV-responsiveness in parsley, are probably mediating the constitutive expression from the heterologous construct. The results with pea protoplasts imply that protoplastation of pea leaf cells itself induces de-repression as a result of stress to the protoplasts. This notion was strengthened by the finding that mRNA levels of the endogenous chalcone synthase were drastically increased as the result of the protoplastation procedure. PMID:10580282

  3. 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 μg/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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

    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.

  5. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    Science.gov (United States)

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  6. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Iva Bozic

    2015-01-01

    Full Text Available Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

  7. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Science.gov (United States)

    Bozic, Iva; Savic, Danijela; Jovanovic, Marija; Bjelobaba, Ivana; Laketa, Danijela; Nedeljkovic, Nadezda; Stojiljkovic, Mirjana; Pekovic, Sanja; Lavrnja, Irena

    2015-01-01

    Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation. PMID:26413464

  8. Plants defective for soluble starch synthase IV (SSIV) activity, methods for obtaining the same, ans uses thereof

    OpenAIRE

    Planchot, Veronique; Mérida, Ángel; d'Hulst, Christophe; Roldán, Isaac; Wattebled, Fabrice; Delvallé, David; Lucas, M. Mercedes

    2008-01-01

    The use of a plant which is modified so as to be rendered defective for Soluble Starch Synthase IV (SSIV), for obtaining starch granules having an increased granule size and a similar starch amylose content, as compared to the same plant that is not defective for Soluble Starch Synthase IV (SSIV).

  9. 21 CFR 582.5449 - Manganese citrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Manganese citrate. 582.5449 Section 582.5449 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5449 Manganese citrate. (a) Product. Manganese citrate. (b) Conditions of use....

  10. 21 CFR 184.1449 - Manganese citrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Manganese citrate. 184.1449 Section 184.1449 Food... Specific Substances Affirmed as GRAS § 184.1449 Manganese citrate. (a) Manganese citrate (Mn3(C6H5O7)2, CAS... manganese carbonate from manganese sulfate and sodium carbonate solutions. The filtered and...

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

    OpenAIRE

    Ehsan Karimi; Jaafar, Hawa Z. E.; Ali Ghasemzadeh

    2012-01-01

    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 t...

  12. Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture.

    Science.gov (United States)

    Kumari, Indu; Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2016-06-01

    Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices. PMID:26207800

  13. A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.

    Science.gov (United States)

    Huang, Xinyi; Hernick, Marcy

    2011-10-15

    Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors. PMID:21729692

  14. Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei.

    Science.gov (United States)

    Vega, Claudia; Moreno-Carranza, Bibiana; Zamorano, Miriam; Quintanar-Stéphano, Andrés; Méndez, Isabel; Thebault, Stéphanie; Martínez de la Escalera, Gonzalo; Clapp, Carmen

    2010-12-01

    Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction. PMID:20943859

  15. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  16. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs; Nouveaux procedes pour mesurer les activites synthase et phosphatase de la bisphosphoglycerate mutase. Interet pour le developpement de drogues therapeutiques

    Energy Technology Data Exchange (ETDEWEB)

    Ravel, P.; Garel, M.C. [Hopital Henri-Mondor, 94 - Creteil (France); Toullec, D. [Laboratoire Glaxo Wellcome, 91- Les Ulis (France)

    1997-12-31

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  17. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  18. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    International Nuclear Information System (INIS)

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

  19. Electrodeposition of Sn-Zn-Cu alloys from citrate solutions

    International Nuclear Information System (INIS)

    The first stage of this study involves the development of stable baths for electrodepositing Sn-Zn-Cu alloys; these developments are based on thermodynamic models of citrate baths and experiments exploring the stability of the baths. The effects of the sodium citrate (complexation agent) concentration and the pH of the solution on the stability of the baths were examined experimentally. The stability of the baths was determined through spectrophotometric analysis. Stable baths designed for the electrodeposition of Sn-Zn-Cu alloys were produced in the range of dominant citrate complexes with highly negative charge (the reduction process is strongly inhibited by high activation energy). Voltammetric studies and potentiostatic deposition were conducted to analyse the co-deposition of tin, zinc, and copper, and the co-evolution of hydrogen. The effect of the solution pH, the concentration of sodium citrate and the hydrodynamic conditions on the electrodeposition process and the composition of the deposits were examined. The deposits were analysed using chemical (μXRF) as also grain size and phase analysis (X-ray diffraction). The possibility of electrodepositing Sn-Zn-Cu alloys from citrate solutions was confirmed. The tin content of these coatings varied from 60 to 96 wt.%, the copper content varied from 3.5 to 20.5 wt.%, and the zinc content reached up to approximately 37.5 wt.%

  20. Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs

    Directory of Open Access Journals (Sweden)

    Thông Hua-Huy

    2016-02-01

    Full Text Available Inhaled nitric oxide (iNO is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats. Rat pups, post-natal day (P 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group or 20 ppm (iNO-20 ppm group, or in room air (control group. Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity. At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7. Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO.

  1. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    Science.gov (United States)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

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

    Science.gov (United States)

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

    2002-01-01

    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

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

    Science.gov (United States)

    Volke, A; Wegener, G; Vasar, E; Volke, V

    2006-01-01

    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. PMID:16541190

  4. Synthesis of alkyl-ether glycerophospholipids in rat glomerular mesangial cells: evidence for alkyldihydroxyacetone phosphate synthase activity

    International Nuclear Information System (INIS)

    We studied the ability of rat glomerular mesangial cells and their microsomal fractions to incorporate 1-[14C]hexadecanol to glycerophospholipids via an O-alkyl ether linkage and assessed the presence and activity of the required enzyme: alkyl-dihydroxy acetone phosphate synthase. Suspensions of cultured mesangial cells incorporated 1-[14C]hexadecanol to the phosphatidyl ethanolamine and phosphatidyl choline lipid pools, via a bond resistant to acid and base hydrolysis. When cell homogenates or microsomal fractions were incubated with palmitoyl-DHAP and 1-[14C]hexadecanol, alkyl-DHAP and 1-O-alkyl glycerol were formed (alkyl:hexadecyl). The activity of the enzyme responsible for the O-alkyl product formation was calculated to be 2.5 +/- 0.3 and 544 +/- 50 pmoles/min/mg protein for mesangial cell homogenates and mesangial cell microsomes, respectively. These observations provide evidence that mesangial cells may elaborate either linked lipid precursors de novo for the biosynthesis of O-alkyl glycerophospholipids

  5. Atorvastatin enhance efficacy of mesenchymal stem cells treatment for swine myocardial infarction via activation of nitric oxide synthase.

    Directory of Open Access Journals (Sweden)

    Lei Song

    Full Text Available BACKGROUND: In a swine model of acute myocardial infarction (AMI, Statins can enhance the therapeutic efficacy of mesenchymal stem cell (MSCs transplantation. However, the mechanisms remain unclear. This study aims at assessing whether atorvastatin (Ator facilitates the effects of MSCs through activation of nitric oxide synthase (NOS, especially endothelial nitric oxide synthase (eNOS, which is known to protect against ischemic injury. METHODS AND RESULTS: 42 miniswines were randomized into six groups (n = 7/group: Sham operation; AMI control; Ator only; MSC only, Ator+MSCs and Ator+MSCs+NG-nitrol-L-arginine (L-NNA, an inhibitor of NOS. In an open-heart surgery, swine coronary artery ligation and reperfusion model were established, and autologous bone-marrow MSCs were injected intramyocardium. Four weeks after transplantation, compared with the control group, Ator+MSCs animals exhibited decreased defect areas of both "perfusion" defined by Single-Photon Emission Computed Tomography (-6.2±1.8% vs. 2.0±5.1%, P = 0.0001 and "metabolism" defined by Positron Emission Tomography (-3.00±1.41% vs. 4.20±4.09%, P = 0.0004; Ejection fraction by Magnetic Resonance Imaging increased substantially (14.22±12.8% vs. 1.64±2.64%, P = 0.019. In addition, indices of inflammation, fibrosis, and apoptosis were reduced and survivals of MSCs or MSC-derived cells were increased in Ator+MSCs animals. In Ator or MSCs alone group, perfusion, metabolism, inflammation, fibrosis or apoptosis were reduced but there were no benefits in terms of heart function and cell survival. Furthermore, the above benefits of Ator+MSCs treatment could be partially blocked by L-NNA. CONCLUSIONS: Atorvastatin facilitates survival of implanted MSCs, improves function and morphology of infarcted hearts, mediated by activation of eNOS and alleviated by NOS inhibitor. The data reveal the cellular and molecular mechanism for anti-AMI therapy with a combination of statin and

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

    Science.gov (United States)

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

    2014-12-01

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

  7. Mechanism of influence of phosphorylation on serine 124 on a decrease of catalytic activity of human thymidylate synthase.

    Science.gov (United States)

    Jarmuła, Adam; Fraczyk, Tomasz; Cieplak, Piotr; Rode, Wojciech

    2010-05-15

    Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the 'phosphorylation effect' is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125-134) and the patch (residues 189-192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF. PMID:20430630

  8. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Directory of Open Access Journals (Sweden)

    Micaela S Sordelli

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  9. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  10. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

    Directory of Open Access Journals (Sweden)

    Cathy W C Bi

    Full Text Available Fo Shou San (FSS is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong and Angelicae Sinensis Radix (ASR; Danggui in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs, and it also stimulated the production of nitric oxide (NO as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+ levels in HUVECs, and the Ca(2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+ and a reduction of ROS.

  11. Distinct parts of leukotriene C{sub 4} synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein

    Energy Technology Data Exchange (ETDEWEB)

    Strid, Tobias; Svartz, Jesper; Franck, Niclas; Hallin, Elisabeth; Ingelsson, Bjoern; Soederstroem, Mats [Division of Cell biology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-58185 Linkoeping (Sweden); Hammarstroem, Sven, E-mail: sven.hammarstrom@liu.se [Division of Cell biology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-58185 Linkoeping (Sweden)

    2009-04-17

    Leukotriene C{sub 4} is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C{sub 4} synthase (LTC{sub 4}S) participate in its biosynthesis. We report evidence that LTC{sub 4}S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC{sub 4}S. FLAP bound to the N-terminal part/first hydrophobic region of LTC{sub 4}S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC{sub 4}S. Fluorescent FLAP- and LTC{sub 4}S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC{sub 4}S 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 LTC{sub 4}S. Direct interaction of 5-LO and LTC{sub 4}S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC{sub 4}S.

  12. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    Science.gov (United States)

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  13. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    International Nuclear Information System (INIS)

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression

  14. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

    Science.gov (United States)

    Proudfoot, L; Nikolaev, A V; Feng, G J; Wei, W Q; Ferguson, M A; Brimacombe, J S; Liew, F Y

    1996-10-01

    Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages. PMID:8855295

  15. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  16. Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

    Science.gov (United States)

    Li, Ying-Hua; Yan, Zhong-Qun; Brauner, Annelie; Tullus, Kjell

    2002-01-01

    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-κB (NF-κB) 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-κB. Results We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (≥10 ng/ml) and time dependent manner (p < 0.05). This effect was further enhanced by IFN-γ (≥10 IU/ml, p < 0.05), but was attenuated by budesonide (10-4–10-10 M) and dexamethasone (10-4–10-6 M) (p < 0.05). The mRNA and protein levels of iNOS were also induced in response to LPS and attenuated by steroids. LPS triggered NF-κB activation, a mechanism responsible for the iNOS expression. 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. PMID:12323081

  17. Changes in Phytochemical Synthesis, Chalcone Synthase Activity and Pharmaceutical Qualities of Sabah Snake Grass (Clinacanthus nutans L.) in Relation to Plant Age

    OpenAIRE

    Ali Ghasemzadeh; Alireza Nasiri; Jaafar, Hawa Z. E.; Ali Baghdadi; Izham Ahmad

    2014-01-01

    In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old). The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74) was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF) production were observed between the three plant growth periods and the different plant parts. The highest conten...

  18. Inhibition of glycogen synthase kinase 3β promotes autophagy to protect mice from acute liver failure mediated by peroxisome proliferator-activated receptor α

    OpenAIRE

    Ren, F.; Zhang, L; Zhang, X; Shi, H; T. Wen; Bai, L.; S. Zheng; Y. Chen; Chen, D.; Li, L.; Duan, Z

    2016-01-01

    Our previous studies have demonstrated that inhibition of glycogen synthase kinase 3β (GSK3β) activity protects mice from acute liver failure (ALF), whereas its protective and regulatory mechanism remains elusive. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that inhibition of GSK3β mediates autophagy to inhibit liver inflammation and protect against ALF. In ALF mice model induced by d-gala...

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

    OpenAIRE

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

    1984-01-01

    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 ...

  20. Artificial citrate operon confers mineral phosphate solubilization ability to diverse fluorescent pseudomonads.

    Science.gov (United States)

    Adhikary, Hemanta; Sanghavi, Paulomi B; Macwan, Silviya R; Archana, Gattupalli; Naresh Kumar, G

    2014-01-01

    Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200-1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration. PMID:25259527

  1. Artificial citrate operon confers mineral phosphate solubilization ability to diverse fluorescent pseudomonads.

    Directory of Open Access Journals (Sweden)

    Hemanta Adhikary

    Full Text Available Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1 and citrate transporter (citC genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP buffered medium, which was sufficient to release 200-1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.

  2. Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum

    DEFF Research Database (Denmark)

    Sørensen, Jens Laurids; Hansen, Frederik Teilfeldt; Sondergaard, Teis Esben;

    2012-01-01

    Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster...

  3. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    Science.gov (United States)

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression. PMID:16651461

  4. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

    DEFF Research Database (Denmark)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet;

    2014-01-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five...... for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids. © 2014 John Wiley & Sons Ltd....

  5. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

    OpenAIRE

    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng

    2016-01-01

    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitr...

  6. Regulation of delta-aminolevulinate synthase activity during the development of oxidative stress.

    Science.gov (United States)

    Kaliman, P A; Barannik, T V

    1999-06-01

    Activities of rat liver delta-aminolevulinate synthetase (delta-ALAS), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH), GSH content in the liver, and the absorption spectrum of blood serum were investigated after CoCl2, HgCl2, or beta-adrenoblocker (propranolol) injection and after CoCl2 and propranolol co-administration. Inhibition of the activity of the key heme biosynthesis enzyme delta-ALAS was most pronounced and prolonged during the first hours after CoCl2 and CoCl2 plus propranolol injections; this was associated with accumulation of Co2+--protoporphyrin-containing products of hemolysis. Inhibition of delta-ALAS after propranolol injection is not mediated by hemolysis. A decrease in GSH content precedes the induction of heme biosynthesis only in the case of HgCl2 administration, and this was associated with inhibition of GR and G6PDH. The decreased GSH content during the first hours after injection of propranolol and co-administration of CoCl2 and propranolol was not followed by increase in delta-ALAS activity 24 h after the injection. The mechanisms of the increase in the free heme content in the liver during the early stages of oxidative stress and the regulation of the key heme biosynthesis enzyme are discussed. PMID:10395986

  7. Citrate pharmacokinetics and calcium levels during high-flux dialysis with regional citrate anticoagulation

    OpenAIRE

    Kozik-Jaromin, Justyna; Nier, Volker; Heemann, Uwe; Kreymann, Bernhard; Böhler, Joachim

    2009-01-01

    Background. Regional citrate anticoagulation is a very effective anticoagulation method for haemodialysis. However, it is not widely used, primarily due to the risk of hypocalcaemia. We studied citrate and calcium kinetics to better understand safety aspects of this anticoagulation method. Methods. During 15 haemodialysis treatments with a calcium-free dialysis solution, citrate was infused pre-dialyser and calcium was substituted post-dialyser. Systemic and extracorporeal citrate and calcium...

  8. Effects of chronic prenatal ethanol exposure on locomotor activity, and hippocampal weight, neurons, and nitric oxide synthase activity of the young postnatal guinea pig.

    Science.gov (United States)

    Gibson, M A; Butters, N S; Reynolds, J N; Brien, J F

    2000-01-01

    Decreased nitric oxide synthase (NOS)-catalyzed formation of NO from L-arginine may be involved in ethanol teratogenesis involving the hippocampus. This hypothesis was tested by determining the effects of chronic prenatal ethanol exposure on locomotor activity and on hippocampal weight, number of CA1 and CA3 pyramidal cells and dentate gyrus granule cells, and NOS activity of the postnatal guinea pig. Timed, pregnant guinea pigs received one of the following chronic oral regimens throughout gestation: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding, or water. At postnatal day (PD) 10, spontaneous locomotor activity was measured. At PD 12, histological analysis was performed on the hippocampal formation, in which hippocampal CA1 and CA3 pyramidal cells and dentate gyrus granule cells were counted; body, brain, and hippocampal weights were measured; and hippocampal NOS enzymatic activity was determined using a radiometric assay. Chronic prenatal ethanol exposure produced hyperactivity, decreased the brain and hippocampal weights with no change in body weight, decreased the number of hippocampal CA1 pyramidal cells by 25-30%, and had no effect on hippocampal NOS activity compared with the two control groups. These data, together with our previous findings in the fetal guinea pig, demonstrate that chronic prenatal ethanol exposure decreases hippocampal NOS activity in near-term fetal life that temporally precedes the selective loss of hippocampal CA1 pyramidal cells in postnatal life. PMID:10758347

  9. Dual regulation of muscle glycogen synthase during exercise by activation and compartmentalization

    DEFF Research Database (Denmark)

    Prats, Clara; Helge, Jørn W; Nordby, Pernille; Qvortrup, Klaus; Ploug, Thorkil; Dela, Flemming; Wojtaszewski, Jørgen

    2009-01-01

    dephosphorylation at sites 2+2a, 3a, and 3a + 3b. Furthermore, we report the existence of several glycogen metabolism regulatory mechanisms based on GS intracellular compartmentalization. After exhausting exercise, epinephrine-induced protein kinase A activation leads to GS site 1b phosphorylation targeting the...... 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....

  10. Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation.

    Directory of Open Access Journals (Sweden)

    Lisa A Ridnour

    Full Text Available Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1 has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

  11. Antifungal Activity of Salvia miltiorrhiza Against Candida albicans Is Associated with the Alteration of Membrane Permeability and (1,3)-β-D-Glucan Synthase Activity.

    Science.gov (United States)

    Lee, Heung-Shick; Kim, Younhee

    2016-03-28

    Candidiasis has posed a serious health risk to immunocompromised patients owing to the increase in resistant yeasts, and Candida albicans is the prominent pathogen of fungal infections. Therefore, there is a critical need for the discovery and characterization of novel antifungals to treat infections caused by C. albicans. In the present study, we report on the antifungal activity of the ethanol extract from Salvia miltiorrhiza against C. albicans and the possible mode of action against C. albicans. The increase in the membrane permeability was evidenced by changes in diphenylhexatriene binding and release of both 260-nm-absorbing intracellular materials and protein. In addition, inhibition of cell wall synthesis was demonstrated by the enhanced minimal inhibitory concentration in the presence of sorbitol and reduced (1,3)-β-D-glucan synthase activity. The above evidence supports the notion that S. miltiorrhiza has antifungal activity against C. albicans by the synergistic activity of targeting the cell membrane and cell wall. These findings indicate that S. miltiorrhiza displays effective activity against C. albicans in vitro and merits further investigation to treat C. albicansassociated infections. PMID:26699747

  12. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action.

    Science.gov (United States)

    Lauer, T; Preik, M; Rassaf, T; Strauer, B E; Deussen, A; Feelisch, M; Kelm, M

    2001-10-23

    The plasma level of NO(x), i.e., the sum of NO(2)- and NO(3)-, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO(x) concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO(2)- and NO(3)- concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO(2)- or NO(3)-. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1-10 microg/min) dose-dependently augmented venous NO(2)- levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO(2)-. Intraarterial infusion of NO(2)- had no effect on FBF. NOS inhibition (N(G)-monomethyl-l-arginine; 4-12 micromol/min) dose-dependently reduced basal NO(2)- and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO(3)- and total NO(x) remained unchanged as did systemic arterial NO(2)- and NO(3)- levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P plasma NO(3)- and/or total NO(x) are generally useful markers of endogenous NO production and demonstrate that only NO(2)- reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive. PMID:11606734

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

    Science.gov (United States)

    Mourad, G; King, J

    1992-11-01

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

  14. Effects of aspirin on number,activity and inducible nitric oxide synthase of endothelial progenitor cells from peripheral blood

    Institute of Scientific and Technical Information of China (English)

    Tu-gang CHEN; Jun-zhu CHEN; Xu-dong XIE

    2006-01-01

    Aim:To investigate whether aspirin has an influence on endothelial progenitor cells (EPC).Methods:Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation,then cells were plated on fibronectin-coated culture dishes.After 7 d of culture,attached cells were stimulated with aspirin (to achieve final concentrations of 1,2,5,and 10 mmol/L) for 3,6,12,and 24 h.EPC were characterized as adherent cells that were double positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine low density lipoprotein (DiLDL) uptake and lectin binding by direct fluorescent staining.EPC proliferation and migration were assayed using a 3- (4,5-dimethyl-2 thiazoyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay.respectively.An EPC adhesion assay was performed by replating the EPC on fibronectin-coated dishes,and then adherent cells were counted.In vitro vasculogenesis activity was assayed by using an in vitro vasculogenesis kit. Inducible nitric oxide synthase (iNOS) was assayed by Westem blotting.Results:Incubation of isolated human MNC with aspirin decreased the number of EPC.Aspirin also decreased the proliferative,migratory,adhesive,and in vitro Vasculogenesis capacity of EPC,and also their iNOS levels in a concentration-and time-dependent manner.Conclusion:Aspirin decreases (1) the number of EPC; (2) the proliferative,migratory,adhesive and in vitro vasculogenesis capacities of EPC;and (3) iNOS levels in EPC.

  15. Impairments in cognition and neural precursor cell proliferation in mice expressing constitutively active glycogen synthase kinase-3

    Directory of Open Access Journals (Sweden)

    Marta ePardo

    2015-03-01

    Full Text Available ABSTRACTBrain glycogen synthase kinase-3 (GSK3 is hyperactive in several neurological conditions that involve impairments in both cognition and neurogenesis. This raises the hypotheses that hyperactive GSK3 may directly contribute to impaired cognition, and that this may be related to deficiencies in neural precursor cells (NPC. To study the effects of hyperactive GSK3 in the absence of disease influences, we compared adult hippocampal NPC proliferation and performance in three cognitive tasks in male and female wild-type mice and GSK3 knockin mice, which express constitutively active GSK3. NPC proliferation was ~40% deficient in both male and female GSK3 knockin mice compared with wild-type mice. Environmental enrichment (EE increased NPC proliferation in male, but not female, GSK3 knockin mice and wild-type mice. Male and female GSK3 knockin mice exhibited impairments in novel object recognition, temporal order memory, and coordinate spatial processing compared with gender-matched wild-type mice. EE restored impaired novel object recognition and temporal ordering in both sexes of GSK3 knockin mice, indicating that this repair was not dependent on NPC proliferation, which was not increased by EE in female GSK3 knockin mice. Acute 1 hr pretreatment with the GSK3 inhibitor TDZD-8 also improved novel object recognition and temporal ordering in male and female GSK3 knockin mice. These findings demonstrate that hyperactive GSK3 is sufficient to impair adult hippocampal NPC proliferation and to impair performance in three cognitive tasks in both male and female mice, but these changes in NPC proliferation do not directly regulate novel object recognition and temporal ordering tasks.

  16. 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.

  17. The time-dependent effect of provinolsTM on brain NO synthase activity in L-NAME-induced hypertension

    Czech Academy of Sciences Publication Activity Database

    Jendeková, L.; Kojšová, S.; Andriantsitohaina, R.; Pecháňová, Olga

    2006-01-01

    Roč. 55, č. S1 (2006), S31-S37. ISSN 0862-8408 Grant ostatní: VEGA(SK) 2/6148/26; VEGA(SK) 1/342906 Institutional research plan: CEZ:AV0Z50110509 Keywords : red wine polyphenols * oxidative damage * nitric oxide synthase Subject RIV: ED - Physiology Impact factor: 2.093, year: 2006

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

    Directory of Open Access Journals (Sweden)

    Seyffarth Gunter

    2004-06-01

    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.

  19. 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)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB 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.

  20. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism[S

    Science.gov (United States)

    Pinkosky, Stephen L.; Filippov, Sergey; Srivastava, Rai Ajit K.; Hanselman, Jeffrey C.; Bradshaw, Cheryl D.; Hurley, Timothy R.; Cramer, Clay T.; Spahr, Mark A.; Brant, Ashley F.; Houghton, Jacob L.; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S.

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  1. Glutamate-induced activation of nitric oxide synthase is impaired in cerebral cortex in vivo in rats with chronic liver failure.

    Science.gov (United States)

    Rodrigo, Regina; Erceg, Slaven; Rodriguez-Diaz, Jesus; Saez-Valero, Javier; Piedrafita, Blanca; Suarez, Isabel; Felipo, Vicente

    2007-07-01

    It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy. PMID:17286583

  2. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

    OpenAIRE

    Proudfoot, L; Nikolaev, A. V.; Feng, G.J.; Wei, W Q; Ferguson, M A; Brimacombe, J S; Liew, F. Y.

    1996-01-01

    Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant red...

  3. Impairments in cognition and neural precursor cell proliferation in mice expressing constitutively active glycogen synthase kinase-3

    OpenAIRE

    Marta ePardo; King, Margaret K.; EMMA ePEREZ-COSTAS; Miguel eMelendez-Ferro; Ana eMartinez; Eleonore eBeurel; Richard Scott Jope

    2015-01-01

    ABSTRACTBrain glycogen synthase kinase-3 (GSK3) is hyperactive in several neurological conditions that involve impairments in both cognition and neurogenesis. This raises the hypotheses that hyperactive GSK3 may directly contribute to impaired cognition, and that this may be related to deficiencies in neural precursor cells (NPC). To study the effects of hyperactive GSK3 in the absence of disease influences, we compared adult hippocampal NPC proliferation and performance in three cognitive ta...

  4. Bisphosphonate Inhibition of a Plasmodium Farnesyl Diphosphate Synthase and a General Method for Predicting Cell-Based Activity from Enzyme Data

    OpenAIRE

    Mukkamala, Dushyant; No, Joo Hwan; Cass, Lauren M.; Chang, Ting-Kai; Oldfield, Eric

    2008-01-01

    We screened 26 bisphosphonates against a farnesyl diphosphate synthase from Plasmodium vivax, finding a poor correlation between enzyme and cell growth inhibition (R2 = 0.06). To better predict cell activity data, we then used a combinatorial descriptor search in which pIC50(cell) = a pIC50(enzyme) + bB + cC + d, where B and C are descriptors (such as SlogP), and a—d are coefficients. R2 increased from 0.01 to 0.74 (for a leave-two-out test set of 26 predictions). The method was then further ...

  5. Dinuclear nickel complexes modeling the structure and function of the acetyl CoA synthase active site

    OpenAIRE

    Ito, Mikinao; Kotera, Mai; Matsumoto, Tsuyoshi; Tatsumi, Kazuyuki

    2009-01-01

    A dinuclear nickel complex with methyl and thiolate ligands, Ni(dadtEt)Ni(Me)(SDmp) (2), has been synthesized as a dinuclear Nid–Nip-site model of acetyl-CoA synthase (ACS) (dadtEt is N,N′-diethyl-3,7-diazanonane-1,9-dithiolate; Dmp is 2,6-dimesitylphenyl). Complex 2 was prepared via 2 methods: (i) ligand substitution of a dinuclear Ni(II)–Ni(II) cation complex [Ni(dadtEt) Ni(tmtu)2] (OTf)2(1) with MeMgBr and KSDmp (tmtu is tetramethylthiourea), (ii) methyl transfer from methylcobaloxime Co(d...

  6. Mean platelet volume measurement, EDTA or citrate?

    Science.gov (United States)

    Dastjerdi, Mansour Siavash; Emami, Tajolmolouk; Najafian, Alireza; Amini, Masoud

    2006-10-01

    Most laboratories use EDTA for anticoagulation of whole blood prior to automated cell counting but due to platelet swelling, mean platelet volume (MPV) values may increase with its use. MPV changes may be less with acid citrate based anticoagulation. As MPV is a marker of platelet function and its precise measurement is important in a number of clinical situations, this study was performed to assess if EDTA and citrate based anticoagulated blood samples can be used interchangeably for MPV measurement. In this cross sectional descriptive study, EDTA and citrate based anticoagulated blood samples of the same patients were assessed by auto-analyzer within 1 h of sampling. In the 61 evaluated patients, there was a close correlation between MPV as measured by EDTA and citrate, but mean MPV measured from EDTA samples was 0.66 fL (9%) more than citrate. There was also a significant negative correlation between platelets count and MPV by both methods. The results of our study reveal that MPV can be measured accurately by both methods of anticoagulation; EDTA and citrate if analysis be performed within 1 h of sampling. PMID:17607580

  7. Chronic hyperammonemia reduces the activity of neuronal nitric oxide synthase in cerebellum by altering its localization and increasing its phosphorylation by calcium-calmodulin kinase II.

    Science.gov (United States)

    El-Mlili, Nisrin; Rodrigo, Regina; Naghizadeh, Bahareh; Cauli, Omar; Felipo, Vicente

    2008-08-01

    Impaired function of the glutamate-nitric oxide-cGMP pathway contributes to cognitive impairment in hyperammonemia and hepatic encephalopathy. The mechanisms by which hyperammonemia impairs this pathway remain unclear. Understanding these mechanisms would allow designing clinical treatments for cognitive deficits in hepatic encephalopathy. The aims of this work were: (i) to assess whether chronic hyperammonemia in vivo alters basal activity of neuronal nitric oxide synthase (nNOS) in cerebellum and/or its activation in response to NMDA receptor activation and (ii) to analyse the molecular mechanisms by which hyperammonemia induces these alterations. It is shown that hyperammonemia reduces both basal activity of nNOS and its activation following NMDA receptor activation. Reduced basal activity is because of increased phosphorylation in Ser847 (by 69%) which reduces basal activity of nNOS by about 40%. Increased phosphorylation of nNOS in Ser847 is because of increased activity of calcium-calmodulin-dependent protein kinases (CaMKII) which in turn is because of increased phosphorylation at Thr286. Inhibiting CaMKII with KN-62 normalizes phosphorylation of Ser847 and basal NOS activity in hyperammonemic rats, returning to values similar to controls. Reduced activation of nNOS in response to NMDA receptor activation in hyperammonemia is because of altered subcellular localization of nNOS, with reduced amount in post-synaptic membranes and increased amount in the cytosol. PMID:18498443

  8. Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase (SPS) Activity in Two Tomato Species

    Institute of Scientific and Technical Information of China (English)

    WANG Li; CUI Na; ZHAO Xiao-cui; FAN Hai-yan; LI Tian-lai

    2014-01-01

    To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase (SPS) activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography (HPLC). The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase (AI) and neutral invertase (NI) in Micro-Tom (Solanum lycopersicum) fruit, while inSolanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.

  9. Nitric Oxide synthases and atrial fibrillation

    Directory of Open Access Journals (Sweden)

    CynthiaAnnCarnes

    2012-04-01

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

  10. Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues

    Directory of Open Access Journals (Sweden)

    Franklin Renty B

    2006-04-01

    Full Text Available Abstract Background In prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines. Results The immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples. Conclusion The results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells

  11. Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

    DEFF Research Database (Denmark)

    Glintborg, Dorte; Højlund, Kurt; Andersen, Nicoline Resen;

    2008-01-01

    CONTEXT: Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. SUBJECTS AND METHODS: We...... investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were...... metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry. RESULTS: Reduced insulin-mediated glucose disposal (P < 0.05) was associated with a lower insulin-stimulated GS activity in PCOS patients (P < 0.05), compared with controls. This was, in part, explained by absent insulin...

  12. Renal Localization of {sup 67}Ga Citrate in Noninfectious Nephritis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kang Wook; Jeong, Min Soo; Rhee, Sunn Kgoo; Kim, Sam Yong; Shin, Young Tai; Ro, Heung Kyu [Chungnam University College of Medicine, Deajeon (Korea, Republic of)

    1992-07-15

    {sup 67}Ga citrate scan has been requested for detection or follow-up of inflammatory or neoplastic disease. Visualization of {sup 67}Ga citrate in the kidneys at 48 and 72 hr post injection is usually interpreted as evidence of renal pathology. But precise mechanisms of abnormal {sup 67}Ga uptake in kidneys were unknown. We undertook a study to determine the clinical value of {sup 67}Ga citrate imaging of the kidneys in 68 patients with primary or secondary nephropathy confirmed by renal biopsy and 66 control patients without renal disease. Renal uptake in 48 to 72 hr images was graded as follows: Grade 0=background activity;1=faint uptake greater than background; 2=definite uptake, but less than lumbar vertebrae;3 same uptake as lumbar vertebrae, but less than liver; 4=same or higher uptake than liver. The results were as follows. 1) 42 of 68(62%) patients with noninfectious nephritis showed grade 2 or higher {sup 67}Ga renal uptake but only 10 percent of control patients showed similar uptake. 2) In 14 patients with systemic lupus erythematosus, 8 of 9 (89%) patients with lupus nephritis exhibited marked renal uptake. 3) 36 of 41 patients (88%) with combined nephrotic syndrome showed Grade 2 or higher renal uptake. 4) Renal {sup 67}Ga uptake was correlated with clinical severity of nephrotic syndrome determined by serum albumin level, 24 hr urine protein excretion and serum lipid levels. 5) After complete remission of nephrotic syndrome, renal uptake in all 8 patients who were initially Grade 3 or 4, decreased to Grade 1 or 0. In conclusion, we think that the mechanism of renal {sup 67}Ga uptake in nephrotic syndrome might be related to the pathogenesis of nephrotic syndrome. In systemic lupus erythematosus, {sup 67}Ga citrate scan is useful in predicting renal involvement.

  13. Elevated CO2-induced production of nitric oxide (NO) by NO synthase differentially affects nitrate reductase activity in Arabidopsis plants under different nitrate supplies.

    Science.gov (United States)

    Du, Shaoting; Zhang, Ranran; Zhang, Peng; Liu, Huijun; Yan, Minggang; Chen, Ni; Xie, Huaqiang; Ke, Shouwei

    2016-02-01

    CO2 elevation often alters the plant's nitrate reductase (NR) activity, the first enzyme acting in the nitrate assimilation pathway. However, the mechanism underlying this process remains unknown. The association between elevated CO2-induced alterations of NR activity and nitric oxide (NO) was examined in Col-0 Arabidopsis fed with 0.2-10 mM nitrate, using NO donors, NO scavenger, and NO synthase (NOS) inhibitor. The noa1 mutant, in which most NOS activity was lost, and the NR activity-null mutant nia1 nia2 were also used to examine the above association. In response to CO2 elevation, NR activity increased in low-nitrate Col-0 plants but was inhibited in high-nitrate Col-0 plants. NO scavenger and NOS inhibitor could eliminate these two responses, whereas the application of NO donors mimicked these distinct responses in ambient CO2-grown Col-0 plants. Furthermore, in both low- and high-nitrate conditions, elevated CO2 increased NOS activity and NO levels in Col-0 and nia1 nia2 plants but had little effect on NO level and NR activity in noa1 plants. Considering all of these findings, this study concluded that, in response to CO2 elevation, either the NR activity induction in low-nitrate plants or the NR activity inhibition in high-nitrate plants is regulated by NOS-generated NO. PMID:26608644

  14. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    Science.gov (United States)

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. PMID:27372278

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

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

  17. Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats

    OpenAIRE

    Pereira B.; Costa-Rosa L.F.B.P.; Bechara E.J.H.; Newsholme P.; Curi R.

    1998-01-01

    Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced b...

  18. Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Dong Ju Son

    2014-08-01

    Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

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

    Science.gov (United States)

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

    2016-03-24

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

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

    Directory of Open Access Journals (Sweden)

    Ehsan Karimi

    2012-11-01

    Full Text Available 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 μg 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    OpenAIRE

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

    2016-01-01

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

  3. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    Science.gov (United States)

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective. PMID:25676153

  4. Effect of simulated microgravity and centrifugation on nitric oxide synthase activity of osteocyte-like cell line MLO-Y4

    Science.gov (United States)

    Sun, Lian-Wen; Yang, Xiao; Fan, Yu-Bo

    Bone is a highly mechanosensitive tissue, which can adapt functionally to varying levels of mechanical loads throughout a lifetime. Osteocytes are thought to be the most mechanically sensitive bone cell population. In order to understand the mechanism of microgravity-induced bone loss, it's very important to research the behavior of osteocytes under microgravity. In this study, rotary cell culture system was used to simulate microgravity. Nitric oxide synthase (NOS) activity in osteocyte-like cell MLO-Y4 was investigated under simulated microgravity. And the effect of centrifugation on NOS activity in sedentary and rotary culture cell was also investi-gated. The cultured cells were divided into four groups, including sedentary control (CON), sedentary control and centrifugation (CONC), rotary culture (RT), rotary and centrifugation (RTC). In CONC and RTC, NOS activity was determined after centrifugation (1100g 5min). The results showed NOS activity decreased significantly in RT compared with CON. However, this difference disappeared after centrifugation. On the other hand, NOS activity increased significant in RTC compared with RT while there was no difference between CON and CONC. These results indicate the normal centrifugation could counter the effect of simulated micro-gravity on NOS activity. However, it has no effect on the cells cultured under 1G. In general, osteocytes under simulated microgravity are more sensitive to centrifugation than that under 1G.

  5. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-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 μL 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.

  6. Rhizobacteria activates (+)-δ-cadinene synthase genes and induces systemic resistance in cotton against beet armyworm (Spodoptera exigua).

    Science.gov (United States)

    Zebelo, Simon; Song, Yuanyuan; Kloepper, Joseph W; Fadamiro, Henry

    2016-04-01

    Gossypol is an important allelochemical produced by the subepidermal glands of some cotton varieties and important for their ability to respond to changing biotic stress by exhibiting antibiosis against some cotton pests. Plant growth-promoting rhizobacteria (PGPR) are root-colonizing bacteria that increase plant growth and often elicit defence against plant pathogens and insect pests. Little is known about the effect of PGPR on cotton plant-insect interactions and the potential biochemical and molecular mechanisms by which PGPR enhance cotton plant defence. Here, we report that PGPR (Bacillus spp.) treated cotton plants showed significantly higher levels of gossypol compared with untreated plants. Similarly, the transcript levels of the genes (i.e. (+)-δ-cadinene synthase gene family) involved in the biosynthesis of gossypol were higher in PGPR-treated plants than in untreated plants. Furthermore, the levels of jasmonic acid, an octadecanoid-derived defence-related phytohormone and the transcript level of jasmonic acid responsive genes were higher in PGPR-treated plants than in untreated plants. Most intriguingly, Spodoptera exigua showed reduced larval feeding and development on PGPR-treated plants. These findings demonstrate that treatment of plants with rhizobacteria may induce significant biochemical and molecular changes with potential ramifications for plant-insect interactions. PMID:26715260

  7. Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Dobritzsch, D.; Gojkovic, Zoran; Andersen, Birgit;

    2003-01-01

    In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P...

  8. Preliminary X-ray crystallographic analysis of 2-methylcitrate synthase from Salmonella typhimurium

    International Nuclear Information System (INIS)

    2-Methylcitrate synthase from S. typhimurium was cloned, overexpressed, purified and crystallized. The crystals diffracted X-rays to 2.4 Å resolution and belonged to the triclinic space group P1 (unit-cell parameters a = 92.068, b = 118.159, c = 120.659 Å, α = 60.84, β = 67.77, γ = 81.92°). Self-rotation function calculations suggested the presence of a putative decamer of protein subunits in the asymmetric unit. Analysis of the genomic sequences of Escherichia coli and Salmonella typhimurium has revealed the presence of several homologues of the well studied citrate synthase (CS). One of these homologues has been shown to code for 2-methylcitrate synthase (2-MCS) activity. 2-MCS catalyzes one of the steps in the 2-methylcitric acid cycle found in these organisms for the degradation of propionate to pyruvate and succinate. In the present work, the gene coding for 2-MCS from S. typhimurium (StPrpC) was cloned in pRSET-C vector and overexpressed in E. coli. The protein was purified to homogeneity using Ni–NTA affinity chromatography. The purified protein was crystallized using the microbatch-under-oil method. The StPrpC crystals diffracted X-rays to 2.4 Å resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 92.068, b = 118.159, c = 120.659 Å, α = 60.84, β = 67.77, γ = 81.92°. Computation of rotation functions using the X-ray diffraction data shows that the protein is likely to be a decamer of identical subunits, unlike CSs, which are dimers or hexamers

  9. 21 CFR 184.1195 - Calcium citrate.

    Science.gov (United States)

    2010-04-01

    ... Food Chemicals Codex, 3d ed. (1981), pp. 49 and 50, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the National Academy Press, 2101... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium citrate. 184.1195 Section 184.1195 Food...

  10. 21 CFR 184.1625 - Potassium citrate.

    Science.gov (United States)

    2010-04-01

    .... (b) The ingredient meets the specifications of the Food Chemicals Codex, 3d ed. (1981), p. 242, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium citrate. 184.1625 Section 184.1625...

  11. 21 CFR 184.1751 - Sodium citrate.

    Science.gov (United States)

    2010-04-01

    ...) The ingredient meets the specifications of the Food Chemicals Codex, 3d ed. (1981), pp. 283-284, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium citrate. 184.1751 Section 184.1751 Food...

  12. 14 N NQR spectrum of sildenafil citrate

    Science.gov (United States)

    Stephenson, David; Singh, Nadia

    2015-04-01

    The 14N nuclear quadrupole resonance (NQR) spectrum of sildenafil citrate tablets has been recorded allowing the quadrupole coupling constants and asymmetry parameters of all six unique nitrogen atoms in its structure to be determined. A density function calculation gives results that are largely in agreement with the experimental values.

  13. 14N NQR spectrum of sildenafil citrate

    International Nuclear Information System (INIS)

    The 14N nuclear quadrupole resonance (NQR) spectrum of sildenafil citrate tablets has been recorded allowing the quadrupole coupling constants and asymmetry parameters of all six unique nitrogen atoms in its structure to be determined. A density function calculation gives results that are largely in agreement with the experimental values

  14. 21 CFR 522.800 - Droperidol and fentanyl citrate injection.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Droperidol and fentanyl citrate injection. 522.800... § 522.800 Droperidol and fentanyl citrate injection. (a) Specifications. Droperidol and fentanyl citrate injection is a sterile solution containing 20 milligrams of droperidol and 0.4 milligram of fentanyl...

  15. 21 CFR 172.370 - Iron-choline citrate complex.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Iron-choline citrate complex. 172.370 Section 172... CONSUMPTION Special Dietary and Nutritional Additives § 172.370 Iron-choline citrate complex. Iron-choline citrate complex made by reacting approximately equimolecular quantities of ferric hydroxide, choline,...

  16. 21 CFR 573.580 - Iron-choline citrate complex.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Iron-choline citrate complex. 573.580 Section 573.580 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Food Additive Listing § 573.580 Iron-choline citrate complex. Iron-choline citrate complex made...

  17. Ventricular tachycardia after administration of sildenafil citrate: a case report

    Directory of Open Access Journals (Sweden)

    Rasmussen Jeppe G

    2007-08-01

    Full Text Available Abstract Background It has not previously been reported that sildenafil citrate causes malignant arrhythmias in humans. Case presentation A 41-year-old man developed sustained ventricular tachycardia following sildenafil citrate administration. Conclusion It cannot be dismissed that this patient experienced ventricular tachycardia as an adverse effect of sildenafil citrate administration.

  18. Ventricular tachycardia after administration of sildenafil citrate: a case report

    OpenAIRE

    Rasmussen Jeppe G; Toft Egon; Frøbert Ole

    2007-01-01

    Abstract Background It has not previously been reported that sildenafil citrate causes malignant arrhythmias in humans. Case presentation A 41-year-old man developed sustained ventricular tachycardia following sildenafil citrate administration. Conclusion It cannot be dismissed that this patient experienced ventricular tachycardia as an adverse effect of sildenafil citrate administration.

  19. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Hopperton, Kathryn E., E-mail: kathryn.hopperton@mail.utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Duncan, Robin E., E-mail: robin.duncan@uwaterloo.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Bazinet, Richard P., E-mail: richard.bazinet@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Archer, Michael C., E-mail: m.archer@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada)

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  20. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    International Nuclear Information System (INIS)

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from 14C-labeled acetate to those supplied exogenously as 14C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare utilization of

  1. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}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 E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} 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{beta}-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{beta}-mediated phosphorylated nuclear factor-{kappa}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 PGE{sub 2} 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.

  2. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  3. Effect of simulated microgravity on nitric oxide synthase activity of osteocyte-like cell line MLO-Y4 in response to fluid shear stress

    Science.gov (United States)

    Sun, Lian-Wen; Yang, Xiao; Fan, Yu-Bo

    It is well known that microgravity could induce bone loss. However, the mechanism remains poorly understood. Osteocytes are extremely sensitive to fluid shear stress, even more than osteobleasts. The effect of simulated microgravity on osteocytes in response to fluid shear was investigated in this study in order to see if the mechanosensibility of osteocytes changed under simulated microgravity. The osteocyte-like cell line, MLO-Y4, was cultured and divided into four groups, including control (CON), control and shear (CONS), rotary (RT), rotary and shear (RTS). In RT and RTS, the cells were cultured in the rotary cell culture system to simulate microgravity condition. After 5 days, the cells in RTS and CONS were subjected to flow shear for 15 min. Then nitric oxide synthase (NOS) activity in the cells was measured using assay kit. The results showed that NOS activity in respond to fluid shear decreased significantly in RTS compared with CONS. In addition, there was significant difference in NOS activity between CONS and CON while no significant difference between RTS and RT. These indicates that the mechanosensibility of osteocytes decreased under simulated microgravity and this maybe the partly causes of the poor effect of exercise to counter microgravity-induced-bone loss. However, further research need to be done to support this finding.

  4. Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593.

    Science.gov (United States)

    He, Huoguang; Wu, Bin; Xiong, Min; Li, Yang; Wu, Wenhua; Wang, Xingguo

    2011-10-01

    Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5' terminus. PMID:21939372

  5. Role of interferon regulatory factor-1 and mitogen-activated protein kinase pathways in the induction of nitric oxide synthase-2 in retinal pigmented epithelial cells.

    Science.gov (United States)

    Faure, V; Hecquet, C; Courtois, Y; Goureau, O

    1999-02-19

    Bovine retinal pigmented epithelial cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Experiments were performed to investigate the involvement of interferon regulatory factor-1 (IRF-1) on NOS-2 induction and its regulation by NOS-2 inhibitors such as pyrrolidine dithiocarbamate (PDTC), an antioxidant, or protein kinase inhibitors. Analysis by transitory transfections showed that LPS, alone or with IFN-gamma, stimulated activity of the murine NOS-2 promoter fragment linked upstream of luciferase and its suppression by PDTC and by the different protein kinase inhibitors, genistein (tyrosine kinase inhibitor), PD98059 (mitogen-actived protein (MAP) kinase kinase inhibitor), and SB 203580 (p38 MAP inhibitor). Using specific antibodies, we have confirmed that extracellular signal-regulated kinases and p38 MAP kinase were activated by LPS and IFN-gamma in retinal pigmented epithelial cells. Analysis by reverse transcriptase-polymerase chain reaction, Western blot, and electrophoretic mobility shift assay demonstrated that IFN-gamma alone or combined with LPS induced an accumulation of IRF-1 mRNA and protein and IRF-1 DNA binding. Transfections assays with the IRF-1 promoter showed an induction of this promoter with IFN-gamma, potentiated by LPS. The decrease of LPS/IFN-gamma-induced IRF-1 promoter activity, IRF-1 synthesis, and IRF-1 activation, by PDTC, genistein, PD98059, and SB 203580, could explained in part the inhibition of the NOS-2 induction by these compounds. Our results demonstrate that IRF-1 is necessary for NOS-2 induction by LPS and IFN-gamma and that its synthesis requires the involvement of a redox-sensitive step, the activation of tyrosine kinases, and extracellular signal-regulated kinases 1/2 and p38 MAP kinases. PMID:9988718

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  7. Citrate Anticoagulation for CRRT in Children: Comparison with Heparin

    Directory of Open Access Journals (Sweden)

    Sara Nicole Fernández

    2014-01-01

    Full Text Available Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P=0.028. 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  8. Citrate Anticoagulation for CRRT in Children: Comparison with Heparin

    Science.gov (United States)

    Fernández, Sara Nicole; Santiago, Maria José; López-Herce, Jesús; García, Miriam; Del Castillo, Jimena; Alcaraz, Andrés José; Bellón, Jose María

    2014-01-01

    Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P = 0.028). 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I) of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin. PMID:25157369

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

    Science.gov (United States)

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

    2012-06-01

    In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K(m)(glutamate) and k(cat) values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity. PMID:22447897

  10. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB.

    Science.gov (United States)

    Yu, Zhiyuan; Zhang, Wenzheng; Kone, Bruce C

    2002-01-01

    Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1beta- and lipopolysaccharide (LPS)+interferon (IFN)-gamma-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor kappaB (NF-kappaB) p65 and p50 complexed with STAT3 in the DNA-protein complex. The direct interaction of STAT3 and NF-kappaB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-kappaB p65 fusion protein and in vitro -translated STAT3alpha. Overexpression of STAT3 dramatically inhibited IL-1beta- or LPS+IFN-gamma-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-kappaB-dependent promoter devoid of STAT-binding elements without affecting NF-kappaB DNA-binding activity. Thus STAT3, via direct interactions with NF-kappaB p65, serves as a dominant-negative inhibitor of NF-kappaB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli. PMID:12057007

  11. Chiral hydroxylation at the mononuclear nonheme Fe(II center of 4-(S hydroxymandelate synthase--a structure-activity relationship analysis.

    Directory of Open Access Journals (Sweden)

    Cristiana M L Di Giuro

    Full Text Available (S-Hydroxymandelate synthase (Hms is a nonheme Fe(II dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.

  12. Phylogenetic diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff), detection of polyketide synthase gene and their antagonistic activity analysis.

    Science.gov (United States)

    Wang, Ya; Gao, Bo Liang; Li, Xi Xi; Zhang, Zhi Bin; Yan, Ri Ming; Yang, Hui Lin; Zhu, Du

    2015-11-01

    The biodiversity of plant endophytic fungi is enormous, numerous competent endophytic fungi are capable of providing different forms of fitness benefits to host plants and also could produce a wide array of bioactive natural products, which make them a largely unexplored source of novel compounds with potential bioactivity. In this study, we provided a first insights into revealing the diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff.) from China using rDNA-ITS phylogenetic analysis. Here, the potential of fungi in producing bioactive natural products was estimated based on the beta-ketosynthase detected in the polyketide synthase (PKS) gene cluster and on the bioassay of antagonistic activity against two rice phytopathogens Thanatephorus cucumeris and Xanthomonas oryzae. A total of 229 endophytic fungal strains were validated in 19 genera. Among the 24 representative strains, 13 strains displayedantagonistic activity against the phytopathogens. Furthermore, PKS genes were detected in 9 strains, indicating their potential for synthesising PKS compounds. Our study confirms the phylogenetic diversity of endophytic fungi in O. rufipogon G. and highlights that endophytic fungi are not only promising resources of biocontrol agents against phytopathogens of rice plants, but also of bioactive natural products and defensive secondary metabolites. PMID:26466878

  13. Effects of L-arginine on serum nitric oxide, nitric oxide synthase and mucosal Na+-K+-A TPase and nitric oxide synthase activity in segmental small-bowel autotransplantation model

    Institute of Scientific and Technical Information of China (English)

    Ting-Liang Fu; Wen-Tong Zhang; Qiang-Pu Chen; Yong Gao; Yu-Hong Hu; Dian-Liang Zhang

    2005-01-01

    AIM: To explore a simple method to create intestinal autotransplantation in rats and growing pigs and to investigate the effect of L-arginine supplementation on serum nitric oxide (NO), nitric oxide synthase (NOS) and intestinal mucosal NOS and Na+-K+-ATPase activity during cold ischemia-reperfusion (IR) in growing pigs.METHODS: In adult Wistar rat models of small bowel autotransplantation, a fine tube was inserted into mesenteric artery via the abdominal aorta. The superior mesenteric artery and vein were occluded. Isolated terminal ileum segment was irrigated with Ringer'ssolution at 4 ℃ and preserved in the same solution at 0-4 ℃ for 60 min. Then, the tube was removed and reperfusion was established. In growing pig models, a terminal ileum segment, 50 cm in length, was isolated and its mesenteric artery was irrigated via a needle with lactated Ringer's solution at 4 ℃. The method and period of cold preservation and reperfusion were described above. Ten white outbred pigs were randomly divided into control group and experimental group. L-arginine (150 mg/kg) was continuously infused for 15 min before reperfusion and for 30 min after reperfusion in the experimental group. One, 24, 48, and 72 h after reperfusion, peripheral vein blood was respectively collected for NO and NOS determination. At the same time point, intestinal mucosae were also obtained for NOS and Na+-K+-ATPase activity measurement.RESULTS: In adult rat models, 16 of 20 rats sustained the procedure, three died of hemorrhage shock and one of deep anesthesia. In growing pig models, the viability of small bowel graft remained for 72 h after cold IR in eight of 10 pigs. In experimental group, serum NO level at 1 and 24 h after reperfusion increased significantly when compared with control group at the same time point (152.2±61.4 μmol/L vs60.8±31.6 μmol/L, t= 2.802,P = 0.02<0.05; 82.2±24.0 μmol/L vs 54.0±24.3 μmol/L, t = 2.490, P = 0.04<0.05). Serum NO level increased significantly at 1

  14. Effect of the structural and morphological properties of Cu/ZnO catalysts prepared by citrate method on their activity toward methanol synthesis from CO2 and H2 under mild reaction conditions

    OpenAIRE

    Karelovic, Alejandro; Bargibant, Alice; Fernandez Rojas, Camila; Ruiz, Patricio

    2012-01-01

    Methanol synthesis from CO2 + H2 was studied at mild reaction conditions (140–250 °C and 7 bar) over Cu/ZnO catalysts prepared by citrate method. The copper content and calcination temperatures were varied so as to obtain a wide range in copper particle size (2–12 nm). Methanol formation rates vary between 0.84 and 2.98 × 10−3 s−1 at 180 °C. Methanol selectivity can attain 100% at temperatures lower than 160 °C. At higher temperatures, CO formation by reverse water gas shift reaction is highl...

  15. A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes.

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K

    2010-09-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%-70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding. PMID:20595348

  16. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

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

  17. Pyrolytic citrate synthesis and ozone annealing

    International Nuclear Information System (INIS)

    A pyrolytic procedure is described that via a citrate synthesis allowed us to obtain very fine grained YBCO powders that, after a first furnace thermal treatment in ozone, results already to contain a large amount of superconducting microcrystals. A second identical thermal treatment gives a final product strongly textured, as shown by magnetic torque measurements. Complementary structural and diamagnetic measurement show the high quality of these sintered pellets. The role covered by both the pyrolytic preparation and the ozone annealing are discussed

  18. Pyrolitic citrate synthesis and ozone annealing

    International Nuclear Information System (INIS)

    A pyrolytic procedure that via a citrate synthesis allowed to obtain very fine grained YBCO powders that, after a first furnace thermal treatment in ozone, result already to contain a large amount of superconducting microcystals is described. A second identical thermal treatment gives a final product strongly textured, as shown by magnetic torque measurements. Complementary structural and diamagnetic measurements show the high quality of these sintered pellets. The role covered by both the pyrolytic preparation and the ozone annealing are discussed

  19. Photodynamical Properties of Various Phthalocyanine Citrates

    Czech Academy of Sciences Publication Activity Database

    Drobek, M.; Klusoň, Petr; Hejda, S.; Krýsa, J.; Rakušan, J.

    Prague: ICT Prague Press, 2010 - (Krýsa, J.), s. 68 ISBN 978-80-7080-750-7. [European Meeting on Solar Chemistry and Photocatalysis: Environmental Applications /6./. Prague (CZ), 13.06.2010-16.06.2010] R&D Projects: GA AV ČR KAN400720701; GA MŠk 1M0577 Institutional research plan: CEZ:AV0Z40720504 Keywords : phthalocyanine citrates * photodynamical properties Subject RIV: CA - Inorganic Chemistry

  20. Sildenafil Citrate in Fetal Growth Restriction

    OpenAIRE

    Panda, Subrat; Das, Ananya; Md Nowroz, Hossain

    2014-01-01

    Background Pregnancies with early onset fetal growth restriction have poor perinatal outcome. Sildenafil citrate (PDE -5 inhibitor) as a vasodilator increases utero-placental blood flow and potentiates fetal growth. Case Presentation In this study, a case was examined and Sildenafil was administered for her. It was found that Sildenafil improved the uterine blood flow with a favorable fetal outcome at delivery. Conclusion Sildenafil, as a vasodilator has emerged as a potential management opti...

  1. Evaluation of the Activities of Pyrimethamine Analogs against Plasmodium vivax and Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase Using In Vitro Enzyme Inhibition and Bacterial Complementation Assays▿

    OpenAIRE

    Bunyarataphan, Sasinee ; Leartsakulpanich, Ubolsree; Taweechai, Supannee; Tarnchompoo, Bongkoch; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth

    2006-01-01

    Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from ...

  2. Abnormal splenic uptake of gallium-67 citrate in a case of infectious mononucleosis

    International Nuclear Information System (INIS)

    A case of fever of undetermined origin (FUO) demonstrated abnormal intense concentration of Ga-67 citrate in the spleen. Initial workup demonstrated two negative mono spot tests for infectious mononucleosis (IM) but, on hospital day 2, a repeat mono test was positive. The diagnosis was subsequently confirmed by rising heterophilic antibody titers. Six weeks after hospital discharge, repeat Ga-67 citrate imaging demonstrated absence of abnormal splenic activity. To our knowledge, this phenomenon has not been previously described in IM. In the appropriate clinical setting, intense splenic uptake of Ga-67 should alert the clinician to include the possibility of IM in the differential diagnosis

  3. Leukotriene-C4 Synthase, a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels, Is Required for Neointimal Hyperplasia*

    Science.gov (United States)

    Zhang, Wei; Zhang, Xuexin; González-Cobos, José C.; Stolwijk, Judith A.; Matrougui, Khalid; Trebak, Mohamed

    2015-01-01

    Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca2+ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation. PMID:25540197

  4. Study on structure-activity relationship of mutation-dependent herbicide resistance acetohydroxyacid synthase through 3D-QSAR and mutation

    Institute of Scientific and Technical Information of China (English)

    YU ZhiHong; NIU CongWei; BAN ShuRong; WEN Xin; XI Zhen

    2007-01-01

    Seventy-four sulfonylureas were synthesized and tested for their inhibitory activity against the whole enzyme of E. Coli acetohydroxyacid synthase (AHAS, EC 2.2.1.6) isoenzyme Ⅱ, and 3D-QSAR analyses were performed based on these inhibitory activities. The binding conformation of chlorimuron-ethyl, a commercial herbicide of AHAS, in the crystal structure of AHAS complex was extracted and used as template to build the initial three-dimensional structure of other sulfonylureas, and then all structures were fully geometry optimized. After systematic optimization of the alignment rule, molecular orientation, grid space and attenuation factor, two satisfactory models with excellent performances (CoMFA: q2 = 0.735, r2 = 0.954, n = 7, r 2pred = 0.832; CoMSIA: q2 = 0.721, r2 = 0.913, n = 8, r 2pred = 0.844) were established. By mapping the 3D contour maps of CoMFA and CoMSIA models into the possible inhibitory active site in the crystal structure of catalytic subunit of yeast AHAS, a plausible binding model for AHAS, with best fit QSAR in the literature so far, was proposed. Moreover, the results of 3D-QSAR were further utilized to interpret resistance of site-directed mutants. A relative activity index (RAI) for AHAS enzyme mutant was defined for the first time to relate the 3D-QSAR and resistance of mutants. This study, for the first time, demonstrated that combination of 3D-QSAR and enzyme mutation can be used to decipher the molecular basis of ligand-receptor interaction mechanism. This study refined our understanding of the ligand-receptor interaction and resistance mechanism in AHAS-sulfonylurea system, and provided basis for designing new potent herbicides to combat the herbicide resistance.

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

    Science.gov (United States)

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

    2016-01-22

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

  6. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-01

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  7. Clinical and biochemical studies on the mechanism of gallium-67 citrate accumulation in tumor tissues

    International Nuclear Information System (INIS)

    A series of 67Ga scintigraphies, taken at Kobe University Hospital and Tenri Hospital from 1970 to 1974, were investigated statistically and histologically. Positive imaging of the lesion was obtained in 87% of 196 cases of primary lung cancer; 94%, 83% and 82% were positive in squamous cell carcinoma, undifferentiated carcinoma, and adenocarcinoma. In a comparative study on inflammatory lesions such as pneumonia or lung abcess, positive imaging was also obtained frequently during the active state but no positive image was noted in the inactive state. Since cyclic AMP is reported to play a role as a potent growth regulator, the effect of cyclic AMP as well as that of theophylline on 67Ga citrate binding in vitro was examined using a Millipore filter. Although cyclic AMP released 67Ga citrate bound to the cytosols of a rat liver, theophylline stimulated 67Ga citrate binding. In order to prove the working hypothesis that cyclic AMP phosphodiesterase binds 67Ga citrate, this enzyme was purified to a specific activity of 2330 mU/mg by an elaborate method such as ammonium sulfate treatment (30 - 50%) and stepwise Hydroxyapatite fractionation. The specific radioactivity of 67Ga citrate bound to this enzyme preparation (500 mM potassium phosphate buffer, pH 7.5) was 70-fold greater than that of transferrin and 240-fold more than that of cyclic AMP-binding protein purified as 50 mM potassium phosphate buffer, pH 7.5 eluted through Hydroxyapatite. It was also confirmed that the lysosome purified from the more rapidly growing hepatoma (7316 A) had a radioactivity two-fold higher than that of the slower growing hepatoma (7794 A) using an in vitro 67Ga citrate binding method. (auth.)

  8. M-CSF from Cancer Cells Induces Fatty Acid Synthase and PPARβ/δ Activation in Tumor Myeloid Cells, Leading to Tumor Progression

    Directory of Open Access Journals (Sweden)

    Jonghanne Park

    2015-03-01

    Full Text Available We investigate crosstalk between cancer cells and stromal myeloid cells. We find that Lewis lung carcinoma cells significantly induce PPARβ/δ activity in myeloid cells in vitro and in vivo. Myeloid cell-specific knockout of PPARβ/δ results in impaired growth of implanted tumors, and this is restored by adoptive transfer of wild-type myeloid cells. We find that IL-10 is a downstream effector of PPARβ/δ and facilitates tumor cell invasion and angiogenesis. This observation is supported by the finding that the CD11blowIL-10+ pro-tumoral myeloid cell is scarcely detected in tumors from myeloid-cell-specific PPARβ/δ knockout mice, where vessel densities are also decreased. Fatty acid synthase (FASN is shown to be an upstream regulator of PPARβ/δ in myeloid cells and is induced by M-CSF secreted from tumor cells. Our study gives insight into how cancer cells influence myeloid stromal cells to get a pro-tumoral phenotype.

  9. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: dhkim@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  10. Ontogeny of nitric oxide synthase I and III protein expression and enzymatic activity in the guinea pig hippocampus.

    Science.gov (United States)

    Kimura, K A; Reynolds, J N; Brien, J F

    1999-09-01

    60. NOS enzymatic activity increased throughout prenatal and postnatal life, and attained highest activity in the adult. The developmental profile of NOS III protein expression was similar to that for NOS enzymatic activity. There was differential expression of NOS I protein, which was low in the GD 50 fetus and increased rapidly during fetal development to attain adult level by GD 62. These data suggest that the guinea pig is a reliable animal model in which to investigate the roles of NO in normal hippocampal development and in mediating neuronal injury in this brain region. PMID:10521566

  11. Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

    Directory of Open Access Journals (Sweden)

    Hunter Randy

    2008-01-01

    Full Text Available Abstract Background Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS; however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1 the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS-induced iNOS activity and nitric oxide (NO generation by microglia; (2 the differential role of p38 mitogen-activated protein kinase (p38 MAPK, c-Jun NH(2-terminal kinase (JNK, and phosphoinositide 3-kinase (PI3K on LPS-induced NO generation; and (3 the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. Methods Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH-positive neurons were counted in mesencephalic neuron-microglia mixed cultures. Results Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-20

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

  13. Trapping and release of citrate-capped gold nanoparticles

    International Nuclear Information System (INIS)

    An electrical method to trap and release charged gold nanoparticles onto and from the surface of gold electrodes modified by an alkanethiol self-assembled monolayer (SAM) is presented. To form electrodes coated with gold nanoparticles (GNPs), amine-terminated SAMs on gold electrodes were immersed in a solution of negatively charged citrate-capped GNPs. Accumulation of GNPs on the electrode surface was monitored by a decrease in the impedance of the SAM-modified electrode and by an increase in the electrochemical activity at the electrode as shown through cyclic voltammetry (CV). Electrostatic interactions between the GNPs and the amine-terminated SAM trap the GNPs on the electrode surface. Application of a subsequent negative bias to the electrode initiated a partial release of the GNPs from the electrode surface. Impedance spectroscopy, cyclic voltammetry, ultraviolet-visible (UV-Vis) spectroscopy and atomic force microscopy (AFM) were used to monitor and confirm the attraction of GNPs to and release from the aminealkanethiolated gold electrodes. This work describes a method of trapping and release for citrate-capped GNPs that could be used for on-demand nanoparticle delivery applications such as in assessing and modeling nanoparticle toxicology, as well as for monitoring the functionalization of gold nanoparticles.

  14. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    International Nuclear Information System (INIS)

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 ± 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK)

  15. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    Science.gov (United States)

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  16. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    Science.gov (United States)

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  17. Changes in phytochemical synthesis, chalcone synthase activity and pharmaceutical qualities of sabah snake grass (Clinacanthus nutans L.) in relation to plant age.

    Science.gov (United States)

    Ghasemzadeh, Ali; Nasiri, Alireza; Jaafar, Hawa Z E; Baghdadi, Ali; Ahmad, Izham

    2014-01-01

    In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old). The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74) was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF) production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW]) and total phenolic (TP) (18.21 mg/g DW) were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW) and gallic acid (5.96 mg/g DW) were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50) values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP) assay showed a higher activity in 6-month-old buds (488 μM of Fe(II)/g) than in 1-year-old buds (453 μM of Fe(II)/g), in contrast to the DPPH result. Significant correlations (p < 0.05) were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity against

  18. Changes in Phytochemical Synthesis, Chalcone Synthase Activity and Pharmaceutical Qualities of Sabah Snake Grass (Clinacanthus nutans L. in Relation to Plant Age

    Directory of Open Access Journals (Sweden)

    Ali Ghasemzadeh

    2014-10-01

    Full Text Available In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old. The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74 was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW] and total phenolic (TP (18.21 mg/g DW were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW and gallic acid (5.96 mg/g DW were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50 values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP assay showed a higher activity in 6-month-old buds (488 μM of Fe(II/g than in 1-year-old buds (453 μM of Fe(II/g, in contrast to the DPPH result. Significant correlations (p < 0.05 were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity

  19. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    Science.gov (United States)

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. PMID:22770942

  20. Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution Atividade de invertases e sacarose sintase em plantas de cafeeiro pulverizadas com solução de sacarose

    OpenAIRE

    José Carlos da Silva; José Donizeti Alves; Amauri Alves de Alvarenga; Marcelo Murad Magalhães; Dárlan Einstein do Livramento; Daniela Deitos Fries

    2003-01-01

    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...

  1. Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell line

    OpenAIRE

    Cruz, M. Teresa; Gonçalo, Margarida; Figueiredo, Américo; Carvalho, Arsélio P.; Duarte, Carlos B.; Lopes, M. Celeste

    2004-01-01

    Nuclear factor kappa B (NF-kB) and activating protein-1 (AP-1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin-derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO4) and increases the ...

  2. Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chih

    2011-05-01

    Full Text Available Abstract Objectives Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl-5-hydroxy-2-(4-hydroxyphenyl-7-methoxy-4H-chromen-4-one, one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS-activated macrophages. Methods We used nitrate and prostaglandin E2 (PGE2 assays to examine inhibitory effect of aciculatin on nitric oxide (NO and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis. Results Aciculatin remarkably decreased the LPS (1 μg/mL-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM. Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs. Conclusion Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

  3. Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle.

    Science.gov (United States)

    Momken, Iman; Fortin, Dominique; Serrurier, Bernard; Bigard, Xavier; Ventura-Clapier, Renée; Veksler, Vladimir

    2002-01-01

    Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle. PMID:12123418

  4. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

    Science.gov (United States)

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2014-04-17

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. PMID:24456689

  5. The SH2B1 obesity locus is associated with myocardial infarction in diabetic patients and with NO synthase activity in endothelial cells

    Science.gov (United States)

    Prudente, Sabrina; Morini, Eleonora; Larmon, Jay; Andreozzi, Francesco; Pietro, Natalia Di; Nigro, Angela; Gervino, Ernest V; Mannino, Gaia Chiara; Bacci, Simonetta; Hauser, Thomas H; Bellacchio, Emanuele; Formoso, Gloria; Pellegrini, Fabio; Proto, Vittoria; Menzaghi, Claudia; Frittitta, Lucia; Pandolfi, Assunta; Sesti, Giorgio; Doria, Alessandro; Trischitta, Vincenzo

    2013-01-01

    Objective Obesity and cardiovascular disease recognize a common metabolic soil and may therefore share part of their genetic background. Genome-wide association studies have identified variability at the SH2B1 locus as a predictor of obesity. We investigated whether SNP rs4788102, which captures the entire SH2B1 variability, is associated with coronary artery disease (CAD) and/or myocardial infarction (MI) in patients with type 2 diabetes mellitus (T2DM). Design and Setting SNP rs4788102 was typed in 2,015 White subjects with T2DM from three CAD case-control studies [n=740 from the Gargano Hearth Study (GHS, Italy); n=818 from the Joslin Hearth Study (JHS, Boston); n=457 from the University of Catanzaro (CZ, Italy)]. Results SNP rs4788102 (G/A) was not associated with CAD (overall allelic OR=1.06, 95% CI=0.93-1.21; p=0.37). On the contrary, it was associated with MI in GHS (1.42, 1.12-1.81; p=0.004) and in the three samples analyzed together (1.21, 1.04-1.41; p=0.016). Insulin stimulated nitric oxide synthase (NOS) activity in human vein endothelial cells from G/G (n=4, p=0.03) but not the G/A (n=5, p=0.83) genotype. Of the SNPs in perfect LD with rs4788102, one (rs7498665) affects amino acid polarity (Ala484Thr) and falls into a highly conserved protein segment of SH2B1 containing a class II SH3 domain binding site. Conclusions Variability at the SH2B1 obesity locus is associated with MI in diabetic patients and with reduced insulin-stimulated NOS activity in human endothelial cells. Further studies are needed to replicate this association and dissect the biology underlying this finding. PMID:21907990

  6. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation

    OpenAIRE

    DU, XUELIANG; Edelstein, Diane; Obici, Silvana; Higham, Ninon; Zou, Ming-Hui; Brownlee, Michael

    2006-01-01

    Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals...

  7. Galactinol synthase enzyme activity influences raffinose family oligosaccharides (RFO) accumulation in developing chickpea (Cicer arietinum L.) seeds.

    Science.gov (United States)

    Gangola, Manu P; Jaiswal, Sarita; Kannan, Udhaya; Gaur, Pooran M; Båga, Monica; Chibbar, Ravindra N

    2016-05-01

    To understand raffinose family oligosaccharides (RFO) metabolism in chickpea (Cicer arietinum L.) seeds, RFO accumulation and corresponding biosynthetic enzymes activities were determined during seed development of chickpea genotypes with contrasting RFO concentrations. RFO concentration in mature seeds was found as a facilitator rather than a regulating step of seed germination. In mature seeds, raffinose concentrations ranged from 0.38 to 0.68 and 0.75 to 0.99 g/100 g, whereas stachyose concentrations varied from 0.79 to 1.26 and 1.70 to 1.87 g/100 g indicating significant differences between low and high RFO genotypes, respectively. Chickpea genotypes with high RFO concentration accumulated higher concentrations of myo-inositol and sucrose during early seed developmental stages suggesting that initial substrate concentrations may influence RFO concentration in mature seeds. High RFO genotypes showed about two to three-fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentrations. RFO biosynthetic enzymes activities correspond with accumulation of individual RFO during seed development. PMID:26953100

  8. Urinary Citrate: A view in Chronic Renal Failure

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    SANTHOSH KUMAR.N

    2013-12-01

    Full Text Available Aim & Objective: To evaluate the 24 hour urinary citrate levels in chronic renal failure and healthy controls and to define the role of urinary citrates in the chronic renal failures. Materials and Methods: The 24 hours urinary citrates, Blood urea, Serum creatinine, Na+, K+were evaluated in 25 chronic renal failure patients and25 healthy subjects taken as controls. In both groups participants were on their usual diet. In addition, none of the participant was taking any drugs that could interfere with the citrate excretion. Results: The mean 24 hour urinary citrate excretion in patients and healthy controls was 296.3 ± 8.543mg and 323.9 ± 4.304mg respectively. Using previously defined values of normal urinary citrates as more than 320 mg.The difference in 24 hour urinary citrateexcretion in all patients and healthy control was statistically significant (

    citrate excretion in recurrent renal failures and healthy controls. Uniformly low citrate excretion in patients indicates that low citrate levels may be a feature seen in predisposing factor for renal failure

  9. Geranyl diphosphate synthase from mint

    Science.gov (United States)

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

    1999-01-01

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

  10. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-03-02

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

  11. Regional age-related changes in neuronal nitric oxide synthase (nNOS, messenger RNA levels and activity in SAMP8 brain

    Directory of Open Access Journals (Sweden)

    Guidon Gérard

    2006-12-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8 mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS. To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx- levels, in three brain areas (n = 7 animals in each group. Calibrated reverse transcriptase (RT and real-time polymerase chain reaction (PCR and biochemical procedures were used. Results We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Conclusion Concomitant variations occurring in NO levels

  12. The activation by glucose of liver membrane nitric oxide synthase in the synthesis and translocation of glucose transporter-4 in the production of insulin in the mice hepatocytes.

    Directory of Open Access Journals (Sweden)

    Suman Bhattacharya

    Full Text Available INTRODUCTION: Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated. METHODS: Grated liver suspension (GLS was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO was measured by methemoglobin method. Glucose transporter-4 (Glut-4 was measured by immunoblot technique using Glut-4 antibody. RESULTS: Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells. CONCLUSION: It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-04-24

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

  14. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

    International Nuclear Information System (INIS)

    Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be context

  15. Crystallization of Δ{sup 1}-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    Energy Technology Data Exchange (ETDEWEB)

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota [Neutron Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Shoyama, Yukihiro; Morimoto, Satoshi, E-mail: morimoto@phar.kyushu-u.ac.jp [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2005-08-01

    Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å{sup 3} Da{sup −1} assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

  16. Crystallization of Δ1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    International Nuclear Information System (INIS)

    Δ1-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å3 Da−1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

  17. Response of patients with indolent systemic mastocytosis to tamoxifen citrate.

    Science.gov (United States)

    Butterfield, Joseph H; Chen, Dong

    2016-01-01

    We examined whether tamoxifen citrate at 20mg/day for 1 year had a beneficial effect on laboratory findings, bone marrow mastocytosis, common clinical symptoms, or quality-of-life assessment for 5 women and 2 men with indolent systemic mastocytosis. Tamoxifen was well tolerated. We found significant reductions in the platelet count, serum alkaline phosphatase, and 24-h urinary excretion of N-methylhistamine and significant increases in serum lactate dehydrogenase and (excluding 2 patients taking aspirin) in 24-h urinary excretion of 11β-prostaglandin F2α. Overall, no change occurred in percent involvement of bone marrow by mastocytosis. Symptom scores were mild and did not change during the treatment. The 36-Item Short Form Health Survey scores for quality of life physical and mental components showed no marked changes. Tamoxifen, an older, nonhematotoxic medication, has limited activity in systemic mastocytosis at the dosage used in this study. PMID:26612479

  18. Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

    International Nuclear Information System (INIS)

    Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p

  19. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation

    DEFF Research Database (Denmark)

    Ramachandran, Roshni; Bhatt, Deepak Kumar; Ploug, Kenneth Beri; Hay-Schmidt, Anders; Jansen-Olesen, Inger; Gupta, Saurabh; Olesen, Jes

    2014-01-01

    BACKGROUND AND AIM: Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene...

  20. Diffuse abdominal gallium-67 citrate uptake in salmonella infections

    International Nuclear Information System (INIS)

    Two pediatric patients with salmonella infections (one with typhoid fever and the second with salmonella C2 gastroenteritis), had a diffuse abdominal uptake of Ga-67 citrate. The possible explanation for this finding is discussed. Salmonella infection should be included as a cause in the differential diagnosis of diffuse accumulation of Ga-67 citrate

  1. Citrate increases glass transition temperature of vitrified sucrose preparations

    NARCIS (Netherlands)

    Kets, E.P.W.; Lipelaar, P.J.; Hoekstra, F.A.; Vromans, H.

    2004-01-01

    The aim of this study was to investigate the effect of sodium citrate on the properties of dried amorphous sucrose glasses. Addition of sodium citrate to a sucrose solution followed by freeze-drying or convective drying resulted in a glass transition temperature (T-g) that was higher than the well-s

  2. Injectable citrate-modified Portland cement for use in vertebroplasty.

    Science.gov (United States)

    Wynn-Jones, Gareth; Shelton, Richard M; Hofmann, Michael P

    2014-11-01

    The injectability of Portland cement (PC) with several citrate additives was investigated for use in clinical applications such as vertebroplasty (stabilization of a fractured vertebra with bone cement) using a syringe. A 2-wt % addition of sodium or potassium citrate with PC significantly improved cement injectability, decreased cement setting times from over 2 h to below 25 min, while increasing the compressive strength to a maximum of 125 MPa. Zeta-potential measurements indicated that the citrate anion was binding to one or more of the positively charged species causing charged repulsion between cement particles which dispersed aggregates and caused the liquefying effect of the anion. Analysis of the hydrating phases of PC indicated that the early strength producing PC phase (ettringite) developed within the first 2 h of setting following addition of the citrate anion, while this did not occur in the control cement (PC only). Within 24 h ettringite developed in PC as well as calcium-silicate-hydrate (C-S-H), the major setting phase of PC, whereas cements containing citrate did not develop this phase. The evidence suggested that in the presence of citrate the cements limited water supply appeared to be utilized for ettringite formation, producing the early strength of the citrate cements. The present study has demonstrated that it is possible to modify PC with citrate to both improve the injectability and crucially reduce the setting times of PC while improving the strength of the cement. PMID:24711245

  3. 21 CFR 520.622b - Diethylcarbamazine citrate syrup.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate syrup. 520.622b Section... Diethylcarbamazine citrate syrup. (a)(1) Specifications. Each milliliter of syrup contains 60 milligrams of... veterinarian. (b)(1) Specifications. Each milliliter of syrup contains 60 milligrams of...

  4. 21 CFR 520.622c - Diethylcarbamazine citrate chewable tablets.

    Science.gov (United States)

    2010-04-01

    ....622c, see the List of CFR Sections Affected, which appears in the Finding Aids section of the printed... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate chewable tablets. 520... Diethylcarbamazine citrate chewable tablets. (a) Specifications. Each chewable tablet contains 30, 45, 60, 120,...

  5. 21 CFR 520.622a - Diethylcarbamazine citrate tablets.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate tablets. 520.622a... Diethylcarbamazine citrate tablets. (a) Sponsors. (1) See 015579 in § 510.600(c) of this chapter for use of 50, 200, and 400 milligram tablets for prevention of heartworm disease in dogs and as an aid in the...

  6. 21 CFR 520.623 - Diethylcarbamazine citrate, oxibendazole chewable tablets.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate, oxibendazole chewable tablets. 520.623 Section 520.623 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... § 520.623 Diethylcarbamazine citrate, oxibendazole chewable tablets. (a) Specifications. Each...

  7. Structural Basis for Norovirus Inhibition and Fucose Mimicry by Citrate

    Energy Technology Data Exchange (ETDEWEB)

    Hansman, Grant S.; Shahzad-ul-Hussan, Syed; McLellan, Jason S.; Chuang, Gwo-Yu; Georgiev, Ivelin; Shimoike, Takashi; Katayama, Kazuhiko; Bewley, Carole A.; Kwong, Peter D. (NIAID)

    2012-01-20

    Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 {angstrom} and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 {mu}M). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 {mu}M) and H type 2 trisaccharide (390 {mu}M), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

  8. Study on the Antimicrobial Properties of Citrate-Based Biodegradable Polymers

    Directory of Open Access Journals (Sweden)

    Lee-Chun eSu

    2014-07-01

    Full Text Available Citrate-based polymers possess unique advantages for various biomedical applications since citric acid is a natural metabolism product, which is biocompatible and antimicrobial. In polymer synthesis, citric acid also provides multiple functional groups to control the crosslinking of polymers and active binding sites for further conjugation of biomolecules. Our group recently developed a number of citrate-based polymers for various biomedical applications by taking advantage of their controllable chemical, mechanical, and biological characteristics. In this study, various citric acid derived biodegradable polymers were synthesized and investigated for their physicochemical and antimicrobial properties. Results indicate that citric acid derived polymers reduced bacterial proliferation to different degrees based on their chemical composition. Among the studied polymers, poly(octamethylene citrate (POC showed approximately 70-80% suppression to microbe proliferation, owing to its relatively higher ratio of citric acid contents. Crosslinked urethane-doped polyester elastomers (CUPEs and biodegradable photoluminescent polymers (BPLPs also exhibited significant bacteria reduction of ~20% and ~50% for Staphylococcus aureus and Escherichia coli, respectively. Thus, the intrinsic antibacterial properties in citrate-based polymers enable them to inhibit bacteria growth without incorporation of antibiotics, silver nanoparticles, and other traditional bacteria-killing agents suggesting that they are unique beneficial materials for wound dressing, tissue engineering, and other potential medical applications where antimicrobial property is desired.

  9. Enhancing uranium solubilization in soils by citrate, EDTA, and EDDS chelating amendments

    International Nuclear Information System (INIS)

    Highlights: ► The aim was to optimize uranium solubilization for the purposes of remediation. ► The most important factor in uranium solubilization was found to be the pH. ► Citrate treatment was the most efficient, with 63% of uranium solubilization. ► The uranium extraction yield with EDDS amendment was greater than with EDTA. - Abstract: A systematic study was made of the effects of three soil amendments on the solubilization of uranium from a granitic soil. The aim was to optimize solubilization so as to enhance bioavailability for the purposes of remediation. The three amendments tested were with citrate, EDTA, and EDDS as chelating agents. The effects of pH, chelator concentration, and leaching time were studied. The most important factor in uranium solubilization was found to be the pH. In the absence of chelating agents, the greatest solubilization was obtained for alkaline conditions, with values representing about 15% of the total uranium activity in the bulk soil. There were major differences in uranium solubilization between the different amendments. The citrate treatment was the most efficient at acidic pH, particularly with the greatest concentration of citrate tested (50 mmol kg−1) after 6 days of treatment. Under these conditions, the uranium concentration in solution was greater by a factor of 356 than in the control suspension, and represented some 63% of the uranium concentration in the bulk soil. Under alkaline conditions, the EDTA and EDDS treatments gave the greatest uranium activity concentrations in solution, but these concentrations were much lower than those with the citrate amendment, and were not very different from the control results. The uranium extraction yield with EDDS amendment was greater than with EDTA.

  10. Enhancing uranium solubilization in soils by citrate, EDTA, and EDDS chelating amendments

    Energy Technology Data Exchange (ETDEWEB)

    Lozano, J.C. [Departamento de Fisica Fundamental, Universidad de Salamanca, 37008 Salamanca (Spain); Blanco Rodriguez, P. [Natural Radioactivity Group, Universidad de Extremadura, Avda. Elvas s/n, 06071 Badajoz (Spain); Vera Tome, F., E-mail: fvt@unex.es [Natural Radioactivity Group, Universidad de Extremadura, Avda. Elvas s/n, 06071 Badajoz (Spain); Calvo, C. Prieto [Departamento de Fisica Fundamental, Universidad de Salamanca, 37008 Salamanca (Spain)

    2011-12-30

    Highlights: Black-Right-Pointing-Pointer The aim was to optimize uranium solubilization for the purposes of remediation. Black-Right-Pointing-Pointer The most important factor in uranium solubilization was found to be the pH. Black-Right-Pointing-Pointer Citrate treatment was the most efficient, with 63% of uranium solubilization. Black-Right-Pointing-Pointer The uranium extraction yield with EDDS amendment was greater than with EDTA. - Abstract: A systematic study was made of the effects of three soil amendments on the solubilization of uranium from a granitic soil. The aim was to optimize solubilization so as to enhance bioavailability for the purposes of remediation. The three amendments tested were with citrate, EDTA, and EDDS as chelating agents. The effects of pH, chelator concentration, and leaching time were studied. The most important factor in uranium solubilization was found to be the pH. In the absence of chelating agents, the greatest solubilization was obtained for alkaline conditions, with values representing about 15% of the total uranium activity in the bulk soil. There were major differences in uranium solubilization between the different amendments. The citrate treatment was the most efficient at acidic pH, particularly with the greatest concentration of citrate tested (50 mmol kg{sup -1}) after 6 days of treatment. Under these conditions, the uranium concentration in solution was greater by a factor of 356 than in the control suspension, and represented some 63% of the uranium concentration in the bulk soil. Under alkaline conditions, the EDTA and EDDS treatments gave the greatest uranium activity concentrations in solution, but these concentrations were much lower than those with the citrate amendment, and were not very different from the control results. The uranium extraction yield with EDDS amendment was greater than with EDTA.

  11. PET/CT Imaging of Tuberculosis with 68ga-Citrate

    Directory of Open Access Journals (Sweden)

    Mariza Vorster

    2015-02-01

    Full Text Available Objective: The impact of tuberculosis (TB on mortality and morbidity is indisputable worldwide and even more so in countries with a high prevalence of Human Immunodeficiency Virus (HIV co-infection. The development of a non-invasive diagnostic tool that is capable of early and accurate detection, staging and follow-up evaluation of tuberculosis is crucial in minimizing its devastating effects. We evaluated PET/CT imaging with a novel tracer, 68Ga-citrate,in this setting. Methods: Thirteen patients with tuberculosis were included in this prospective pilot study and were imaged with 68Gacitrate. A diagnosis of TB was reached with bacteriological or histopathology studies (n=8 or based on a combination of clinical data, biochemistry and imaging (n=5. PET images were acquired at 60 minutes (and 120 minutes where possible and analyzed qualitatively (relative to the liver and semi-quantitatively (using SUVmax and change in SUVmax. PET findings were also compared to that of CT. Results: All 13 patients demonstrated abnormal tracer accumulation in the lungs or extra-pulmonary or both. 68Ga-citrate accumulated in every lung lesion noted on CT in six cases (46%. In seven cases (54% some of the lung lesions noted on CT were not 68Ga-citrate avid, which is suggestive of non-active tuberculosis lesions. Ten patients (77% demonstrated extrapulmonary involvement, which included various lymph node groups, skeletal lesions, pleural-, splenic- and gastro-intestinal tract involvement. More extra-pulmonary lesions were detected on PET compared to CT in eight cases (80%. The results of dual-time point imaging varied significantly amongst study participants. Conclusion: Pulmonary and extra-pulmonary tuberculosis lesions demonstrate 68Ga-citrate accumulation; with more extrapulmonary lesions detected on PET compared to CT. 68Ga-citrate PET may also provide a way of distinguishing active from inactive lesions for treatment response evaluation.

  12. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-05

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

  13. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  14. STRUCTURAL ANALYSIS AND MOLECULAR DYNAMICS STUDY OF PHB SYNTHASE

    Directory of Open Access Journals (Sweden)

    T. Femlin Blessia

    2012-02-01

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

  15. Synthesis and characterization of biomimetic citrate-based biodegradable composites.

    Science.gov (United States)

    Tran, Richard T; Wang, Liang; Zhang, Chang; Huang, Minjun; Tang, Wanjin; Zhang, Chi; Zhang, Zhongmin; Jin, Dadi; Banik, Brittany; Brown, Justin L; Xie, Zhiwei; Bai, Xiaochun; Yang, Jian

    2014-08-01

    Natural bone apatite crystals, which mediate the development and regulate the load-bearing function of bone, have recently been associated with strongly bound citrate molecules. However, such understanding has not been translated into bone biomaterial design and osteoblast cell culture. In this work, we have developed a new class of biodegradable, mechanically strong, and biocompatible citrate-based polymer blends (CBPBs), which offer enhanced hydroxyapatite binding to produce more biomimetic composites (CBPBHAs) for orthopedic applications. CBPBHAs consist of the newly developed osteoconductive citrate-presenting biodegradable polymers, crosslinked urethane-doped polyester and poly (octanediol citrate), which can be composited with up to 65 wt % hydroxyapatite. CBPBHA networks produced materials with a compressive strength of 116.23 ± 5.37 MPa comparable to human cortical bone (100-230 MPa), and increased C2C12 osterix gene and alkaline phosphatase gene expression in vitro. The promising results above prompted an investigation on the role of citrate supplementation in culture medium for osteoblast culture, which showed that exogenous citrate supplemented into media accelerated the in vitro phenotype progression of MG-63 osteoblasts. After 6 weeks of implantation in a rabbit lateral femoral condyle defect model, CBPBHA composites elicited minimal fibrous tissue encapsulation and were well integrated with the surrounding bone tissues. The development of citrate-presenting CBPBHA biomaterials and preliminary studies revealing the effects of free exogenous citrate on osteoblast culture shows the potential of citrate biomaterials to bridge the gap in orthopedic biomaterial design and osteoblast cell culture in that the role of citrate molecules has previously been overlooked. PMID:23996976

  16. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-10

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

  17. Na/K citrate versus sodium bicarbonate in prevention of contrast-induced nephropathy.

    Science.gov (United States)

    Abouzeid, Sameh Mohamed; ElHossary, Hossam E

    2016-05-01

    Contrast-induced nephropathy (CIN) is one of the important complications of radiographic procedures, especially in patients with chronic kidney disease. It is also one of the common causes of acute kidney injury. The pathogenesis is postulated to be the effect of oxygen- free radicals and hyperosmolar stress on the renal medulla. It is reported that the production of superoxide is most active at acid environment. K/Na citrate is well known as a urine alkalinization medium, and this has been evaluated earlier with standard hydration for reduction of CIN and was stated to be efficient. We aimed to determine the efficacy of Na/K citrate in reducing the frequency of CIN in comparison to sodium bicarbonate in patients after coronary angiography. Two hundred and ten patients with renal dysfunction [estimated glomerular filtration rate (eGFR), 60 mL/min/1.73 m(2) or less] who underwent elective or emergency coronary angiography (CAG) with/without percutaneous coronary intervention (PCI) at our institution were enrolled into the study. The patients were randomized into two groups, Group 1-Taking Na/K citrate and Group 2-Taking sodium bicarbonate. Radiographic contrast agent iohexol was used. Change in creatinine, percent change in creatinine, percent change in eGFR, change in serum potassium, and urine pH were all compared between the two groups. There was no significant difference for prevention of CIN when comparing the Na/K citrate with sodium bicarbonate solution in patients exposed to CAG with or without PCI. Mean absolute change in eGFR after 48 h after administration of contrast between sodium bicarbonate group and Na/K citrate group was -0.60 ± 1.58 versus -0.71 ± 1.38. Serum potassium decreased postprocedure in the sodium bicarbonate group than in the citrate group (3.90 ± 0.33 vs. 4.14 ± 0.39). Both agents are equally effective in reducing the incidence of CIN, but the citrate would possibly be a safer option for patients at risk of hypokalemia. PMID:27215244

  18. Calcium(II)(3) (3,5-Diisopropylsalicylate)(6)(H(2)O)(6) Activates Nitric Oxide Synthase: An Accounting for its Action in Decreasing Platelet Aggregation.

    Science.gov (United States)

    Donham, D C; Sorenson, J R

    2000-01-01

    Purposes of these studies were first; to determine whether or not Calcium(II)(3) (3,5- diisopropylsalicylate)(6)(H(2)O)(6) [Ca(II)(3)(3,5-DIPS)(6)], a lipophilic calcium complex, could decrease activated-platelet aggregation, and second; to determine whether or not it is plausible that Ca(II)(3)(3,5-DIPS)(6) decreases activated-platelet aggregation by facilitating the synthesis of Nitric Oxide (NO) by Nitric Oxide Synthase (NOS). The influence of Ca(II)(3)(3,5-DIPS)(6) on the initial rate of activated-platelet aggregation was determined by measuring the decrease in rate of increase in transmission at 550 nm for a suspension of Thrombin-CaCl(2) activated platelets following the addition of 0, 50, 100, 250, or 500 muM Ca(II)(3)(3,5-DIPS)(6). To establish that the Ca(lI)(3)(3,5- DIPS)(6)-mediated decrease in aggregation was due to activation of NOS, the effect of L-NMMA, an inhibitor of NOS, on the inhibition of platelet aggregation by Ca(II)(3)(3,5-DIPS)(6) was determined using a suspension of activated platelets contaimng 0 or 250 muM Ca(II)(3)(3,5-DIPS)(6) without or with 1 mM L-NMMA. An in vitro Bovine Brain NOS reaction mixture, containing CaCl(2) for the activation of Phosphodiesterase-3' ,5'-Cyclic Nucleotide Activator required for the activation of NOS, was used to determine whether or not Ca(II)(3)(3,5-DIPS)(6) could be used as a substitute for the addition of Ca. The decrease in absorbance at 340 nm, lambda maximum for NADPH, was measured to determine NOS activity following the addition of NOS to the complete reaction mixture containing either CaCl(2), Ca(II)(3)(3,5-DIPS)(6), or neither Ca compound. Increasing the concentration of Ca(II)(3)(3,5-DIPS)(6) caused a concentration related decrease in activated platelet aggregation. The addition of L-NMMA to activated platelets, in the absence of Ca(II)(3)(3,5-DIPS)(6), caused a 129% increase in initial rate of platelet aggregation. The initial rate of platelet aggregation decreased 74% with the addition of 250 mu

  19. Folic Acid Reverses Nitric Oxide Synthase Uncoupling and Prevents Cardiac Dysfunction in Insulin Resistance: Role of Ca2+/Calmodulin-Activated Protein Kinase II

    OpenAIRE

    Roe, Nathan D.; He, Emily Y.; Wu, Zhenbiao; Ren, Jun

    2013-01-01

    Nitric oxide synthase (NOS) may be uncoupled to produce superoxide rather than nitric oxide (NO) under pathological conditions such as diabetes mellitus and insulin resistance, leading to cardiac contractile anomalies. Nonetheless, the role of NOS uncoupling in insulin resistance-induced cardiac dysfunction remains elusive. Given that folic acid may produce beneficial effect for cardiac insufficiency partially through its NOS recoupling capacity, this study was designed to evaluate the effect...

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

    OpenAIRE

    Silverman, P M; Eoyang, L

    1987-01-01

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

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

    OpenAIRE

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

    2012-01-01

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

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

    OpenAIRE

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

    2013-01-01

    Cellulose is the most abundant biopolymer on Earth, primarily formed by vascular plants, but also by some bacteria. Bacterial extracellular polysaccharides, such as cellulose and alginate, are an important component of biofilms, which are multicellular, usually sessile, aggregates of bacteria. Biofilms exhibit a greater resistance to antimicrobial treatments compared with isolated bacteria and thus are a particular concern to human health. Cellulose synthases synthesize cellulose by polymeriz...

  3. N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-γ (IFN-γ)

    OpenAIRE

    Platten, Michael; Wick, Wolfgang; Wischhusen, Jörg; WELLER, MICHAEL

    2001-01-01

    Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-γ (IFN-γ), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes.Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-γ (200 ...

  4. Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell line

    OpenAIRE

    Cruz, MT; Gonçalo, Margarida; A. Figueiredo; Carvalho, AP; Duarte, CB

    2004-01-01

    Nuclear factor kappa B (NF-kB) and activating protein-1 (AP-1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin-derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO(4)) and increases the expression of the i...

  5. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  6. Na/K citrate versus sodium bicarbonate in prevention of contrast-induced nephropathy

    OpenAIRE

    Sameh Mohamed Abouzeid; Hossam E ElHossary

    2016-01-01

    Contrast-induced nephropathy (CIN) is one of the important complications of radiographic procedures, especially in patients with chronic kidney disease. It is also one of the common causes of acute kidney injury. The pathogenesis is postulated to be the effect of oxygen- free radicals and hyperosmolar stress on the renal medulla. It is reported that the production of superoxide is most active at acid environment. K/Na citrate is well known as a urine alkalini- zation medium, and this has been...

  7. 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)

    Elisa Mitiko Kawamato

    2002-09-01

    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

  8. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan); Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  9. Effect of curcumin on nitric oxide synthase expression in Iipopolysaccharide-activated microglia cells and the anti-oxidative effect of curcumin

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND:It has been demonstrated that curcumin can increase the activities of various anti-oxidase in blood and tissue,effectively eliminate various free radicals,reduce the production of peroxisome,and alleviate oxidative stress reaction.Whether it has the same effect on microglia? OBJECTIVE:To observe the effects of curcumin on the expressions of inducible nitric oxide synthase (iNOS),nuclear factor-κB(YF-κB),and superoxide dismutase (SOD) in microglial cell line BV stimulated by lipopolysaccharide(LPS).DESIGN:An observational comparative study.SETTING:Research Room of Biochemistry,Medical College of Nantong University.MATERIALS:Mice microglia cell line BV,iNOS and NF-κ B reporter gene plasmids were presented by Dr.Bhat.NR.from the Medical University of South Carolina(USA).Curcumin was produced by the Xi'an Branch of China Chengdu Scholar Bio-Tech.Co.,Ltd.;LPS (E.Coli 026:B6).anti-mice iNOS monoclonal antibody,horseradish peroxidase labeled goat-anti-mice IgG were the products of Sigma Company (USA).METHODS:The experiments were carried out in the Research Room of Biochemistry,Medical College of Nantong University from May 2006 to April 2007.①Detection of iNOS:The cells Were seeded onto 24-well plate at the density of 1*105,After the cells had adhered to the cover glasses,the cells were grouped as negative control group(the primary antibody was replaced by phosphate bufffered solution PBS);normal control group (the cells were normally cultured);LPS-treated group(the cells were treated with LPS for 24 hours);curcumin+LPS group(the cells were treated with curcumin for 1 hour and LPS for 24 hours).The expressions of iNOS protein were detected with immunocytochemical staining.②Detennination of iNOS and NF-κ B gene activities:According to the introduction of the kit for transfection,jNOS or NF-κ B report gene plasmids were transiently transfected with Lipofectamine TM 2000 liposomes into the cells in the 24-well plate for 24 hours.The cells were divided

  10. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Science.gov (United States)

    Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

    2015-08-01

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

  12. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    OpenAIRE

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of...

  13. Starvation for ilvB operon leader amino acids other than leucine or valine does not increase acetohydroxy acid synthase activity in Escherichia coli.

    OpenAIRE

    Tsui, P; Freundlich, M

    1985-01-01

    Eleven different amino acids are encoded in the ilvB leader mRNA. Starvation for leucine or valine, but not for any of the other nine amino acids, resulted in high levels of acetohydroxy acid synthase I. These results are discussed in terms of a report (C.A. Hauser and G.W. Hatfield, Proc. Natl. Acad. Sci. U.S.A. 81:76-79, 1984) which suggests that threonine and alanine, in addition to leucine and valine, are involved in the regulation of the ilvB operon.

  14. Injectable citrate-modified Portland cement for use in vertebroplasty

    OpenAIRE

    Wynn-Jones, Gareth; Shelton, Richard M.; Hofmann, Michael P

    2014-01-01

    The injectability of Portland cement (PC) with several citrate additives was investigated for use in clinical applications such as vertebroplasty (stabilization of a fractured vertebra with bone cement) using a syringe. A 2-wt % addition of sodium or potassium citrate with PC significantly improved cement injectability, decreased cement setting times from over 2 h to below 25 min, while increasing the compressive strength to a maximum of 125 MPa. Zeta-potential measurements indicated that the...

  15. Why sildenafil and sildenafil citrate monohydrate crystals are not stable?

    OpenAIRE

    Sawatdee, Somchai; Pakawatchai, Chaveng; Nitichai, Kwanjai; Srichana, Teerapol; PHETMUNG, Hirihattaya

    2015-01-01

    Sildenafil citrate was crystallized by various techniques aiming to determine the behavior and factors affecting the crystal growth. There are only 2 types of sildenafil obtaining from crystallization: sildenafil (1) and sildenafil citrate monohydrate (2). The used techniques were (i) crystallization from saturated solutions, (ii) addition of an antisolvent, (iii) reflux and (iv) slow solvent evaporation method. By pursuing these various methods, our work pointed that the best formation of cr...

  16. THE POSSIBLE EFFECT OF SILDENAFIL CITRATE AND FENUGREEK SEED POWDER ON ENHANCING SERUM TESTOSTERONE LEVELS IN ADULT MALE ALBINO RATS

    International Nuclear Information System (INIS)

    Sildenafil citrate is a phosphodiesterase 5 inhibitor (PDE5) that increases cyclic guanosine monophosphate (cGMP) which improves vasodilatation and there is a hypothesis that fenugreek seeds have 3 mechanisms by which it may enhance serum testosterone levels. The present study aimed to evaluate the effect of sildenafil citrate and fenugreek seeds alone or in combination on serum testosterone levels in normal adult male albino rats. Besides, total protein, albumin, globulin, bilirubin levels and alanine transaminase, aspartate transaminase and alkaline phosphatase activities were evaluated. The present study claimed that single or combined treatment with sildenafil and fenugreek seed powder (FSP) may enhance serum testosterone levels in adult male albino rats

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

    OpenAIRE

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

    1990-01-01

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

  18. Nitric oxide synthase in the pineal gland

    OpenAIRE

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

    1996-01-01

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

  19. Nitric Oxide Synthases in Heart Failure

    OpenAIRE

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

    2013-01-01

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

  20. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  1. Electroretinography and immunohistochemistry of retina in rabbits treated with sildenafil citrate

    Directory of Open Access Journals (Sweden)

    A.V.C. Amaral

    2015-12-01

    Full Text Available Sildenafil citrate is a type-5 phosphodiesterase inhibitor (PDE-5, able to inhibit type-6 phosphodiesterase (PDE-6 as well, providing clinical benefits and paraeffects, some of them potentially related to the retina. The effects of the sildenafil on the retrobulbar and retinal circulation were studied in 27 adult male rabbits of the White New Zealand breed. The electric activity of the retina was evaluated before and at the end of the treatments, and immunohistochemistry studies were conducted. An amplitude increase of the b wave was found in the mixed response of cones and rods after 7 days of treatment with sildenafil citrate. However, in the other evaluations and periods, the values did not differ from the basal ones. Through immunohistochemistry, no significant decrease of the expression of PDE-5 and PDE-6 proteins was observed. Based on the results obtained, it is possible to admit that the sildenafil citrate did not change the expression of PDE-5 and PDE-6, neither the electroretinographic activity of the retina of male rabbits of the White New Zealand breed.

  2. SFH2 regulates fatty acid synthase activity in the yeast Saccharomyces cerevisiae and is critical to prevent saturated fatty acid accumulation in response to haem and oleic acid depletion.

    Science.gov (United States)

    Desfougères, Thomas; Ferreira, Thierry; Bergès, Thierry; Régnacq, Matthieu

    2008-01-01

    The yeast Saccharomyces cerevisiae is a facultative anaerobic organism. Under anaerobiosis, sustained growth relies on the presence of exogenously supplied unsaturated fatty acids and ergosterol that yeast is unable to synthesize in the absence of oxygen or upon haem depletion. In the absence of exogenous supplementation with unsaturated fatty acid, a net accumulation of SFA (saturated fatty acid) is observed that induces significant modification of phospholipid profile [Ferreira, Régnacq, Alimardani, Moreau-Vauzelle and Bergès (2004) Biochem. J. 378, 899-908]. In the present paper, we focus on the role of SFH2/CSR1, a hypoxic gene related to SEC14 and its involvement in lipid metabolism upon haem depletion in the absence of oleic acid supplementation. We observed that inactivation of SFH2 results in enhanced accumulation of SFA and phospholipid metabolism alterations. It results in premature growth arrest and leads to an exacerbated sensitivity to exogenous SFA. This phenotype is suppressed in the presence of exogenous oleic acid, or by a controlled expression of FAS1, one of the two genes encoding FAS. We present several lines of evidence to suggest that Sfh2p and oleic acid regulate SFA synthase in yeast at different levels: whereas oleic acid acts on FAS2 at the transcriptional level, we show that Sfh2p inhibits fatty acid synthase activity in response to haem depletion. PMID:17803462

  3. Regulation of oxidative enzyme activity and eukaryotic elongation factor 2 in human skeletal muscle: influence of gender and exercise

    DEFF Research Database (Denmark)

    Roepstorff, Carsten; Schjerling, P.; Vistisen, Bodil;

    2005-01-01

    AIM: To investigate gender-related differences in the responses of oxidative enzymes and eukaryotic elongation factor-2 (eEF2) to exercise. METHODS: The influence of exercise (90 min, 60%VO(2peak)) on citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HAD) activity and mRNA content......, together with eEF2 expression and phosphorylation at rest, were assessed in skeletal muscle of untrained (UT) and endurance trained (ET) females and males. RESULTS: Citrate synthase and HAD mRNA were higher in females than in males (27% and 48%, respectively, P < 0.05) whereas CS and HAD activity did not...... differ between females and males (NS). In females only, CS activity was enhanced (P < 0.05) by 90 min exercise. Resting CS mRNA content did not differ between UT and ET but, nevertheless, CS activity was 56% higher in ET than in UT volunteers (P < 0.001). HAD mRNA and activity were not influenced by...

  4. Smoking cessation early in pregnancy and birth weight, length, head circumference, and endothelial nitric oxide synthase activity in umbilical and chorionic vessels: an observational study of healthy singleton pregnancies

    DEFF Research Database (Denmark)

    Andersen, Malene R; Simonsen, Ulf; Uldbjerg, Niels;

    2009-01-01

    BACKGROUND: Reduced production of the vasodilator nitric oxide (NO) in fetal vessels in pregnant smokers may lower the blood flow to the fetus and result in lower birth weight, length, and head circumference. The present study measured endothelial NO synthase (eNOS) activity in fetal umbilical and...... chorionic vessels from nonsmokers, smokers, and ex-smokers and related the findings to the fetal outcome. METHODS AND RESULTS: Of 266 healthy, singleton pregnancies, 182 women were nonsmokers, 43 were smokers, and 41 stopped smoking early in pregnancy. eNOS activity and concentration were quantified in...... endothelial cells of the fetal vessels. Cotinine, lipid profiles, estradiol, l-arginine, and dimethylarginines that may affect NO production were determined in maternal and fetal blood. Serum cotinine verified self-reported smoking. Newborns of smokers had a lower weight (P< or =0.001) and a smaller head...

  5. Preparation of lead titanate zirconate from metal citrates

    International Nuclear Information System (INIS)

    Lead titanate zirconate (PZT) preparation from its metal constituent citrates have been investigated. Metal citrates were obtained by forced precipitation using a dehydration alcohol mixture. Salt solutions of lead nitrate and octahydrated zirconyl chloride, and titanium tetrachloride were treated separately with citric acid and ammonium hydroxide. Zirconium, titanium and lead oxides resulted from thermal decomposition of corresponding citrates at 5000 C, 4500 C and 2500 C, respectively. Lead titanate (PT) and lead zirconate (P Z) were obtained by calcining at 4500 C and 5000 C, respectively, after adequate heating of citrates mechanically mixed in ethyl ether. PZT samples were obtained with different starting stoichiometry. Rhombohedral PZT-1 53/47 sample was prepared from co precipitating zirconyl ammonium and ammonium lead citrates in presence of ethanolic titanium oxide dispersion, and calcinating at 8000 C. Rhombohedral PZT-q 52/48 sample was obtained from heating at 5000 C for 2 hours a mixture of metal citrates coprecipitated by dehydration mixture of acetone-ethanol-formic acid (2:1:0,06). Tetragonal PZT-m stoichiometry 53/47 sample were obtained by calcining at after 6000 C for 2 hours after heating a mechanically mixed metal citrates. PT phase arose at 4000 C. PZT-m powders obtained in a range of 4000 C-8000 C were isostatically pressed, and sintered at 11000 C and 12000 C in saturated Pb O atmosphere. Rhombohedral sintered PZT was obtained with 7,78 g.cm-3 at 12000 C. (author). 123 refs, 53 figs, 32 tabs

  6. Alkali absorption and citrate excretion in calcium nephrolithiasis

    Science.gov (United States)

    Sakhaee, K.; Williams, R. H.; Oh, M. S.; Padalino, P.; Adams-Huet, B.; Whitson, P.; Pak, C. Y.

    1993-01-01

    The role of net gastrointestinal (GI) alkali absorption in the development of hypocitraturia was investigated. The net GI absorption of alkali was estimated from the difference between simple urinary cations (Ca, Mg, Na, and K) and anions (Cl and P). In 131 normal subjects, the 24 h urinary citrate was positively correlated with the net GI absorption of alkali (r = 0.49, p < 0.001). In 11 patients with distal renal tubular acidosis (RTA), urinary citrate excretion was subnormal relative to net GI alkali absorption, with data from most patients residing outside the 95% confidence ellipse described for normal subjects. However, the normal relationship between urinary citrate and net absorbed alkali was maintained in 11 patients with chronic diarrheal syndrome (CDS) and in 124 stone-forming patients devoid of RTA or CDS, half of whom had "idiopathic" hypocitraturia. The 18 stone-forming patients without RTA or CDS received potassium citrate (30-60 mEq/day). Both urinary citrate and net GI alkali absorption increased, yielding a significantly positive correlation (r = 0.62, p < 0.0001), with the slope indistinguishable from that of normal subjects. Thus, urinary citrate was normally dependent on the net GI absorption of alkali. This dependence was less marked in RTA, confirming the renal origin of hypocitraturia. However, the normal dependence was maintained in CDS and in idiopathic hypocitraturia, suggesting that reduced citrate excretion was largely dietary in origin as a result of low net alkali absorption (from a probable relative deficiency of vegetables and fruits or a relative excess of animal proteins).

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

    Science.gov (United States)

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

    2016-03-15

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

  8. Cellulose synthase complexes: structure and regulation

    Directory of Open Access Journals (Sweden)

    Lei eLei

    2012-04-01

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

  9. Arsenic mobilization by citrate and malate from a red mud-treated contaminated soil.

    Science.gov (United States)

    Castaldi, Paola; Silvetti, Margherita; Mele, Elena; Garau, Giovanni; Deiana, Salvatore

    2013-01-01

    The mobility and bioavailability of As in the soil-plant system can be affected by a number of organic acids that originate from the activity of plants and microorganisms. In this study we evaluated the ability of citrate and malate anions to mobilize As in a polluted subacidic soil (UP soil) treated with red mud (RM soil). Both anions promoted the mobilization of As from UP and RM soils, with citrate being more effective than malate. The RM treatment induced a greater mobility of As. The amounts of As released in RM and UP soils treated with 3.0 mmol L citric acid solution were 2.78 and 1.83 μmol g respectively, whereas an amount equal to 1.73 and 1.06 μmol g was found after the treatment with a 3.0 mmol L malic acid solution. The release of As in both soils increased with increasing concentration of organic acids, and the co-release of Al and Fe in solution also increased. The sequential extraction showed that Fe/Al (oxi)hydroxides in RM were the main phases involved in As binding in RM soil. Two possible mechanisms could be responsible for As solubilization: (i) competition of the organic anions for As adsorption sites and (ii) partial dissolution of the adsorbents (e.g., dissolution of iron and aluminum oxi-hydroxides) induced by citrate or malate and formation of complexes between dissolved Fe and Al and organic anions. This is the first report on the effect of malate and citrate on the As mobility in a polluted soil treated with RM. PMID:23673944

  10. Rationale and study design of a three-period, 58-week trial of ferric citrate as a phosphate binder in patients with ESRD on dialysis.

    Science.gov (United States)

    Umanath, Kausik; Sika, Mohammed; Niecestro, Robert; Connelly, Carolyn; Schulman, Gerald; Koury, Mark J; Lewis, Julia B; Dwyer, Jamie P

    2013-01-01

    Chronic kidney disease associated mineral and bone disorders arise as a result of aberrant bone mineral metabolism in patients with advancing levels of renal dysfunction and end-stage renal disease. One of the cornerstones of treatment is the use of phosphate-binding agents. We describe the rationale and study design for a clinical trial to assess the safety and efficacy of ferric citrate as a phosphate binder. This trial is a three-period, international, multicenter, randomized, controlled clinical trial to assess the safety and efficacy of ferric citrate as a phosphate binder, consisting of a 2-week washout period, a 52-week safety assessment period in which subjects are randomized to ferric citrate or active control, and a 4-week efficacy assessment period in which subjects randomized to ferric citrate in the safety assessment period are randomized to ferric citrate or placebo. Eligible subjects include end-stage renal disease patients who have been treated with thrice-weekly hemodialysis or peritoneal dialysis for at least 3 months in dialysis clinics in the United States and Israel. Primary outcome measure will be the effect of ferric citrate vs. placebo on the change in serum phosphorus. Safety assessments will be performed by monitoring adverse events, concomitant medication use, and sequential blood chemistries (including iron parameters, phosphorus, and calcium). This three-period trial will assess the efficacy of ferric citrate as a phosphate binder. If proven safe and efficacious, ferric citrate will likely provide an additional phosphate binder to treat chronic kidney disease associated mineral and bone disorders. PMID:22702490

  11. Theoretical Study on Sulfur Dioxide Absorption with Citrate Solution

    Institute of Scientific and Technical Information of China (English)

    薛娟琴; 洪涛; 王召启; 李林波

    2006-01-01

    The citrate absorption of SO2 is currently one of the most successful and economic methods to harness sulfur dioxide pollution.In order to theoretically elucidate the mechanism of SO2 absorption by citrate solution and provide theoretical instruction for experiments and industrial process, the theory of multi-buffer solution, combined with computer numerical calculation methods, was applied to study the distribution parameters of the components of the citrate solution in the process of SO2 absorption and the following results were obtained: (1) HCi2- and H2Ci- in the citrate solution played the dominant role in the absorption and desorption processes; (2) Through the calculation for the buffer capacity of citrate solution, it was found that the pH of the absorption and desorption solution should be in the range of 2~8, while at pH=4.5 the buffer capacity reached its maximum. Some valuable parameters were obtained, which are instructive to the ensuing experiments and industrial design.

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

    Science.gov (United States)

    Fu, Yi; Zhu, Yi

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

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

  14. Alkali absorption and citrate excretion in calcium nephrolithiasis

    Science.gov (United States)

    Sakhaee, K.; Williams, R. H.; Oh, M. S.; Padalino, P.; Adams-Huet, B.; Whitson, P.; Pak, C. Y.

    1993-01-01

    The role of net gastrointestinal (GI) alkali absorption in the development of hypocitraturia was investigated. The net GI absorption of alkali was estimated from the difference between simple urinary cations (Ca, Mg, Na, and K) and anions (Cl and P). In 131 normal subjects, the 24 h urinary citrate was positively correlated with the net GI absorption of alkali (r = 0.49, p mEq/day). Both urinary citrate and net GI alkali absorption increased, yielding a significantly positive correlation (r = 0.62, p reduced citrate excretion was largely dietary in origin as a result of low net alkali absorption (from a probable relative deficiency of vegetables and fruits or a relative excess of animal proteins).

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

    Science.gov (United States)

    Kulmacz, R J

    1989-08-25

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

  16. Efficacy of preventing hemodialysis catheter infections with citrate lock.

    Science.gov (United States)

    Silva, Jorge; Antunes, Jorge; Carvalho, Telmo; Ponce, Pedro

    2012-10-01

    Prevalent use of tunneled dialysis catheters can reach 30%. Infection remains the most serious catheter-related problem. Catheter locks are increasingly used for prevention, but are not yet recommended either by the Food and Drug Association or European Medicines Agency, on the basis of increasing bacterial resistance or lock toxicity. The aim was to test safety and effectiveness of citrate. A prospective, interventional study was conducted to assess the safety and efficacy of a 30% citrate lock in preventing catheter-related bacteremia (CRB). A total of 157 prevalent tunneled catheters were locked with citrate and prospectively followed during a 1-year period. The primary endpoint was first CRB diagnosed according to two of the diagnostic criteria for Catheter Infection of Centers for Disease Control and Prevention (CDC), namely definite and probable infection. The CDC criterion of possible but not proved infection was not considered. This citrate lock cohort (n = 157) had 10 episodes of CRB. We observed 0.49 CRB episodes/1000 patient-days and the mean infection-free catheter day was 130.6 ± 100.9. No clinically relevant adverse events were observed. No proved tunnel or exit site infection was observed and no patients died because of CRB. Catheter obstruction episodes were reported on 69 occasions out of 14 catheters. These results were compared with an historical cohort from a previous study of catheter locking with low-dose gentamicin and did not show significant difference in efficacy. Citrate lock is effective in preventing CRB. No toxicity was observed. The use of citrate lock may have advantages over antibiotic locks: no reported bacterial resistance, lower industrial cost, and less manipulation. PMID:22515732

  17. Formation of colloidal silver nanoparticles: Capping action of citrate

    International Nuclear Information System (INIS)

    Colloidal silver sols of long-time stability are formed in the γ-irradiation of 1.0 x 10-4 M AgClO4 solutions, which also contain 0.3 M 2-propanol, 2.5 x 10-2 M N2O, and sodium citrate in various concentrations. The reduction of Ag+ in these solutions is brought about by the 1-hydroxyalkyl radical generated in the radiolysis of 2-propanol; citrate does not act as a reductant but solely as a stabilizer of the colloidal particles formed. Its concentration is varied in the range from 5.0 x 10-5 to 1.5 x 10-3 M, and the size and size distribution of the silver particles are studied by electron microscopy. At low citrate concentration, partly agglomerated large particles are formed that have many imperfections. In an intermediate range (a few 10-4 M), well-separated particles with a rather narrow size distribution and little imperfections are formed, the size slightly decreasing with increasing citrate concentration. At high citrate concentrations, large lumps of coalesced silver particles are present, due to destabilization by the high ionic strength of the solution. These findings are explained by two growth mechanisms: condensation of small silver clusters (type-1 growth), and reduction of Ag+ on silver particles via radical-to-particle electron transfer (type-2 growth). The particles formed in the intermediate range of citrate concentration were studied by high-resolution electron microscopy and computer simulations. They constitute icosahedra and cuboctahedra

  18. Effect of sildenafil citrate on penile erection of rhesus macaques

    Institute of Scientific and Technical Information of China (English)

    Xun-BinHuang; Cheng-LiangXiong; Cheng-GaoYu; Jie-LingZhou; Ji-YunShen

    2004-01-01

    Aim: To examine the effect of sildenafil citrate on penile erection of male rhesus macaque. Methods:Twenty Macaca mulatta were divided into the sildenafil treated and the control groups of l0 animals each. The penile size, the corpus cavernosal electromyogram (EMG) and the intra-corpus cavernosal pressure (ICP) were determined. Results: The diameter of penis and the ICP were significantly increased and the corpus cavernosal EMG significantly reduced in the sildenafil group. Conclusion: Sildenafil citrate increases the penile size and ICP and reduces the corpus cavernosal EMG in male rhesus macaque. (Asian J Androl 2004 Sep; 6: 233-235)

  19. Gastric fluid pH in patients receiving sodium citrate.

    Science.gov (United States)

    Viegas, O J; Ravindran, R S; Shumacker, C A

    1981-07-01

    Gastric fluid pH was measured following induction of anesthesia and placement of an endotracheal tube in 30 surgical patients undergoing elective operations. None of the patients received an anticholinergic drug before surgery. Fifteen patients who had been given 15 ml of sodium citrate 15 to 20 minutes before induction of anesthesia had a mean pH of 6.2 +/- 0.8. The control group, which also consisted of 15 patients, had a mean pH of 2.1 +/- 1.4. The increase in gastric pH noted following sodium citrate would result in reduced pulmonary reaction should aspiration occur. PMID:7195668

  20. Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

    International Nuclear Information System (INIS)

    Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP+). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP+ as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP+-induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP+ from apoptosis via the GSK-3beta signaling pathway.

  1. Hemodiafiltration using pre-dilutional on-line citrate dialysate: A new technique for regional citrate anticoagulation: A feasibility study

    Directory of Open Access Journals (Sweden)

    Radhouane Bousselmi

    2015-01-01

    Full Text Available A prospective, observational, feasibility study was carried out on four patients with end-stage renal failure undergoing bicarbonate hemodialysis to study the feasibility of an on-line hemodiafiltration technique using a citrate dialysate with pre-dilutional infusion of citrate as a technique for regional citrate anticoagulation. All patients had contraindication to systemic heparin anticoagulation. The dialysis technique consisted of an on-line hemodiafiltration with a citrate dialysate without calcium using a Fresenius 4008S dialysis machine and Fresenius Polysulfone F60 dialyzers. The infusion solution was procured directly from the dialysate and was infused into the arterial line. To avoid the risk of hypocalcemia, calcium gluconate was infused to the venous return line. The study was carried out in two stages. During the first stage, the citrate infusion rate was 80 mL/min and the calcium infusion rate was 9 mmol/h. At the second stage, the rates were 100 mL/min and 11 mmol/h, respectively. The primary endpoint of this study was the incidence of thrombosis in the extracorporeal blood circuit and/or the dialyzer. A total of 78 sessions were conducted. All the sessions were well tolerated clinically and there were no major incidents in any of the four patients. At the first stage of the study, there were five incidences of small clots in the venous blood chamber, an incidence of extracorporeal blood circuit thrombosis of 12.5%. At the second stage of the study, no cases of extracorporeal blood circuit or dialyzer thrombosis were noted. Hemodiafiltration with on-line citrate dialysate infusion to the arterial line is safe and allows an effective regional anticoagulation of the extracorporeal blood circuit without the need for systemic anticoagulation.

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

    Science.gov (United States)

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

    2003-03-25

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

  3. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    International Nuclear Information System (INIS)

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  4. Polymeric architectures of bismuth citrate based on dimeric building blocks

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Four bismuth complexes, (H2En)[Bi2(cit)2(H2O)4/3]·(H2O)x (1), (H2En)3[Bi2(cit)2Cl4]·(H2O)x (2), (HPy)2[Bi2(cit)2(H2O)8/5]·(H2O)x (3) and (H2En)[Bi2(cit)2](H2O)x (4) [cit = citrate4-; En = ethylenediamine; Py = pyridine] have been synthesized and crystallized. The crystal structures reveal that the basic building blocks in all of these complexes are bismuth citrate dimeric units which combine to form polymeric architectures. The embedded protonated ethylenediamine and pyridine moieties in the polymeric frameworks have been identified by X-ray crystallography and solid-state cross polarization/magic angle spinning (CP/MAS) 13C NMR. Based on the framework of complex 1, a structural model of a clinically used antiulcer drug, ranitidine bismuth citrate (RBC) was generated. The behavior of the protonated amine-bismuth citrate complexes in acidic aqueous solution has been studied by electrospray ionization-mass spectrometry (ESI-MS).

  5. Tumour imaging using technetium-99m-citrate

    International Nuclear Information System (INIS)

    Sixteen patients with soft tissue malignancy or fibroadenoma of the breast (Group A) were imaged using 99mTc-citrate. Majority of the patients (n=14) has new untreated lesions. Appreciable skeletal uptake of the tracer was serendipitously noticed in all cases. One of these had widespread bone metastases seen almost identically in 99mTc-citrate and 99mTc-MDP studies. Accordingly, 10 patients (Group B) having more than 40 malignant lesions on the bone scan underwent 99mTc-citrate study. In group A, accumulation of the tracer was seen in all malignant breast nodules and axillary lymphnode mass (n=4), medullary carcinoma of the thyroid along with its metastasis and a carcinoid (n=4) and an ovarian malignancy. Uptake and outflow pattern could differentiate fibroadenoma (n=3) from carcinoma of the breast. No significant uptake was seen in liver secondaries (n>10), lymphoma lesions (n=5), papillary carcinoma of thyroid, renal cell and embryonal cell carcinoma. In group B patients, the radiotracer accumulated well in the metastatic lesions while there was distinctly lesser uptake in normal/degenerated joints compared to the bone scan. The study shows potential of the tracer in imaging soft tissue malignancies. Bone scanning with 99mTc-citrate is an interesting possibility since mechanism of its uptake appears to be different to 99mTc-MDP. (author)

  6. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens

    2007-01-01

    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...

  7. {sup 14}N NQR spectrum of sildenafil citrate

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, David, E-mail: david.stephenson@sta.uwi.edu; Singh, Nadia [University of the West Indies, Chemistry Department (Trinidad and Tobago)

    2015-04-15

    The {sup 14}N nuclear quadrupole resonance (NQR) spectrum of sildenafil citrate tablets has been recorded allowing the quadrupole coupling constants and asymmetry parameters of all six unique nitrogen atoms in its structure to be determined. A density function calculation gives results that are largely in agreement with the experimental values.

  8. Enzyme Basis for pH Regulation of Citrate and Pyruvate Metabolism by Leuconostoc oenos

    NARCIS (Netherlands)

    Ramos, Ana; Lolkema, Juke S.; Konings, Wilhelmus; Santos, Helena

    1995-01-01

    Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oenos was investigated. 13C nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspensions. The utilization

  9. 76 FR 19997 - Determination That FENTORA (Fentanyl Citrate) Buccal Tablet, 300 Micrograms, Was Not Withdrawn...

    Science.gov (United States)

    2011-04-11

    ... HUMAN SERVICES Food and Drug Administration Determination That FENTORA (Fentanyl Citrate) Buccal Tablet... determined that FENTORA (fentanyl citrate) buccal tablet, 300 micrograms (mcg), was not withdrawn from sale... drug applications (ANDAs) for fentanyl citrate buccal tablet, 300 mcg, if all other legal...

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

    Science.gov (United States)

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

    2012-07-01

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

  11. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    DEFF Research Database (Denmark)

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.;

    2012-01-01

    resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99...

  12. Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

    Science.gov (United States)

    Thumelin, S; Kohl, C; Girard, J; Pégorier, J P

    1999-06-01

    The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such

  13. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

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

  14. The cystathionine-β-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels.

    Science.gov (United States)

    Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian; Chatterjee, Abhishek; Ait-Tihyaty, Maria; Ullman, Buddy; Jardim, Armando

    2016-06-01

    The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools. PMID:26853689

  15. Colon Transit Scintigraphy by 67Ga-Citrate for Idiopathic Constipation

    Directory of Open Access Journals (Sweden)

    H. Javadi

    2005-06-01

    Full Text Available Background/Objective: Segmental colonic transit studies are important in patients with severe constipation. This study is the first Iranian preliminary survey of colonic transit scintigraphy using 67Ga-citrate as a new method in constipated patients with normal radiographic and colonoscopic evaluations. Patients and Methods: Thirteen patients with idiopathic constipation underwent colon transit scintigraphy. After oral administration of 6-7 MBq 67Ga-citrates, serial abdominal images were taken up to 72 hours. Pattern classification wa s performed visually according to the distribution of radioactivity. Scintigraphic parameters such as geometric mean center (GMC of segmental retention of tracer, as well as mean ac tivity profiles and colo nic tracer half-clearance time were calculated Results: Three patterns of colonic transit scintigraph y were recognized. Nine patients had the normal pattern, i.e. excellent propagation of ac tivity. Three patients had the colonic inertia pattern with marked retention of activity in th e transverse colon and splenic flexure at 48 hours. One patient had significant retention of activity in the rectosigmoid at 72 hours, defined as functional rectosigmoid obstruction (FRSO. No significant difference was seen in GMC24h between the normal pattern and colonic inertia (P=0.053, but GMC48h and GMC72h markedly differed between the two groups (P=0.0 16 and 0.025 respectively. The mean half clearance time (MCT of the two groups was di fferent (P=0.017. Our results are well compatible with scintigraphic diagnostic criteria in different patterns of colonic transit defined by other studies with different radiotracer. Conclusion: Oral 67Ga-citrate colon transit scintigraphy is a feasible method to evaluate idiopathic constipation and seems to be a suitable surrogate for radio-opaque markers.

  16. Properties of phosphorylated thymidylate synthase.

    Science.gov (United States)

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

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  17. Metformin-clomiphene citrate vs. clomiphene citrate alone: Polycystic ovarian syndrome

    Directory of Open Access Journals (Sweden)

    Aqueela Ayaz

    2013-01-01

    Full Text Available Background: Polycystic ovary syndrome (PCOS is the commonest endocrinopathy in women that is associated with reproductive and metabolic disorders. Objectives: We compared the ovulation and conception rates after the treatment with clomiphene citrate (CC alone and in combination with metformin in infertile patients presented with polycystic ovarian syndrome (PCOS. Materials and Methods: This randomized controlled trial of independent cases and controls was conducted at the Department of Obstetrics and Gynecology, Hera General Hospital, Makkah, Saudi Arabia from February 01 to December 31, 2008. The 42 subjects diagnosed as PCOS were divided into group A and B (21 subjects in each for management with CC + metformin and CC alone, respectively. Group A received 500 mg three times a day of metformin continuously from the first cycle for 6 months or till pregnancy was confirmed. In both groups CC was started at a dose of 50 mg from day-2 till day-6 of the menstrual cycle. The dose of CC was increased to 100 mg in second and 150 mg in third cycle, and then remained 150 mg for the remaining three cycles. With ovulation the dose of CC was unaltered in both groups. Data were analyzed using Statistical Package for the Social Sciences (SPSS version 16. Results: More than 50% females in both groups were had body mass index > 25. Group A achieved high rate of regular cycles, ovulation success, and conception than group B (71.4% vs. 38.1%; P = 0.03, (76.2% vs. 38.1%; P = 0.021, and (66.6% vs. 28.6%; P = 0.01, respectively. Conclusion: Management with metformin + CC increased the ovulation and conception rates.

  18. Sodium citrate-assisted anion exchange strategy for construction of Bi2O2CO3/BiOI photocatalysts

    International Nuclear Information System (INIS)

    Highlights: • Heterostructured Bi2O2CO3/BiOI microspheres were prepared via anion exchange. • Sodium citrate-assisted anion exchange for construction of composite photocatalysts. • Bi2O2CO3/BiOI composites show high visible light photocatalytic activity. - Abstract: Bi2O2CO3/BiOI heterojuncted photocatalysts were constructed through a facile partial anion exchange strategy starting from BiOI microspheres and urea with the assistance of sodium citrate. The content of Bi2O2CO3 in the catalysts was regulated by modulating the amount of urea as a precursor, which was decomposed to generate CO32− in the hydrothermal process. Citrate anion plays a key role in controlling the morphology and composition of the products. The Bi2O2CO3/BiOI catalysts display much higher photocatalytic activity than pure BiOI and Bi2O2CO3 towards the degradation of rhodamine B (RhB) and bisphenol A (BPA). The enhancement of photocatalytic activity of the heterojuncted catalysts is attributed to the formation of p–n junction between p-BiOI and n-Bi2O2CO3, which is favorable for retarding the recombination of photoinduced electron-hole pairs. Moreover, the holes are demonstrated to be the main active species for the degradation of RhB and BPA

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

    Science.gov (United States)

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

    2015-11-25

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

  20. Regional citrate anticoagulation in critically ill patients during continuous blood purification

    Institute of Scientific and Technical Information of China (English)

    龚德华; 季大玺; 徐斌; 谢红浪; 刘云; 黎磊石

    2003-01-01

    Objectives To evaluate the safety and define the contraindication of regional citrate anticoagulation treatment on various critically ill patients being treated by continuous blood purification, who also had bleeding tendencies. Methods Forty critically ill patients being treated by continuous blood purification (CBP) were involved in this study. Due to their bleeding tendencies, regional citrate anticoagulation treatment was given to all of them. Those with hepatic function impairment (n=10) were classified as Group A, those with hypoxemia were classified as Group B (n=10), and the others as Group C (n=20). Blood samples were collected before treatment, and at 4, 12, 24, 36, and 48 hour intervals during CBP. These samples then were used arterial blood gas analysis, whole blood activated clotting time (WBACT) pre- and post-filter, and serum ionized calcium examination. Results WBACT pre-filter showed little fluctuant through the 48hr period of CBP, and WBACT post-filter showed obvious prolongation than that of the pre-filter (P<0.05) at all time points. Metabolic acidosis was found in Group A patients before CBP, and improved during CBP. Normal acid-base conditions of patients were disturbed and deteriorated in Group B during CBP, but not in Group C. Serum ionized calcium was maintained at a normal range during CBP in Group A and C patients, but declined significantly in Group B patients (vs. pre-treatment, P<0.05). Conclusions Regional citrate anticoagulation can be safely used in conjunction with CBP treatment for patients with hepatic function impairment , but may induce acidosis and a decline in serum ionized calcium when used with hypoxemic patients.

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

    Science.gov (United States)

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

    2016-04-15

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

  2. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase

    DEFF Research Database (Denmark)

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-01-01

    ? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a...... sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears...

  3. Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Raji, V. [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India); Kumar, Jatish [Division of photochemistry and photobiology, National Institute for Interdisciplinary Sciences and Technology (CSIR), Thiruvananthapuram 695 019, Kerala (India); Rejiya, C.S.; Vibin, M.; Shenoi, Vinesh N. [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India); Abraham, Annie, E-mail: annieab2@yahoo.co.in [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India)

    2011-08-15

    Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV-visible-NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I-control cells, Group II-cells treated with laser light alone, Group III-cells treated with unconjugated AuNP and further laser irradiation and Group IV-anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.

  4. Electrochemical stability of citrate-capped gold nanoparticles electrostatically assembled on amine-modified glassy carbon

    International Nuclear Information System (INIS)

    The stability of citrate-capped gold nanoparticles assembled on covalently attached ethylenediamine films on glassy carbon was probed using cyclic voltammetry and scanning electron microscopy. The accessible potential windows in 0.01 M HClO4 and phosphate buffer (pH 7.4) were found to be very similar to those for re-constructed polycrystalline bulk gold. At the nanoparticle electrodes, gold is dissolved at high positive potentials and re-deposited at lower potentials resulting in the formation of fewer, larger particles. The electrografted amine layer does not limit the stability range and citrate-capping groups do not stabilise the nanoparticles to oxidation. In addition to the structural stability of the nanoparticle electrodes, preliminary investigations of the stability of their activity for voltammetric analyses were also undertaken. Repeated voltammetric reduction of H2O2 at the nanoparticle electrodes showed excellent reproducibility indicating that the nanoparticle surface maintained a constant activity for the redox process. In contrast, a re-constructed polycrystalline bulk gold electrode did not exhibit a stable response under the same conditions.

  5. ATP citrate lyase knockdown impacts cancer stem cells in vitro

    OpenAIRE

    Hanai, J-i; Doro, N; Seth, P; Sukhatme, V P

    2013-01-01

    ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial–mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-sm...

  6. Subclinical abortions in patients treated with clomiphene citrate

    International Nuclear Information System (INIS)

    Using radioimmunoassay for human chorionic gonadotrophin beta-subunit, 39 treatment cycles of clomiphene citrate therapy were studied prospectively for incidence of subclinical abortions. Eight treatment cycles resulted in clinically recognizable pregnancies and three other treatment cycles ended up with subclinical abortions. The plasma progesterone levels in patients with subclinical abortions at the 13th day after ovulation were lower than those in patients with normal pregnancies. (author)

  7. Studies on Ga-67 citrate uptake in various experimental tumors

    International Nuclear Information System (INIS)

    Ga-67 citrate uptake was autoradiographically studied in Ehrlich's tumor and sarcoma 180 in mice and 3'-Me-DAB hepatoma in rats with a histological comparison. In order to evaluate the relationship between tumor growth and degree of Ga-67 citrate uptake, animals were sacrificed 3, 5, 7 and 10 days after transplantation of tumor cells of Ehrlich's tumor and sarcoma 180. The results were as follows. 1) When Ga-67 citrate was administered to mice having Ehrlich's tumor, sarcoma 180 and to rats having 3'-Me-DAB hepatoma, varying degrees of accumulation of Ga-67 were seen in all experimental tumors. 2) In Ehrlich's tumor and sarcoma 180, RI uptake ratio (Tumor/Normal tissue uptake ratio; T/N ratio) was increased in accordance with tumor growth and was about 5 in these tumors 10 days after transplantation of tumor cells. On the other hand, RI uptake ratio (Tumor/Normal Liver tissue uptake ratio; T/NL ratio) ranged from 1.62 to 3.31 in 3'-Me-DAB hepatoma. 3) In all tumors, remarkable accumulation of Ga-67 was demonstrated in the highly cellular sites of tumors without degeneration and necrosis. 4) In Ehrlich's tumor and sarcoma 180, an accumulation of Ga-67 citrate was also observed in granulation tissues consisting of fibroblasts, fibrocyte, neutrophils and a small number of lymphocytes around the tumor, and it was more intense than that of tumorous tissue in many cases. 5) In 3'-Me-DAB hepatoma, Ga-67 uptake was seen in hepatocellular carcinoma with little fibrous stroma, but not in cholangiocarcinoma with dense fibrous stroma. (author)

  8. Citrate Root Exudation under Zn and P Deficiency

    OpenAIRE

    Bless Aplena Elen Siane

    2012-01-01

    Zinc and phosphorus are essential nutrients with low bioavailability in calcareous soils. Some plants exude organic acids to increase the solubility of these two nutrients. The objective of this study was to examine citrate exudation rates of different lupin (Feodora and Energy) and rapeseed (Dunkeld, Yickadee and Rainbow) cultivars underdeficiencies of Zn and P. The plants were cultivated into three different nutrient solutions (complete, -Zn, and -P)with pH around 7. Under Zn deficiency, ra...

  9. Silver-YBCO composite through citrate gel decomposition

    International Nuclear Information System (INIS)

    Silver-YBCO composite containing upto 75% silver has been prepared by thermal decomposition of citrate gel. In this paper the morphological and structural changes taking place during the decomposition of the gel in the range 100--900 degrees C are presented. Heat treatment at 915 degrees C of the composite powder containing Ag2O above a critical limit has been found to impart superconductivity without any external oxygen annealing. The mechanical and microstructural features of the sintered composite are presented

  10. Electrodeposition of iron-molybdenum coatings from citrate electrolyte

    OpenAIRE

    Ved, M. V.; Sakhnenko, N. D.; Karakurkchi, A. V.; Zyubanova, S. I.

    2014-01-01

    Specifi c features of the electrodeposition of iron–molybdenum coatings from a citrate electrolyte based on iron(III) sulfate and sodium molybdate in dc and unipolar pulsed modes were studied. It was demonstrated that bright compact coatings with varied content of molybdenum can be produced by varying the relative concentrations of salts of the alloy-forming components and the solution pH. The current density ranges providing the high efficiency of the galvanostastic electrolysis were determi...

  11. Electrodeposition of iron-molybdenum-tungsten coatings from citrate electrolytes

    OpenAIRE

    Karakurkchi, A. V.; Ved, M. V.; Sakhnenko, N. D.; Yermolenko, I. Yu.

    2015-01-01

    Specific features of the electrodeposition of iron–molybdenum–tungsten coatings from citrate electrolytes based on iron(III) sulfate in the dc mode and with a unipolar pulsed current were studied. It was shown that varying the relative concentrations of salts of alloy-forming metals and the solution pH makes it possible to obtain lustrous compact coatings with low porosity and various contents of high-melting components. The effect of temperature on the coating composition and current efficie...

  12. PZ, PT and PZT formation from metal citrates

    International Nuclear Information System (INIS)

    Lead zirconate, lead titanate and lead titanate-zirconate were obtained by mechanical mixing of lead, titanium and zirconium citrates in ether and by calcination. The process was analyzed by DTA, TGA, IR, pore size distribution and surface area measurements. The results indicate that the decomposition reaction and formation of PZ, PT occur simultaneaously without formation of intermediate compounds. PZT was formed from 5000C. (author)

  13. The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose

    Directory of Open Access Journals (Sweden)

    Madoka eYonekura

    2013-03-01

    Full Text Available Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter–luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light–dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both POsSPS1::LUC and POsSPS11::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the 2 OsSPS genes.

  14. Sildenafil citrate and uteroplacental perfusion in fetal growth restriction

    Directory of Open Access Journals (Sweden)

    Marzieh Vahid Dastjerdi

    2012-01-01

    Full Text Available Background: To determine whether the phosphodiesterase type 5 inhibitor, Sildenafil citrate, affects uteroplacental perfusion. Materials and Methods: Based on a randomized double-blinded and placebo-controlled trial, forty one pregnant women with documented intrauterine growth retardation at 24-37 weeks of gestation were evaluated for the effect of a single dose of Sildenafil citrate on uteroplacental circulation as determined by Doppler ultrasound study of the umbilical and middle cerebral arteries. Statistical analysis included χ2 -test to compare proportions, and independent-samples t-test and paired student′s t-test to compare continuous variables. Results: Sildenafil group fetuses demonstrated a significant decrease in systolic/diastolic ratios (0.60 [SD 0.40] [95% Cl 0.37-0.84], P=0.000, and pulsatility index (0.12 [SD 0.15] [95% Cl 0.02-0.22], P=0.019 for the umbilical artery and a significant increase in middle cerebral artery pulsatility index (MCA PI (0.51 [SD 0.60] [95% Cl 0.16-0.85], P=0.008. Conclusion: Doppler velocimetry index values reflect decreased placental bed vascular resistance after Sildenafil. Sildenafil citrate can improve fetoplacental perfusion in pregnancies complicated by intrauterine growth restriction. It could be a potential therapeutic strategy to improve uteroplacental blood flow in pregnancies with fetal growth restriction (FGR.

  15. Calcium citrate and vitamin D in the treatment of osteoporosis.

    Science.gov (United States)

    Quesada Gómez, José Manuel; Blanch Rubió, Josep; Díaz Curiel, Manuel; Díez Pérez, Adolfo

    2011-01-01

    The combination of calcium with vitamin D (vitamin D(3) [colecalciferol]) forms the basis of preventive and therapeutic regimens for osteoporosis. A number of studies have suggested that the combination of calcium and vitamin D is effective when administered at respective dosages of at least 1200 mg and 800 IU per day, although efficacy is, as expected, affected by patient compliance. Overall, treatment with this combination appears to be effective in reducing the incidence of non-vertebral and hip fractures. Also, in all drug studies (of antiresorptive and anabolic agents and strontium ranelate) that demonstrated a reduction in risk of osteoporotic fractures, patients also took calcium and vitamin D supplements. An important finding in this regard is that vitamin D levels have been demonstrated to be inadequate in more than half of women treated for osteoporosis in the US and Europe. The capacity of the small intestine to absorb calcium salts depends on the solubility and ionization of the salts. These properties vary for different salts, with fasting calcium citrate absorption being greater than that of calcium lactogluconate and calcium carbonate. Calcium citrate formulations taken between meals may help to prevent abdominal distension and flatulence, as well as minimize the risk of renal calculus formation, thus helping to optimize patient compliance. Therefore, calcium citrate combined with vitamin D is the combination of choice for the prevention or treatment of osteoporosis. PMID:21405146

  16. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

    Science.gov (United States)

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-06-01

    Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases. PMID:17557809

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

    International Nuclear Information System (INIS)

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

  18. Conservation and Role of Electrostatics in Thymidylate Synthase

    OpenAIRE

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

    2015-01-01

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

  19. The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity

    DEFF Research Database (Denmark)

    Gaster, Michael; Petersen, Ingrid; Højlund, Kurt;

    2002-01-01

    (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The...... present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic...

  20. The influence of ferric (III citrate on ATP-hydrolases of Desulfuromonas acetoxidans ІМV В-7384

    Directory of Open Access Journals (Sweden)

    O. Maslovska

    2013-02-01

    Full Text Available Desulfuromonas acetoxidans obtains energy for growth by the anaerobic oxidation of organic compounds with the carbon dioxide formation. It was found that ferrum and manganese are used as terminal electron acceptors in the processes of anaerobic respiration, such as dissimilative Fe3+- and Mn4+-reduction, carried out by these bacteria (Lovely, 1991. D. acetoxidans ІМV B-7384 can be used as anode biocatalyst in microbial fuel cell with high electron recovery through acetate oxidation to the electric current as a result of electron transfer to the anode or 3d-type transition metals, such as ferrum and manganese, in the process of their reduction. Investigation of biochemical changes of D. acetoxidans ІМV B-7384 under the influence of Fe (III compounds is important for optimization of the process of bacterial electricity generation. ATP-hydrolase is located in cytoplasmic membrane, and its subunits are exposed to both the cytoplasm and the external environment. Therefore, the changes of that enzyme activity can be used as an indicator of various stress exposure. Presence of ferric iron ions in the bacterial growth medium could catalyze generation of organic reactive oxygen species, such as peroxyl (ROO- and alkoxyl (RO- radicals. Lipid peroxidation is one of the main reasons of cell damage and it’s following death under the influence of reactive oxygen metabolites. It is known that lipid peroxidation and membrane transport processes are somehow interrelated, but mechanisms of such interaction are still unidentified. In our previous researche we have shown the influence of ferric (III citrate on the intensity of lipid peroxidation of D. аcetoxidans ІМV В-7384. Significant increase of the content of lipid peroxidation products (lipid hydroperoxides, conjugated dienes and malondialdehyde in bacterial cells has been observed under the addition of ferric (III citrate into the cultural medium. The increase of the concentration of lipid

  1. Candesartan ameliorates acute myocardial infarction in rats through inducible nitric oxide synthase, nuclear factor‑κB, monocyte chemoattractant protein‑1, activator protein‑1 and restoration of heat shock protein 72.

    Science.gov (United States)

    Lin, Xuefeng; Wu, Min; Liu, Bo; Wang, Junkui; Guan, Gongchang; Ma, Aiqun; Zhang, Yong

    2015-12-01

    Candesartan, an angiotensin II type 1 receptor antagonist, has a variety of biological activities, including antioxidant, anti‑inflammatory and anticancer activities, with specific pharmacological effects. The present study investigated the mechanisms and protective effect of candesartan on acute myocardial infarction in rats. Male Wistar rats (8‑week‑old) were induced as a model of acute myocardial infarction and treated with candesartan (0.25 mg/kg) for 2 weeks. The present study first measured the activities of casein kinase (CK), the MB isoenzyme of creatine kinase (CK‑MB) and lactate dehydrogenase (LDH), the level of cardiac troponin T (cTnT) and infarct size. Subsequently, western blot analysis was performed to analyze the protein expression levels of inducible nitric oxide synthase (iNOS) and heat shock protein 72 (HSP72) in the rats. An enzyme linked immunosorbent assay was used to detect iNOS and nuclear factor‑κB (NF‑κB) activity. In addition, gene expression levels of monocyte chemotactic protein‑1 (MCP‑1) and activating protein‑1 (AP‑1) were determined using reverse transcription‑quantitative polymerase chain reaction analysis. Finally, the activities of caspase‑3 and caspase‑9 were examined using colorimetric assay kits. In the serum of the rat model of acute myocardial infarction, candesartan significantly increased the activities of CK, CK‑MB and LDH, and the level of cTnT. The infarction size was perfected by candesartan treatment. Candesartan significantly reduced the protein expression and activity of iNOS, the activity of NF‑κB p65, and the gene expression levels of MCP‑1 and AP‑1 in the rat model of acute myocardial infarction. Candesartan increased the protein expression of HSP‑72 in the acute myocardial infarction rat model. However, candesartan did not effect the levels of caspase‑3 or caspase‑9 in the rat model of acute myocardial infarction. These results suggested that candesartan ameliorates

  2. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata. PMID:26750479

  3. A Process-Based Model of TCA Cycle Functioning to Analyze Citrate Accumulation in Pre- and Post-Harvest Fruits

    OpenAIRE

    Etienne, Audrey; GÉNARD, MICHEL; Bugaud, Christophe

    2015-01-01

    Citrate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. The regulation of citrate accumulation throughout fruit development, and the origins of the phenotypic variability of the citrate concentration within fruit species remain to be clarified. In the present study, we developed a process-based model of citrate accumulation based on a simplified representation of the TCA cycle to predict citrate concentration in...

  4. Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume.

    Science.gov (United States)

    McShine, R L; Sibinga, S; Brozovic, B

    1990-01-01

    Platelet counts on whole blood samples collected into tripotassium salt of EDTA, trisodium citrate (Na3 citr), citrate phosphate dextrose adenine formula 1 (CPDA-1) and acid citrate dextrose formula A (ACD-A), all showed a statistically significant drop (P less than 0.01) after 1 h standing at room temperature (RT) as compared with the immediate (within 30 min) counts. After 1 h the enumeration became stable in the EDTA samples but the drop continued up to 4-6 h in those samples taken into citrate. The decreases in citrate were significant (18-30%, P less than 0.001). The addition of EDTA (1.5 mg/ml) to the citrated samples after the sixth hour count created a significant rise (6-22%, P less than 0.01) in the counts between the sixth and the seventh hour. Our observations show that platelet counts in citrated blood samples are lower than those in EDTA and highlight the necessity to present citrated samples mixed wtih dried EDTA when characterization or quality control of blood and blood components is required. Analysis of the mean platelet volume (MPV) showed significantly lower values (6-13%, P less than 0.05) in the citrated samples as compared to the same samples in EDTA, and a significant increase (4-6%, P less than 0.01) on the addition of EDTA to the citrated samples after the sixth hour analysis. PMID:2125542

  5. The Effects of Sildenafil Citrate and Vitamins A, C and E on Testicular Damage in Alloxan-Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Abuzer Tas

    2012-01-01

    Full Text Available Sildenafil citrate is an active ingredient used successfully in the treatment of erection disorders caused by a variety of factors. The purpose of this study is to research the effects of Sildenafil citrate and vitamins A, C and E on testicular degeneration in rats with alloxan diabetes. The rats were divided into II groups; healthy control and diabetic groups. The diabetic rats were divided into X groups with ten animals in each group. Group I consisted of control diabetic rats that were given only distilled water, Group II was the diabetic group treated with glibenclamide; Group III was the diabetic group treated with insulin, Group IV was the diabetic group treated with Sildenafil citrate, Group V was the diabetic group treated with Vitamin A, Group VI was the diabetic group treated with vitamin C, Group VII was the diabetic group treated with vitamin E, Group VIII was the diabetic group treated with Sildenafil citrate and vitamin A, Group IX was the diabetic group treated with Sildenafil citrate and vitamin C and Group X was the diabetic group treated with Sildenafil citrate and vitamin E. Testis tissue samples were collected on the 3, 7 and 15th days of treatment in control and diabetic groups. In the histopathological examination of the testicles of diabetic rats, the seminiferous tubules and interstitium of testes in the control groups were normal and the complete spermatogenic cells in the seminiferous tubules were healthy and uniformly arranged. Severe degeneration and tubular atrophy was observed in the seminiferous tubules on the 3, 7 and 15th days in the group with induced alloxan diabetes given only distilled water (Group I. The lesions on day 3rd in the diabetic group given glibenclamide (Group II were similar to those in the diabetic control group given only distilled water. All of the tubules had been affected by the 3rd day in group three which was given insulin. A small number of spermatogenic cells and giant cells were

  6. Exogenous citrate impairs glucose tolerance and promotes visceral adipose tissue inflammation in mice.

    Science.gov (United States)

    Leandro, João G B; Espindola-Netto, Jair M; Vianna, Maria Carolina F; Gomez, Lilian S; DeMaria, Thaina M; Marinho-Carvalho, Monica M; Zancan, Patricia; Paula Neto, Heitor A; Sola-Penna, Mauro

    2016-03-01

    Overweight and obesity have become epidemic worldwide and are linked to sedentary lifestyle and the consumption of processed foods and drinks. Citrate is a metabolite that plays central roles in carbohydrate and lipid metabolism. In addition, citrate is the additive most commonly used by the food industry, and therefore is highly consumed. Extracellular citrate can freely enter the cells via the constitutively expressed plasma membrane citrate transporter. Within the cytosol, citrate is readily metabolised by ATP-citrate lyase into acetyl-CoA - the metabolic precursor of endogenously produced lipids and cholesterol. We therefore hypothesised that the citrate ingested from processed foods and drinks could contribute to increased postprandial fat production and weight gain. To test our hypothesis, we administered citrate to mice through their drinking water with or without sucrose and monitored their weight gain and other metabolic parameters. Our results showed that mice receiving citrate or citrate+sucrose did not show increased weight gain or an increase in the weight of the liver, skeletal muscles or adipose tissues (AT). Moreover, the plasma lipid profiles (TAG, total cholesterol, LDL and HDL) were similar across all groups. However, the group receiving citrate+sucrose showed augmented fasting glycaemia, glucose intolerance and the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) in their AT. Therefore, our results suggest that citrate consumption contributes to increased AT inflammation and altered glucose metabolism, which is indicative of initial insulin resistance. Thus, citrate consumption could be a previously unknown causative agent for the complications associated with obesity. PMID:26863933

  7. Microsomal prostaglandin E2 synthase-1 is induced by conditional expression of RET/PTC in thyroid PCCL3 cells through the activation of the MEK-ERK pathway.

    Science.gov (United States)

    Puxeddu, Efisio; Mitsutake, Norisato; Knauf, Jeffrey A; Moretti, Sonia; Kim, Hei W; Seta, Karen A; Brockman, Diane; Myatt, Leslie; Millhorn, David E; Fagin, James A

    2003-12-26

    RET/PTC rearrangements are believed to be tumor-initiating events in papillary thyroid carcinomas. We identified microsomal prostaglandin E2 synthase-1 (mPGES-1) as a RET/PTC-inducible gene through subtraction hybridization cloning and expression profiling with custom microarrays. The inducible prostaglandin E2 (PGE2) biosynthetic enzymes cyclooxygenase-2 (COX-2) and mPGES-1 are up-regulated in many cancers. COX-2 is overexpressed in thyroid malignancies compared with benign nodules and normal thyroid tissues. Eicosanoids may promote tumorigenesis through effects on tumor cell growth, immune surveillance, and angiogenesis. Conditional RET/PTC1 or RET/PTC3 expression in PCCL3 thyroid cells markedly induced mPGES-1 and COX-2. PGE2 was the principal prostanoid and up-regulated (by approximately 60-fold), whereas hydroxyeicosatetraenoic acid metabolites were decreased, consistent with shunting of prostanoid biosynthesis toward PGE2 by coactivation of the two enzymes. RET/PTC activated mPGES-1 gene transcription. Based on experiments with kinase inhibitors, with PCCL3 cell lines with doxycycline-inducible expression of RET/PTC mutants with substitutions of critical tyrosine residues in the kinase domain, and lines with inducible expression of activated mutants of H-RAS and MEK1, RET/PTC was found to regulate mPGES-1 through Shc-RAS-MEK-ERK. These data show a direct relationship between activation of a tyrosine kinase receptor oncogene and regulation of PGE2 biosynthesis. As enzymes involved in prostanoid biosynthesis can be targeted with pharmacological inhibitors, these findings may have therapeutic implications. PMID:14555660

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

    Directory of Open Access Journals (Sweden)

    Jennifer M Atkinson

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

  9. Supplementation of clomiphene citrate cycles with Cimicifuga racemosa or ethinyl oestradiol--a randomized trial.

    Science.gov (United States)

    Shahin, Ahmed Y; Ismail, Alaa M; Shaaban, Omar M

    2009-10-01

    The anti-oestrogenic activity of clomiphene citrate (CC) on the cervical mucous and endometrium may be the reason for the relatively low pregnancy rates in CC induction cycles. Various follicular-phase supplements have been tried to improve cycle outcome in these patients. This study compared follicular-phase supplementation with either phytoestrogen (PE) or ethinyl oestradiol (EE) in CC induction cycles for the treatment of unexplained infertility. A total of 134 patients were randomly allocated to each treatment group (67 each). The PE group needed significantly fewer days for adequate follicular maturation, had a thicker endometrium and higher oestradiol concentration at the time of human chorionic gonadotrophin injection (all P < 0.001). The PE group had higher luteal-phase serum progesterone compared with the EE group. No significant difference was found regarding clinical pregnancy rates (14.0% versus 21.1%, respectively). In conclusion, the cycle characteristics in unexplained infertility women treated with clomiphene citrate induction and timed intercourse improved after follicular-phase supplementation with PE compared with EE supplementation. Further studies are needed to confirm the mechanism beyond these effects. PMID:19909590

  10. Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase

    International Nuclear Information System (INIS)

    The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in λgt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases

  11. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K;

    2001-01-01

    . (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of...... animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for...

  12. Gallium-67 citrate imaging in lymphoma

    International Nuclear Information System (INIS)

    Gallium imaging in lymphoma fulfils an important clinical role. The determination of the nature of a residual mass post therapy has prognostic implications for the patient and often changes treatment plans. It has been estimated that approximately 68 per cent of lymphoma patients have a residual mass after treatment but of those only 18 per cent relapse. Morphological imaging techniques such as Xray computed tomography (CT) is unable to determine whether a residual mass after treatment represents tumour or fibrotic and necrotic tissue. Gallium, being a viability agent, is able to accurately identify the amount of viable tumour cells in a mass after treatment allowing assessment of residual masses with increased confidence. This case study presents a 61 year-old lady with a history of high-grade non Hodgkin's lymphoma originating in the thyroid. Gallium imaging was undertaken immediately post surgery, three months after initial therapy and again after a resurgence in symptoms. On all three occasions the Gallium scan showed more extensive disease than that identified on CT. The most recent Gallium scan incorporated CT co registration allowing a concomitant interpretation of anatomy and physiology to be undertaken. In this patient Gallium imaging accurately differentiated active disease from post therapy changes effecting a change in disease status allowing appropriate therapy to be undertaken. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2004-07-01

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

  15. Differential behaviour of four plant polysaccharide synthases in the presence of organic solvents.

    Science.gov (United States)

    Kerry, M E; Gregory, A C; Bolwell, G P

    2001-08-01

    The behaviour of four membrane-bound glycosyl transferases involved in cell wall polysaccharide synthesis has been studied in relation to the effects of a graded series of organic solvents on their activity and type of product formed. Relative enzyme inhibition observed for some solvents was in direct relationship to the hydrophilicity of the product. This was in the order of arabinan synthase > callose synthase> xylan synthase > beta-1,4-glucan synthase. The former two were always inhibited, the xylan synthase rather less so. However, the beta-1,4-glucan synthase showed significant increases in substrate incorporation in the presence of solvents. A graded series of primary alcohols were much more effective in enhancing activity than acetone, ethyl acetate and dimethyl formamide. In the presence of the most effective solvent, methanol, there was considerable activation of beta-1,4-glucan production. This reciprocal nature of the behaviour of the beta-1,4- and beta-1,3-glucan synthases in organic solvent is supportive of recent molecular data that the two types of glucans are catalysed by separate enzyme systems. However, the results reported here do not totally negate the proposition that either enzyme is capable of synthesising the other linkage in minor amounts in vitro. PMID:11430978

  16. Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

    Indian Academy of Sciences (India)

    Sam Annie-JeyachristYn; Arumugam Geetha; Rajagopal Surendran

    2008-03-01

    Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis. In the present investigation, we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis. The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver. Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis. Platelet aggregation was also measured in the presence of the eNOS inhibitor, diphenylene iodinium chloride (DIC). The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects. During the course of platelet aggregation, concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples, whereas the elevation was not significant in platelets of patients with cirrhosis. A parallel increase was observed in the levels of NO and NOS activity. In the presence of the eNOS inhibitor, platelet aggregation was enhanced and accompanied by an elevated calcium level. The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS. Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis. The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance, and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.

  17. Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Guojing Li

    2012-06-01

    Full Text Available Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs. The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

  18. Invertase and sucrose synthase activities in coffee plants sprayed with sucrose solution Atividade de invertases e sacarose sintase em plantas de cafeeiro pulverizadas com solução de sacarose

    Directory of Open Access Journals (Sweden)

    José Carlos da Silva

    2003-01-01

    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%.Uma prática cuja eficiência não foi ainda comprovada cientificamente, é a pulverização dos cafeeiros com solução diluída de sacarose, como fonte de carbono para as plantas. Este trabalho visou estudar o efeito da pulverização de açúcar via folha nos teores endógenos de carboidratos e na atividade das enzimas invertases e sacarose sintase em mudas de cafeeiros (Coffea arabica L. com baixo (baixo e alto (normal nível de reservas de carbono. As pulverizações ocorreram nas concentrações de 0,5 e 1% de sacarose utilizando-se água como testemunha. A aplicação de sacarose a 1% aumentou a concentração de açúcares solúveis totais (AST em plantas depauperadas, como

  19. Ferric Citrate Hydrate as a Phosphate Binder and Risk of Aluminum Toxicity

    OpenAIRE

    Ajay Gupta

    2014-01-01

    Ferric citrate hydrate was recently approved in Japan as an oral phosphate binder to be taken with food for the control of hyperphosphatemia in patients with chronic kidney disease (CKD). The daily therapeutic dose is about 3 to 6 g, which comprises about 2 to 4 g of citrate. Oral citrate solubilizes aluminum that is present in food and drinking water, and opens the tight junctions in the intestinal epithelium, thereby increasing aluminum absorption and urinary excretion. In healthy animals d...

  20. Antibacterial properties and mode of action of new triaryl butene citrate compounds.

    Science.gov (United States)

    El Arbi, Mehdi; Théolier, Jérémie; Pigeon, Pascal; Jellali, Karim; Trigui, Fatma; Top, Siden; Aifa, Sami; Fliss, Ismail; Jaouen, Gérard; Hammami, Riadh

    2014-04-01

    The aim of this study was to evaluate the antibacterial activity of newly synthesized triaryl butene analogues of tamoxifen. Several compounds were synthesized and converted to citrate salts to ensure greater solubility. Four compounds showed significant antibacterial activity at micromolar concentrations against Gram-positive and Gram-negative foodborne pathogens including Listeria monocytogenes, Listeria ivanovii, Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Two compounds at 50 μM, caused only 7.8 and 11% hemolysis. One of these as well as the remaining two caused high K(+) and Na(+) efflux from bacterial cells. Ultrastructural alterations were also visible using transmission electron microscopy, which revealed severe damage of the inner or outer membrane of E. coli. L. ivanovii showed swelling, corrugations and similar damage indicating a loss of cell-wall integrity. Organometallic compounds may offer interesting opportunities for the design of novel classes of antimicrobial compounds. PMID:24602786

  1. Ferric Citrate Reduces Intravenous Iron and Erythropoiesis-Stimulating Agent Use in ESRD.

    Science.gov (United States)

    Umanath, Kausik; Jalal, Diana I; Greco, Barbara A; Umeukeje, Ebele M; Reisin, Efrain; Manley, John; Zeig, Steven; Negoi, Dana G; Hiremath, Anand N; Blumenthal, Samuel S; Sika, Mohammed; Niecestro, Robert; Koury, Mark J; Ma, Khe-Ni; Greene, Tom; Lewis, Julia B; Dwyer, Jamie P

    2015-10-01

    Ferric citrate (FC) is a phosphate binder with shown efficacy and additional effects on iron stores and use of intravenous (iv) iron and erythropoiesis-stimulating agents (ESAs). We provide detailed analyses of changes in iron/hematologic parameters and iv iron/ESA use at time points throughout the active control period of a phase 3 international randomized clinical trial. In all, 441 subjects were randomized (292 to FC and 149 to sevelamer carbonate and/or calcium acetate [active control (AC)]) and followed for 52 weeks. Subjects on FC had increased ferritin and transferrin saturation (TSAT) levels compared with subjects on AC by week 12 (change in ferritin, 114.1±29.35 ng/ml; Ptreatment with FC as a phosphate binder results in increased iron parameters apparent after 12 weeks and reduces iv iron and ESA use while maintaining hemoglobin over 52 weeks, with a safety profile similar to that of available binders. PMID:25736045

  2. Physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry.

    Science.gov (United States)

    Morgunov, Igor G; Kamzolova, Svetlana V

    2015-08-01

    In this study, physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry were studied by an investigation of growth dynamics, the consumption of glycerol, and the fatty acid fractions from waste as well as by measuring the activities of enzymes involved in the metabolism of waste. It was shown that Y. lipolytica realizes concurrent uptake of glycerol and the fatty acid fractions during conversion of glycerol-containing waste, although glycerol was utilized at a higher rate than fatty acids. Under optimal feeding of glycerol-containing waste by portions of 20 g l(-1), the citric acid production and the ratio between citric acid and isocitric acid depended on the strain used. It was revealed that wild strain Y. lipolytica VKM Y-2373 produced citrate and isocitrate with a ratio of 1.7:1, while the mutant strain Y. lipolytica NG40/UV7 synthesized presumably citric acid (122.2 g l(-1)) with a citrate-to-isocitrate ratio of 53:1 and the yield of 0.95 g g(-1). PMID:25846335

  3. Biosynthesis of P(3HB-co-3HV-co-3HHp terpolymer by Cupriavidus necator PHB-4 transformant harboring the highly active PHA synthase gene of Chromobacterium sp. USM2

    Directory of Open Access Journals (Sweden)

    Rathi, D-N.

    2013-01-01

    Full Text Available Aims: This study evaluates potentials of Cupriavidus necator PHB4 transformant harboring the highly activepolyhydroxyalkanoate synthase gene (phaC of a locally isolated Chromobacterium sp. USM2 for its ability toincorporate 3-hydroxyheptanoate (3HHp monomer.Methodology and results: A mixture of fructose and sodium heptanoate fed to the culture gave rise to poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyheptanoate, [P(3HB-co-3HV-co-3HHp] terpolymer synthesis, withtraces of 3HHp monomers confirmed through gas chromatography (GC, proton (1H and carbon (13C NMR spectra.Conclusion, significance and impact of study: This study has revealed that the PHA synthase of Chromobacteriumsp. USM2 has a broad range of substrate specificity. The synthase is able to polymerize 3-hydroxyalkanoate monomershaving 4–7 carbon atoms.

  4. Forsterite Carbonation in Wet Supercritical CO2 and Sodium Citrate

    Science.gov (United States)

    Qiu, L.; Schaef, T.; Wang, Z.; Miller, Q.; McGrail, P.

    2013-12-01

    Lin Qiu1*, Herbert T. Schaef2, Zhengrong Wang1, Quin R.S. Miller3, BP McGrail2 1. Yale University, New Haven, CT, USA 2. Pacific Northwest National Laboratory, Richland, WA, USA 3. University of Wyoming, Laramie, WY, USA Geologic reservoirs for managing carbon emissions (mostly CO2) have expanded over the last 5 years to include unconventional formations including basalts and fractured shales. Recently, ~1000 metric tons of CO2 was injected into the Columbia River Basalt (CRB) in Eastern Washington as part of the Wallula Pilot Project, Big Sky Regional Carbon Partnership. Based on reservoir conditions, the injected CO2 is present as a supercritical fluid that dissolves into the formation water over time, and reacts with basalt components to form carbonate minerals. In this paper, we discuss mineral transformation reactions occurring when the forsterite (Mg2SiO4) is exposed to wet scCO2 in equilibrium with pure water and sodium citrate solutions. Forsterite was selected as it is an important olivine group mineral present in igneous and mafic rocks. Citrate was selected as it has been shown to enhance mineral dissolution and organic ligands are possible degradation products of the microbial communities present in the formational waters of the CRB. For the supercritical phase, transformation reactions were examined by in situ high pressure x-ray diffraction (HXRD) in the presence of supercritical carbon dioxide (scCO2) in contact with water and sodium citrate solutions at conditions relevant to carbon sequestration. Experimental results show close-to-complete dissolution of forsterite in contact with scCO2 equilibrated with pure water for 90 hours (90 bar and 50°C). Under these conditions, thin films of water coated the mineral surface, providing a mechanism for silicate dissolution and transport of cations necessary for carbonate formation. The primary crystalline component initially detected with in situ HXRD was the hydrated magnesium carbonate, nesquehonite [Mg

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

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, P.M.; Eoyang, L.

    1987-06-01

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

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

    International Nuclear Information System (INIS)

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

  7. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer C Ewald

    Full Text Available Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET. We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

  8. Renal uptake of /sup 67/Ga-citrate in renal amyloidosis due to Familiar Mediterranean Fever

    Energy Technology Data Exchange (ETDEWEB)

    Banzo-Marraco, J.; Abos-Olivares, M.D.; Iribar-Ibabe, M.C.; Prats-Rivera, E.; Banzo-Marraco, J.I.; Teijeiro-Vidal, J.; Nerin-Mora, E.; Nerin de la Puerta, I.

    1981-06-01

    Renal uptake of /sup 67/Ga-citrate is described in a patient with biopsy-proven amyloidosis of the kidneys, due to Familiar Mediterranean Fever. After administration 150 MBq (4mCi) /sup 67/Ga-citrate, scans were done at 48, 72, and 120 h. Intense uptake was noted in both kidneys. A renal biopsy done 5 days after the /sup 67/Ga-citrate scan revealed a pattern typical of amyloidosis. Gallium scanning can be useful in patients with fever of unknown origin. Renal amyloidosis can be considered when renal uptake of /sup 67/Ga-citrate associated with nephrotic syndrome is observed.

  9. Comparison of the efficiency of clomiphene citrate and letrozole in combination with metformin in moderately obese clomiphene citrate - resistant polycystic ovarian syndrome patients

    Directory of Open Access Journals (Sweden)

    Bjelica Artur

    2016-01-01

    Full Text Available Introduction. Polycystic ovary syndrome is the most common endocrinopathy in women of reproductiveage. Therapy for those who want to get pregnant involves ovulation induction using clomiphene citrate, metformin, letrozole and gonadotropins. Objective. The aim of the study was to compare the efficacy of combinations of clomiphene citrate-metformin and letrozole-metformin in obese patients who are resistant to clomiphene citrate alone. Methods. The investigation was conducted as a retrospective study involving 60 moderately obese patients with polycystic ovary syndrome. Thirty-one of them received the clomiphene citrate-metformin, and 29 letrozole-metformin therapy. Stimulation was carried out for the procedures of intrauterine insemination (IUI. Results. The age of patients, duration of infertility, and body mass index in both groups were similar. There was statistically significant difference in the thickness of the endometrium in favor of the group having the letrozole-metformin therapy (8.9 ± 1.7 mm compared with the group receiving the clomiphene citrate-metformin treatment (6.3 ± 1.3 mm. The number of follicles was not statistically significantly different. Pregnancy rate in the first cycle of IUI in the clomiphene citrate group was 6.4%, and 17.2% in the letrozole group, which also was not statistically different. After the third IUI cycle, the pregnancy rate was significantly higher in the letrozole group (20.6%, while in the clomiphene citrate group it was (9.6%. Conclusion. This retrospective study demonstrated the advantages of the use of letrozole over clomiphene citrate in combination with metformin in moderately obese patients with polycystic ovary syndrome who are resistant to stimulation with clomiphene citrate alone.

  10. Citrate anticoagulation for continuous renal replacement therapy in small children

    OpenAIRE

    Soltysiak, Jolanta; Warzywoda, Alfred; Kociński, Bartłomiej; Ostalska-Nowicka, Danuta; Benedyk, Anna; Silska-Dittmar, Magdalena; Zachwieja, Jacek

    2013-01-01

    Background Regional citrate anticoagulation (RCA) is one of the methods used to prevent clotting in continuous renal replacement therapy (CRRT). The aim of this study was to describe the outcomes and complications of RCA-CRRT in comparison to heparin anticoagulation (HA)-CRRT in critically ill children. Methods This study was a retrospective review of 30 critically ill children (16 on RCA- and 14 on HA-CRRT) who underwent at least 24 h of CRRT. The mean body weight of the children was 8.69 ± ...

  11. Impact of Creatine on Coordination of Urea and Citrate Cycles

    Czech Academy of Sciences Publication Activity Database

    Kohlíková, E.; Petr, M.; Navrátil, Tomáš; Šenholdová, Z.; Přistoupilová, K.; Přistoupil, T. I.; Heyrovský, Michael; Pelclová, D.

    Praha: IV. interní klinika 1. LF UK Praha, 2006 - (Tvrzická, E.), s. 21-24 ISBN 80-239-7726-1. [Atherosklerosa 2006. Diagnostika, léčba, prevence v dětském i dospělém věku. Praha (CZ), 11.09.2006-13.09.2006] R&D Projects: GA MPO 1H-PK/42; GA MZd NR8107 Institutional research plan: CEZ:AV0Z40400503 Keywords : Atherosclerosis * Citrate cycle * Urea cycle * Blood Subject RIV: CF - Physical ; Theoretical Chemistry

  12. Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid.

    Science.gov (United States)

    Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Stec-Martyna, Emilia; Pawlowska, Zofia; Watala, Cezary

    2016-06-25

    Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1

  13. DHEA and non-alcoholic fat liver disease: increased gene expression of peroxisome proliferation-activated receptor γ (PPARγ and fatty acid synthase (FAS

    Directory of Open Access Journals (Sweden)

    Felipe Natali Almeida

    2014-05-01

    Full Text Available Dehydroespiandrosterone (DHEA is associated with improvements in chronic degenerative diseases, including obesity, insulin resistance, and cardiovascular diseases. Nevertheless, it is observed an increase in its concentration in individuals with liver lipid infiltration, but it is not precise if this condition emerges as a cause or a consequence. In this way, we aimed to identify gene expression alterations in lipid and glucose liver metabolism markers, as well as oxidative stress markers. For this purpose, male Wistar rats, 12-14 months old were treated with subcutaneous injections of DHEA (only dose of 10 mg kg-1; and after 7 days, hepatic gene expression by PCR real time were performed for the following genes:  G6Pase, PEPCK, FAS, PPARγ, malic enzyme, ChREBP, LXR, catalase, GPx, iNOS, NADPH oxidase subunits and PCNA. We observed a tendency of reduction in G6Pase gene expression in treated group (p = 0.08. In addition, it was identified an increase in liver PPARγ and FAS gene expressions, two markers of increased activity of lipogenic pathway. We also observed an increase in iNOS gene expression, a known inductor of systemic and hepatic insulin resistance. In conclusion, our data indicates that the treatment with DHEA can be associated with the development of liver lipid infiltration and hepatic insulin resistance.

  14. Producing biofuels using polyketide synthases

    Science.gov (United States)

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

    2013-04-16

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

  15. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    Science.gov (United States)

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. PMID:23911730

  16. Formulation, Characterization and Physicochemical Evaluation of Potassium Citrate Effervescent Tablets

    Directory of Open Access Journals (Sweden)

    Fatemeh Fattahi

    2013-02-01

    Full Text Available Purpose: The aim of this study was to design and formulation of potassium citrate effervescent tablet for reduction of calcium oxalate and urate kidney stones in patients suffering from kidney stones. Methods: In this study, 13 formulations were prepared from potassium citrate and effervescent base in different concentration. The flowability of powders and granules was studied. Then effervescent tablets were prepared by direct compression, fusion and wet granulation methods. The prepared tablets were evaluated for hardness, friability, effervescent time, pH, content uniformity. To amend taste of formulations, different flavoring agents were used and then panel test was done by using Latin Square method by 30 volunteers. Results: Formulations obtained from direct compression and fusion methods had good flow but low hardness. Wet granulation improves flowability and other physicochemical properties such as acceptable hardness, effervescence time ≤3 minutes, pH<6, friability < 1%, water percentage < 0.5% and accurate content uniformity. In panel test, both of combination flavors; (orange - lemon and (strawberry - raspberry had good acceptability. Conclusion: The prepared tablets by wet granulation method using PVP solution had more tablet hardness. It is a reproducible process and suitable to produce granules that are compressed into effervescent tablets due to larger agglomerates.

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  18. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

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

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat......, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous m......RNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and Fd...

  19. The effects of creatine pyruvate and creatine citrate on performance during high intensity exercise

    Directory of Open Access Journals (Sweden)

    Purpura Martin

    2008-02-01

    Full Text Available Abstract Background A double-blind, placebo-controlled, randomized study was performed to evaluate the effect of oral creatine pyruvate (Cr-Pyr and creatine citrate (Cr-Cit supplementation on exercise performance in healthy young athletes. Methods Performance during intermittent handgrip exercise of maximal intensity was evaluated before (pretest and after (posttest 28 days of Cr-Pyr (5 g/d, n = 16, Cr-Cit (5 g/d, n = 16 or placebo (pla, 5 g/d, n = 17 intake. Subjects performed ten 15-sec exercise intervals, each followed by 45 sec rest periods. Results Cr-Pyr (p Conclusion It is concluded that four weeks of Cr-Pyr and Cr-Cit intake significantly improves performance during intermittent handgrip exercise of maximal intensity and that Cr-Pyr might benefit endurance, due to enhanced activity of the aerobic metabolism.

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

    Science.gov (United States)

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

    2016-04-12

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

  1. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

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

  2. Bioavailability of zinc and phosphorus in calcareous soils as affected by citrate exudation

    NARCIS (Netherlands)

    Duffner, A.; Hoffland, E.; Temminghoff, E.J.M.

    2012-01-01

    Aims Zinc (Zn) and phosphorus (P) deficiency often occurs at the same time and limits crop production in many soils. It has been suggested that citrate root exudation is a response of plants to both deficiencies. We used white lupin (Lupinus albus L.) as a model plant to clarify if citrate exuded by

  3. PREPARATION OF SORBITOL CITRATE POLYESTERS BY REACTIVE EXTRUSION AND APPLICATION AS INHIBITIORS OF CALCIUM CARBONATE PRECIPITATION

    Science.gov (United States)

    Sorbitol citrates were prepared using a vented ZSK-30-twin-screw extruder as part of a program to develop bio-based, water soluble polycarboxylates. A Box-Behnken experimental design was used and included the variables sorbitol, citric acid, sodium citrate, temperature and feed rate. Extent of est...

  4. MECHANISM AND ENERGETICS OF A CITRATE-TRANSPORT SYSTEM OF KLEBSIELLA-PNEUMONIAE

    NARCIS (Netherlands)

    VANDERREST, ME; ABEE, T; MOLENAAR, D; KONINGS, WN

    1991-01-01

    The citrate-transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz, E. & Oesterhelt, D. (1985) EMBO J. 4, 1599 - 1603] has been subcloned in Escherichia coli DH1. Uptake of citrate in E. coli membrane vesicles via this uptake system is an electrogenic process, although the pH grad

  5. 21 CFR 520.763c - Dithiazanine iodide and piperazine citrate suspension.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Dithiazanine iodide and piperazine citrate... § 520.763c Dithiazanine iodide and piperazine citrate suspension. (a) Specifications. Each milliliter of the drug contains 69 milligrams of dithiazanine iodide and 83 milligrams of piperazine base...

  6. Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Pudlik, Agata M.; Lolkema, Juke S.

    2011-01-01

    Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transport

  7. Addition of senna improves quality of colonoscopy preparation with magnesium citrate

    Institute of Scientific and Technical Information of China (English)

    Stergios Vradelis; Evangelos Kalaitzakis; Yalda Sharifi; Otto Buchel; Satish Keshav; Roger W Chapman; Barbara Braden

    2009-01-01

    AIM: To prospectively investigate the effectiveness and patient's tolerance of two low-cost bowel cleansing preparation protocols based on magnesium citrate only or the combination of magnesium citrate and senna. METHODS: A total of 342 patients who were referred for colonoscopy underwent a colon cleansing protocol with magnesium citrate alone ( n = 160) or magnesium citrate and senna granules ( n = 182). The colonoscopist rated the overall efficacy of colon cleansing using an established score on a 4-point scale. Patients were questioned before undergoing colonoscopy for side effects and symptoms during bowel preparation. RESULTS: The percentage of procedures rescheduled because of insufficient colon cleansing was 7% in the magnesium citrate group and 4% in the magnesium citrate/senna group ( P = 0.44). Adequate visualization of the colonic mucosa was rated superior under the citramag/senna regimen ( P = 0.004). Both regimens were well tolerated, and did not significantly differ in the occurrence of nausea, bloating or headache. However, abdominal cramps were observed more often under the senna protocol (29.2%) compared to the magnesium citrate only protocol (9.9%, P < 0.0003). CONCLUSION: The addition of senna to the bowel preparation protocol with magnesium citrate significantly improves the cleansing outcome.

  8. Standardization of the biological control of 67 Ga-citrate in rats and mice

    International Nuclear Information System (INIS)

    67Ga-citrate is frequently used in Nuclear Medicine for tumor diagnosis. This study established the biological distribution parameters in rats with experimental abscesses in the left thigh. The biological model choosen made possible the routine control of 67Ga-citrate giving a realiable result in a relative short-time (60 min.) after the radionuclide administration. (author)

  9. Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F-1-ATP synthase

    OpenAIRE

    Böckmann, R.; Grubmueller, H.

    2002-01-01

    The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from rotating asymmetric gamma- 'stalk' to the three active sites of the F-1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F-1- ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By ...

  10. Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice

    OpenAIRE

    Deng, Liyuan; Elmore, C. Lee; Lawrance, Andrea K.; Matthews, Rowena G.; Rozen, Rima

    2008-01-01

    Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B12-dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we u...

  11. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen.

    OpenAIRE

    Mo, Y; Nagel, C.; Taylor, L P

    1992-01-01

    Chalcone synthase catalyzes the initial step of that branch of the phenylpropanoid pathway that leads to flavonoids. A lack of chalcone synthase activity has a pleiotropic effect in maize and petunia mutants: pollen fertility as well as flavonoid synthesis is disrupted. Both maize and petunia mutants are self-sterile due to a failure to produce a functional pollen tube. The finding that the mutant pollen is partially functional on wild-type stigmas led to the isolation and identification of k...

  12. Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.

    OpenAIRE

    Hrazdina, G; Zobel, A M; Hoch, H. C.

    1987-01-01

    Chalcone synthase [naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), E.C. 2.3.1.74], the key enzyme of flavonoid pathways that was believed to be soluble, has been localized on ribosome-bearing endoplasmic reticulum membranes in the epidermis of buckwheat (Fagopyrum esculentum M.) hypocotyls. Enzyme activity measurement and immunoblots of buckwheat hypocotyl homogenates that were fractionated on linear sucrose density gradients and developed with a spec...

  13. Defining the Potassium Binding Region in an Apple Terpene Synthase*S⃞

    OpenAIRE

    Green, Sol; Christopher J Squire; Nieuwenhuizen, Niels J.; Baker, Edward N.; Laing, William

    2009-01-01

    Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating...

  14. Structure and Mechanism of Human UDP-xylose Synthase

    OpenAIRE

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

    2012-01-01

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

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

    OpenAIRE

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

    2008-01-01

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

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

    DEFF Research Database (Denmark)

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

    1989-01-01

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

  17. Radiolabeled porphyrin versus gallium-67 citrate for the detection of human melanoma in athymic mice

    International Nuclear Information System (INIS)

    We performed the biodistribution and imaging studies of 111In and 67Ga labeled tetra(4-N-methylpyridyl) porphine, (T4NMPYP), and compared it to that of 67Ga citrate in athymic mice bearing a human melanoma xenograft. The biodistribution results of both 111In and 67Ga labeled T4NMPYP (3, 6, 24, and 48 hours) were similar but differed from that of 67Ga citrate (48 hours). The optimum tumor uptake of both radiolabeled porphyrins was at 6 hours postinjection and was lower than the tumor uptake of 67Ga citrate at 48 hours postinjection. Kidney was the only organ showing higher uptake of radiolabeled porphyrin compared to that of 67Ga citrate. The imaging studies performed with 111In T4NMPYP and 67Ga citrate correspond to the biodistribution results. Osteomyelitis present in one mouse showed good localization of 111In T4NMPYP. 15 refs., 3 figs., 5 tabs

  18. Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM

    DEFF Research Database (Denmark)

    Hansen, L; Arden, K C; Rasmussen, S B;

    1997-01-01

    Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active ...

  19. 留叶数对烟叶淀粉积累及相关酶活性的影响%Effects of Leaf Population on Starch Accumulation and Activity of Amylase and Sucrose Synthase in Tobacco Leaves

    Institute of Scientific and Technical Information of China (English)

    宋淑芳; 陈建军; 周冀衡

    2013-01-01

    为探讨烟叶成熟期间淀粉合成机理,分析了4个不同留叶数处理(分别留叶16,18,20,22片)的烟叶成熟期间中、上部叶淀粉积累动态及淀粉酶(AM)、蔗糖合成酶(SS)活性变化规律及其相关性.结果表明,留叶数与成熟期间上部叶的AM活性、淀粉含量无显著的相关性(r=-0.101,P=0.441 >0.05,r =0.221,P=0.089 >0.05),与上部叶的SS活性有显著的正相关(r=0.300*,P =0.020 <0.05);与中部叶的AM活性无显著的相关性(r=-0.179,P =0.172 >0.05),与中部叶的SS活性有极显著的正相关(r=0.395**,P=0.002<0.01),与中部叶的淀粉含量有显著的正相关(r =0.328*,P=0.011 <0.05).留叶数增多时,成熟期间上、中部叶AM活性稍有下降,SS活性显著上升,淀粉含量也有所提高.%To discuss the mechnissm of starch synthesis in flue-cured tobacco leaves during the mature stage, four treatments of leaf poputation( 16,18,20,22) were adopted,the trends of starch accumulation and the activities of amylase and sucrose synthase in middle and upper leaves were analyzed. The results showed that: in the mature stage,there were no significant correlation of leaf population with starch content and the activity of AM in upper leaves(r= -0. 101,P= 0.441 >0. 05 ,r =0. 221 ,P =0.089 >0.05) ,but there was significant correlation of that with the activity of SS(r =0. 300* ,P =0.020 0.05) ,but there was extremely significant correlation of that with the activity of SS( r =0. 395 ** ,P =0.002 <0.01) ,and significant correlation of that with starch con-tent( r =0. 328 * ,P =0.011 < 0.05). When the leaf population was increased, the activity of AM decreased slightly, but the the activity of SS went up markedly,and the starch content rose obviously.

  20. PREPARATION, CHRACTERIZATION AND EVALUATION OF STARCH CITRATE- A NEW MODIFIED STARCH AS A DISINTEGRANT IN TABLET FORMULATIONS

    OpenAIRE

    K. P. R. Chowdary

    2011-01-01

    Starch citrate prepared by reacting potato starch with citric acid at elevated temperatures was found to be a white, crystalline, non-hygroscopic powder. Starch citrate prepared exhibited excellent flow characteristics. Starch citrate was insoluble in water and aqueous fluids of acidic and alkaline pHs. It also exhibited good swelling (1500%) in water. It has no pasting or gelling property when heated at 1000C in water for 30 min. As starch citrate exhibited good swelling in water it is consi...

  1. Model‐Based Assessment of Plasma Citrate Flux Into the Liver: Implications for NaCT as a Therapeutic Target

    OpenAIRE

    Z. Li; Erion, DM; Maurer, TS

    2016-01-01

    Cytoplasmic citrate serves as an important regulator of gluconeogenesis and carbon source for de novo lipogenesis in the liver. For this reason, the sodium‐coupled citrate transporter (NaCT), a plasma membrane transporter that governs hepatic influx of plasma citrate in human, is being explored as a potential therapeutic target for metabolic disorders. As cytoplasmic citrate also originates from intracellular mitochondria, the relative contribution of these two pathways represents critical in...

  2. Spermine and citrate as metabolic biomarkers for assessing prostate cancer aggressiveness.

    Directory of Open Access Journals (Sweden)

    Guro F Giskeødegård

    Full Text Available Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients with a high cancer content (mean: 61.8% were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS. Multivariate analysis (PLS, PLS-DA and absolute quantification (LCModel were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30 and high grade (GS≥7, n = 81 cancer with normal adjacent tissue (n = 47. High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044 and citrate (p = 7.73·10(-4, and an increase in the clinically applied (total choline+creatine+polyamines/citrate (CCP/C ratio (p = 2.17·10(-4. The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71, and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.

  3. Use of Blemaren citrate formula in gout patients with nephrolithiasis

    Directory of Open Access Journals (Sweden)

    M S Eliseev

    2008-01-01

    Results. After completion of a course of Blemaren therapy, there was an 8% reduction in the mean serum UA levels, which correlated with an increase in its daily excretion (by an average of 20%. The highest increase in UA excretion was observed in 20 patients with baseline hypoex-cretion (<700 mg/day: from 226,3 (range 201,6-436,8 to 635,0 (range 272,2-705,6 mg/day (p = 0,01. UA excretion substantially unchanged in patients with normal uricosuria (>700 mg/day. Side effects that could cause the agent to be discontinued were absent. Conclusion. The Blemaren citrate formula used in gout patients with nephrolithiasis causes a significant increase in the renal excretion of UA (p = 0,01, normalizes its metabolic parameters, and shows a high safety, without worsening hepatic and renal functions and electrolyte metabolism.

  4. Effect of Nb on barium titanate prepared from citrate solutions

    Directory of Open Access Journals (Sweden)

    Stojanović Biljana D.

    2002-01-01

    Full Text Available The influence of the addition of dopants on the microstructure development and electrical properties of BaTiO3 doped with 0.2, 0.4, 0.6, 0.8 mol% of Nb and 0.01 mol% of Mn based compounds was studied. Doped barium titanate was prepared using the polymeric precursor method from citrate solutions. The powders calcined at 700°C for 4 hours were analysed by infrared (IR spectroscopy to verify the presence of carbonates, and by X-ray diffraction (XRD for phase formation. The phase composition, microstructure and dielectric properties show a strong dependence on the amount of added niobium.

  5. Inkjet printing of silver citrate conductive ink on PET substrate

    International Nuclear Information System (INIS)

    Highlights: ► A direct synthesis method of silver conductive film on PET substrate was presented. ► A stable particle-free conductive ink was prepared. ► Formation of silver-amine complex reduced the thermal decomposition temperature. ► Conductive patterns for flexible electronics were fabricated by inkjet printing. ► Silver film on PET substrate possessed highest adhesion rating even without polymer. - Abstract: Direct synthesis of silver conductive film on PET substrate by inkjet printing silver citrate conductive ink was presented in this paper. This kind of conductive ink contained silver citrate as silver precursor, 1,2-diaminopropane as complex agent dissolving the silver salt and methanol and isopropanol as a media adjusting the viscosity and surface tension. The formation of silver-amine complex reduced the decomposition temperature from 180 °C to 135 °C, thus the ink could be cured at relatively low temperature. The film reached the lowest resistivity of 17 μΩ cm after cured at 150 °C for 50 min, 3.1 μΩ cm at 230 °C and possessed high reflection and excellent adhesive property. Electrical conductivity, surface morphology and composition were investigated by four-point probe method, scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS). It is demonstrated how the cured condition affects the silver film. Moreover, radio-frequency identification (RFID) antenna was fabricated by inkjet printing, which opens up routes for the flexible electronics fabrication.

  6. Inkjet printing of silver citrate conductive ink on PET substrate

    Energy Technology Data Exchange (ETDEWEB)

    Nie Xiaolei [Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Wang Hong, E-mail: hongwang@tju.edu.cn [Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Zou Jing [Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China)

    2012-11-15

    Highlights: Black-Right-Pointing-Pointer A direct synthesis method of silver conductive film on PET substrate was presented. Black-Right-Pointing-Pointer A stable particle-free conductive ink was prepared. Black-Right-Pointing-Pointer Formation of silver-amine complex reduced the thermal decomposition temperature. Black-Right-Pointing-Pointer Conductive patterns for flexible electronics were fabricated by inkjet printing. Black-Right-Pointing-Pointer Silver film on PET substrate possessed highest adhesion rating even without polymer. - Abstract: Direct synthesis of silver conductive film on PET substrate by inkjet printing silver citrate conductive ink was presented in this paper. This kind of conductive ink contained silver citrate as silver precursor, 1,2-diaminopropane as complex agent dissolving the silver salt and methanol and isopropanol as a media adjusting the viscosity and surface tension. The formation of silver-amine complex reduced the decomposition temperature from 180 Degree-Sign C to 135 Degree-Sign C, thus the ink could be cured at relatively low temperature. The film reached the lowest resistivity of 17 {mu}{Omega} cm after cured at 150 Degree-Sign C for 50 min, 3.1 {mu}{Omega} cm at 230 Degree-Sign C and possessed high reflection and excellent adhesive property. Electrical conductivity, surface morphology and composition were investigated by four-point probe method, scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS). It is demonstrated how the cured condition affects the silver film. Moreover, radio-frequency identification (RFID) antenna was fabricated by inkjet printing, which opens up routes for the flexible electronics fabrication.

  7. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Science.gov (United States)

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. PMID:25447532

  8. Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-kappaB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line.

    OpenAIRE

    Duarte, Carlos B.; M. Celeste Lopes; Américo Figueiredo; M. Teresa Cruz; Margarida Gonçalo; Ana Luísa Vital

    2003-01-01

    AIMS: Nitric oxide (NO) has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in a mouse fetal skin dendritic cell line. METHODS: NO production was assessed by the method of Griess. Expression of the inducible isoform of nitric oxide synthase (iNOS) protein was evaluated by wester...

  9. Product Variability of the ‘Cineole Cassette'Monoterpene Synthases of Related Nicotiana Species

    Institute of Scientific and Technical Information of China (English)

    Anke F(a)hnrich; Katrin Krause; Birgit Piechulla

    2011-01-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassere' comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,and α-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoterpenes of this ‘cineole cassette' with α-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99% identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the ‘cineole cassette′ monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of N.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night (diurnal rhythm).

  10. Solubilization of beta-glucan synthases from the membranes of cultured ryegrass endosperm cells.

    Science.gov (United States)

    Henry, R J; Stone, B A

    1982-06-01

    beta-Glucan synthases were solubilized by treating membrane preparations from suspension-cultured ryegrass (lolium multiflorum) endosperm cells with detergents. Of the seven detergents tested only digitonin and octyl glucoside dissociated active synthases from the membranes. The digitonin-solubilized enzymes produced 1,4-beta-glucans and 1,3:1,4-beta-glucans, whereas the digitonin-insoluble enzymes produced, in addition, 1,3-beta-glucans. Chromatography of the digitonin-solubilized beta-glucan synthases on DEAE-Sepharose resulted in their partial purification. The octyl glucoside-solubilized enzymes produced more 1,3-beta-glucans than did the membrane-bound preparations. These results suggest that the 1,3-beta-glucan synthase is a separate enzyme and is not involved in 1,3:1,4-beta-glucan synthesis. Digitonin not only dissociated synthases from the membranes, but also stimulated synthase activity. This effect may be related to the inhibition by digitonin of glucosyl transfer from UDP-glucose to form steryl glucosides. PMID:6214254

  11. Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

    Science.gov (United States)

    Hu, Xiao-Mei; Shi, Cai-Yun; Liu, Xiao; Jin, Long-Fei; Liu, Yong-Zhong; Peng, Shu-Ang

    2015-02-01

    ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLβ1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLβ1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment. PMID:25120169

  12. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-15

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

  13. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

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

    2014-01-01

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

  15. Effect of sodium citrate on preparation of nano-sized cobalt particles by organic colloidal process

    Institute of Scientific and Technical Information of China (English)

    Huaping ZHU; Hao LI; Huiyu SONG; Shijun LIAO

    2009-01-01

    Nano-sized cobalt particles with the diameter of 2 nm were prepared via an organic colloidal process with sodium formate, ethylene glycol and sodium citrate as the reducing agent, the solvent and the complexing agent, respectively. The effects of sodium citrate on the yield, crystal structure, particle size and size distribution of the prepared nano-sized cobalt particles were then investigated. The results show that the average particle diameter decreases from 200 nm to 2 nm when the molar ratio of sodium citrate to cobalt chloride changes from 0 to 6. Furthermore, sodium citrate plays a crucial role in the controlling of size distribution of the nano-sized particles. The size distribution of the particle without sodium citrate addition is in range from tens of nanometers to 300 or 400 nm, while that with sodium citrate addition is limited in the range of (2±0.25) nm. Moreover, it is found that the addition of sodium citrate as a complex agent could decrease the yield of the nano-sized cobalt particle.

  16. Oxidative capacity and glycogen content increase more in arm than leg muscle in sedentary women after intense training

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai Baastrup; Connolly, Luke; Weihe, Pál;

    2015-01-01

    in m. deltoideus of the HIS group compared to m. vastus lateralis of the SOC group for citrate synthase maximal activity (95±89 vs. 27±34%), citrate synthase protein expression (100±29 vs. 31±44%), 3-hydroxyacyl-CoA dehydrogenase maximal activity (35±43 vs. 3±25%), muscle glycogen content (63±76 vs....... 20±51%) and expression of mitochondrial complex II, III and IV. Additionally, HIS caused higher (P<0.05) increases than MOS in m. deltoideus citrate synthase maximal activity, citrate synthase protein expression, and muscle glycogen content. In conclusion, m. deltoideus has a higher adaptive...

  17. Radiolabeling of a wound-inducible pyridoxal phosphate utilizing protein from tomato: evidence for its identification as ACC synthase

    International Nuclear Information System (INIS)

    Aminocyclopropane 1-carboxylic acid (ACC) synthase, a pyridoxal phosphate utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate limiting step in the biosynthesis of the plant hormone, ethylene. Ethylene, besides being involved in normal plant growth processes, is also produced in response to stress, e.g. wounding, pathogen infection, etc. The authors report the partial purification (400 fold) of ACC synthase from wounded pink tomato pericarp by classical techniques including ammonium sulfate precipitation, ion exchange and phenyl sepharose chromatography. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme and the low levels present in plant tissue. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase enriched fraction was achieved. Evidence that this radiolabeled protein is ACC synthase will be presented. Amino acid sequence determination of putative ACC synthase-derived peptides is underway

  18. Examination of methylphenidate-mediated behavior regulation by glycogen synthase kinase-3 in mice

    OpenAIRE

    Mines, Marjelo A.; Beurel, Eleonore; Jope, Richard S

    2012-01-01

    Abnormalities in dopaminergic activity have been implicated in psychiatric diseases, such as attention deficit hyperactivity disorder (ADHD), and are treated with therapeutic stimulants, commonly methylphenidate or amphetamine. Amphetamine administration increases glycogen synthase kinase-3 (GSK3) activation, which is necessary for certain acute behavioral responses to amphetamine, including increased locomotor activity and impaired sensorimotor gating. Here, we tested if modulating GSK3 by a...

  19. Citrate-selective electrochemical μ-sensor for early stage detection of prostate cancer

    OpenAIRE

    Azzouzi, Sawsen; Patra, Hirak K.; Ben Ali, Mounir; Abbas, Mohammed Nooredeen; Dridi, Cherif; Errachid, Abdelhamid; Turner, Anthony P. F.

    2016-01-01

    The extremely specialised anatomical function of citrate inside the prostate, make it one of the preferred biomarkers for early stage detection of prostate cancer. However, current detection methods are seriously limited due to the very low citrate concentrations that need to be measured in order to follow disease progression. In the present work, we report a novel citrate-selective μ-sensor based on iron (III) phthalocyanine chloride-C-monoamido-Poly-n-Butyl Acrylate (Fe(III)MAPcCl-P-n-BA) m...

  20. Ferric Citrate Transport of Escherichia coli: Functional Regions of the FecR Transmembrane Regulatory Protein

    OpenAIRE

    Welz, Dietrich; Braun, Volkmar

    1998-01-01

    Transcription of the ferric citrate transport genes of Escherichia coli is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR...