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Sample records for malate synthase activity

  1. Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

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    Durchschlag, H.; Zipper, P.

    1985-04-01

    Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0,30,60 h after irradiation. To elucidate the role of OH/sup -/, O/sub 2/, and H/sub 2/O/sub 2/ in the X-ray inactivation of the enzyme, experiments were performed in the absence of presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Asub(r)=3% (corresponding to a D/sub 37/=0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t=0 led to Asub(r)=21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Asub(r)=72% and D/sub 37/=6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate.

  2. Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-chen; WANG Juan; SU Pei-ying; MA Chun-mei; ZHU Shu-hua

    2014-01-01

    Mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) are sequential enzymes in Krebs cycle. mMDH, CS and the complex between mMDH and CS (mMDH+CS) were treated with nitric oxide solution. The roles of notric oxide (NO) on the secondary structures and the interactions between mMDH and CS were studied using circular diehroism (CD) and Fourier transform surface plasmon resonance (FT-SPR), respectivley. The effects of NO on the activities of mMDH, CS and mMDH+CS were also studied. And the regulations by NO on mMDH and CS were simulated by PyMOL software. The results of SPR conifrmed that strong interaction between mMDH and CS existed and NO could signiifcantly regulate the interaction between the two enzymes. NO reduced the mass percents ofα-helix and increased that of random in mMDH, CS and mMDH+CS. NO increased the activities of CS and mMDH+CS, and inhibited the activity of mMDH. Graphic simulation indicated that covalent bond was formed between NO and Asn242 in active site of CS. However, there was no direct bond between NO and mMDH. The increase in activity of mMDH+CS complex depended mostly on the interaction between NO and CS. All the results suggested that the regulations by NO on the activity and interaction between mMDH and CS were accord with the changes in mMDH, CS and mMDH+CS caused by NO.

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

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    Thomas Geoffrey C

    2011-05-01

    Full Text Available Abstract Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA comprises ~530 residues, the G isoform (MSG is ~730 residues, and this third isoform (MSH-halophilic is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C

  4. Anaerobic Induction of Isocitrate Lyase and Malate Synthase in Submerged Rice Seedlings Indicates the Important Metabolic Role of the Glyoxylate Cycle

    Institute of Scientific and Technical Information of China (English)

    Ying LU; Yong-Rui WU; Bin HAN

    2005-01-01

    The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings,we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment.The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions.

  5. Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints

    International Nuclear Information System (INIS)

    Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from ∼4.5 to ∼3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes

  6. 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...

  7. 大肠杆菌苹果酸合酶A的酶学和生理功能研究%Characterization and physiological role of malate synthase A in Escherichia coil

    Institute of Scientific and Technical Information of China (English)

    王程; 王敖; 赵旵军; 朱国萍

    2011-01-01

    Malate synthase is one of the key enzymes in glyoxylate cycle. The aceB gene, encoding malate synthase A ( MSA) , was amplified from the genomic DNA of Escherichia coli MC1655 using PCR with primers designed according to the sequence of E. coli genome. The PCR product was cloned into pET-29b( + ) , resulting the recombinant plasmid pET-MSA. The target protein ( MSA) was overexpressed in E. coli Rosetta ( DE3) under IPTG induction, and then the enzyme was purified to homogeneity. The molecular weight ( MW) of MSA was about 60 kDa. The optimal pH and temperature of MSA were pH 8. 0 and 30 C, respectively. The maximum activity of purified MSA was observed in the presence of Mg2+ . The Km and Vmax values for acetyl-CoA of MSA were 8. 07 μM and 3. 6 μM/min, respectively. Furthermore, the mutant strains, MG: : ΔaceB with MSA deletion and MG: : ΔaceBΔglcB with MSA and malate synthase G ( MSG) deletion, were constructed, respectively. It was observed that the growth rate of the mutant E. coli strain with MSA deletion was remarkably lower than that of the wild-type strain, indicating that MSA was essential for E. coli growth on acetate. Although MSG was able to partially recover the function of MSA , the glyoxylate bypass containing MSA was the much more effective metabolic pathway of glyoxylate.%苹果酸合酶是乙醛酸循环的关键酶之一.E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码.根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29t,(+),构建了重组表达质粒pET-MSA.经IPTG诱导,MSA在E.coli Rosetta(DE3)中获得高效表达.纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃.纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min.此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::AaceB和MG::AaceBAglcB.研究发现缺少MSA的E.coli突变菌株在乙

  8. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. PMID:27039280

  9. Effects of Al(III and Nano-Al13 Species on Malate Dehydrogenase Activity

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    Rong Fu Chen

    2011-05-01

    Full Text Available The effects of different aluminum species on malate dehydrogenase (MDH activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT modified glass carbon electrode (GCE. The results showed that Al(III and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III and Al13 concentration increase. Our study also found that the effects of Al(III and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

  10. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    Science.gov (United States)

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  11. Unique animal prenyltransferase with monoterpene synthase activity

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    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.

  12. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

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    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

  13. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle. PMID:198130

  14. The role of malate in the synthesis of glutamate in Pisum arvense roots

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    Genowefa Kubik-Dorosz

    2014-02-01

    Full Text Available The in vivo and in vitro activities of NADH-dependent glutamate synthase in excised Pisum arvense roots increased several-fold under the influence of malate while pyruvate oxaloacctate. citrate and succinate inhibited this entyme. The plastids isolated from Pisum arvense root,. ahen incubated with glutamine and α-ketoglutarate, released glutamate into the medium Malate clearly stimulated this process. Albizziin (25 mM completely reduced the presence of glutamate in the incubation mixture. These results indicate that reduced pyridine nucleotides arising in P. arvense root plastids during oxidation of malic acid may constitute the indispensable source of electrons for glutamic acid synthesis.

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

    Science.gov (United States)

    TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

  16. The BnALMT1 and BnALMT2 genes from rape encode aluminum-activated malate transporters that enhance the aluminum resistance of plant cells.

    Science.gov (United States)

    Ligaba, Ayalew; Katsuhara, Maki; Ryan, Peter R; Shibasaka, Mineo; Matsumoto, Hideaki

    2006-11-01

    The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.

  17. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B;

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal...... and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  18. 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

  19. SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and other transcription factors are involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 expression.

    Science.gov (United States)

    Tokizawa, Mutsutomo; Kobayashi, Yuriko; Saito, Tatsunori; Kobayashi, Masatomo; Iuchi, Satoshi; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y; Koyama, Hiroyuki

    2015-03-01

    In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

  20. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

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

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    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. Malate and fumarate extend lifespan in Caenorhabditis elegans.

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    Clare B Edwards

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

  3. Development Of New Visible Spectrophotometric Methods For Quantitative Determination Of Almotriptan Malate As An Active Pharmaceutical Ingredient In Formulations

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    U. Viplava Prasad

    2012-06-01

    Full Text Available Purpose: The aim of the investigation was to see the simple and sensitive visible spectrophotometric methods for the determination of the almotriptan malate in bulk and tablet dosage forms. Methods: Two simple, sensitive and cost effective visible spectrophotometric methods (M1-M2 were developed for the estimation of almotriptan malate in bulk and dosage forms. The first method (M1 is based on the formation of blue reduced product by treating drug with Folin Ciocalteu (FC reagent in the presence of sodium carbonate solution with an absorption maximum of 770nm. The second method (M2 is based on the complex formation product by drug with 1, 10-phenanthroline in the presence of Fe (III as an oxidant in phosphoric acid medium with an absorption maximum of 510nm. Results: Beer’s law obeyed in the concentration range of 4-12μg/ml and 1-5 μg/ml for methodM1 and M2 respectively. No interference was observed from the usually existing additives in pharmaceutical formulations and the applicability of the methods was examined by analyzing AXERT tablets containing AM. Conclusion: The reported methods for its assay involve sophisticated equipment, which are very costly and pose problems of maintenance. To overcome these problems, the use of visible spectrophotometric technique is justifiable. The statistical data proved the accuracy, reproducibility and the precision of the proposed methods.

  4. 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...

  5. 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

  6. 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...

  7. Common catabolic enzyme patterns in a microplankton community of the Humboldt Current System off northern and central-south Chile: Malate dehydrogenase activity as an index of water-column metabolism in an oxygen minimum zone

    Science.gov (United States)

    González, R. R.; Quiñones, R. A.

    2009-07-01

    An extensive subsurface oxygen minimum zone off northern and central-south Chile, associated with the Peru-Chile undercurrent, has important effects on the metabolism of the organisms inhabiting therein. Planktonic species deal with the hypoxic and anoxic environments by relying on biochemical as well as physiological processes related to their anaerobic metabolisms. Here we characterize, for the first time, the potential enzymatic activities involved in the aerobic and anaerobic energy production pathways of microplanktonic organisms (catabolic pathways in the oxygen minimum zone. Malate dehydrogenase had the highest oxidizing activity of nicotinamide adenine dinucleotide (reduced form) in the batch of catabolic enzymatic activities assayed, including potential pyruvate oxidoreductases activity, the electron transport system, and dissimilatory nitrate reductase. Malate dehydrogenase correlated significantly with almost all the enzymes analyzed within and above the oxygen minimum zone, and also with the oxygen concentration and microplankton biomass in the water column of the Humboldt Current System, especially in the oxygen minimum zone off Iquique. These results suggest a possible specific pattern for the catabolic activity of the microplanktonic realm associated with the oxygen minimum zone spread along the Humboldt Current System off Chile. We hypothesize that malate dehydrogenase activity could be an appropriate indicator of microplankton catabolism in the oxygen minimum zone and adjacent areas.

  8. Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis.

    Science.gov (United States)

    Sallé-Lefort, Sandrine; Miard, Stéphanie; Nolin, Marc-André; Boivin, Louise; Paré, Marie-Ève; Debigaré, Richard; Picard, Frédéric

    2016-10-01

    Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway. PMID:27499160

  9. 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

  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. 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...

  12. 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.

  13. The role of malate in plant homeostasis

    OpenAIRE

    Finkemeier, Iris; Sweetlove, Lee J.

    2009-01-01

    Malate is a central metabolite of the plant cell with important roles in plant physiology and metabolism. Here, we summarize the most recent advances in our understanding of malate homeostasis in central metabolism, guard cell functioning, and root exudation.

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

    Institute of Scientific and Technical Information of China (English)

    Guang-Jin Yuan; Xiao-Rong Zhou; Zuo-Jiong Gong; Pin Zhang; Xiao-Mei Sun; Shi-Hua Zheng

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB (NF-кB) and tumor necrosis factor-α (TNF-α)expression in the liver.METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT)activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-KB p65, iNOS, eNOS and TNF-αprotein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR).RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-кB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-кB, and TNF-α mRNA expression.CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-αexpression. eNOS activity is reduced, but its mRNA expression is not affected.

  15. 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

  16. [Effect of heavy metals on activity of key enzymes of glyoxylate cycle and content of thiobarbituric acid reactive substances in the germinating soybean Glicine max L.seeds].

    Science.gov (United States)

    Bezdudnaia, E F; Kaliman, P A

    2008-01-01

    The influence of CoCl2 and CdCl2 on the activity of isocytrate lyase, malate synthase and NAD-malate dehydrogenase in the seed lobes and the composition of malondialdehyde products at early stages of germinating of soybean seeds: after first 24-hours, 72 hours and 96 hours are investigated. It is shown that when germinating in the medium containing no metal salts, isocytrate lyase activity is greatly increased during 96 h and malate synthase is increased after 72 h and is decreased after 96 h of germination period. CoCl2 activated isocytrate lyase activity after 72 hours and decreased malate synthase activity after 96 hours. The lengthening of the primary root under such conditions is noted. CdCl2 inhibited isocytrate lyase activity during first 24 hours and suppressed malate synthase activity after 96 hours. During this process the germ growth is suppressed. CoCl2 increased the composition of malondialdehyde products during each period of germination, and CdCl2 increased malondialdehyde content after 72 and 96 hours. The role of glyoxylate cycle enzymes in transforming fatty acids into carbohydrates and in forming the primary root under the process of germination of seed lobes of soybean is discussed. PMID:18710031

  17. 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.

  18. Pressure-related activation of inducible nitric oxide synthase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A lot of reports suggested that inducible nitric oxide synthase (iNOS) has a very different nature from constitutive NOS including endothelial NOS (eNOS) and neural NOS (nNOS). When exposed to cytokines or bacterial products, iNOS could be greatly activated and produces hundreds or thousands fold more NO than it does usually. Whether iNOS activation is arterial pressure related is not clear. In the present experiment, we studied three groups(n=6) of Sprague Dawley (SD) rats with implanted aorta and venous catheters that were maintained on 1 mEq/d, 12.5 mEq/d and 25 mEq/d of sodium intake respectively. Pulsatile arterial pressure signals from the amplifier were sent to a digital computer and the urine samples were taken every other day for nitrate/nitrite excretion (UNOx) assay using Greiss Reaction. After 6 days infusion, the rats were euthanized with an overdose of sodium pentobarbital, and the renal medullas were rapidly removed and frozen on dry ice for iNOS activity assay. Morever separate groups of hypertensive rats including spontaneously hypertensive rat (SHR, n=6) and High NaCl-induced hypertensive rat (NaHR, n=6) were used to measure renal iNOS protein by Western Blotting. The results showed that the mean arterial pressure (MAP) were significantly increased with the increase intake of sodium, the MAP (mmHg) at day 6 were 99.6±3.5,116.65±4.2 and 125.43±4.5, and the iNOS activity (nmol*g-1 protein*min-1) were 122.3±23.4, 342.4±35.6 and 623.9±65.4 in 1 mEq/d, 12.5 mEq/d and 25 mEq/d of sodium intake-rats respectively. At the same time, UNOx at day 6 were also increased, in turn, to 5 865.6±343.0 (for 12.5 mEq/d intake-rats) and (9 642.8±1 045.3) (for 25 mEq/d sodium intake-rats) nmol/d from (3 834.9±234.8) nmol/d of 1 mEq/d sodium intake-rats respectively. Western blotting showed that the renal medullary iNOS protein in SHR and NaHR were increased by 178%±13% and 104%±9% of normal Wistar rats. The data indicates that elevated arterial pressure

  19. 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...

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-17

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

  2. 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)

  3. Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle.

    Science.gov (United States)

    Moreau, F; Romani, R

    1982-11-01

    After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with alpha-ketoglutarate and malate as substrates. Whereas alpha-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD(+), glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

  4. Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

    Science.gov (United States)

    Yao, Jin; Wang, Xiao-Qun; Li, Yu-Jie; Shan, Kun; Yang, Hong; Wang, Yang-Ning-Zhi; Yao, Mu-Di; Liu, Chang; Li, Xiu-Miao; Shen, Yi; Liu, Jing-Yu; Cheng, Hong; Yuan, Jun; Zhang, Yang-Yang; Jiang, Qin; Yan, Biao

    2016-01-01

    The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro MALAT1-CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling. PMID:26964565

  5. Synthesis of novel methotrexate derivatives with inhibition activity of nitric oxide synthase

    Institute of Scientific and Technical Information of China (English)

    Ming Sheng Feng; Ping Guo; Li Xun Jiang; Jing Bo Shi; Yu Ping Cao; Qi Zheng Yao

    2009-01-01

    Seventeen 4-alkylamino/arylamino-substituted methotrexate(MTX)derivatives 6a-14a were designed and synthesized.Their inhibition activities against inducible nitric oxide synthase(iNOS)were evaluated in vitro.The pharmacological results showed that most of the prepared compounds displayed the potent inhibitory effects on iNOS.

  6. "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...

  7. 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 ...

  8. 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

  9. 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.

  10. A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases.

    Science.gov (United States)

    Pieńkowska, J; Wrzesiński, J; Szweykowska-Kulińska, Z

    1998-01-01

    Plant cytoplasmic tyrosine tRNA was pseudouridylated at three different positions: 35, 39 and 55. These pseudouridines were introduced by three different enzymes--pseudouridine synthases. Variants of the Arabidopsis thaliana pre-tRNA(Tyr) were constructed that allow to monitor specifically pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least psi35 and psi55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNA(Tyr) transcribed by T7 RNA polymerase. U35 is converted to psi35 only in an intron-dependent manner, while pseudouridylation of U55 is insensitive to the presence or absence of an intron.

  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. Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne.

    Science.gov (United States)

    Hong, Hoang Thi Kim; Nose, Akihiro; Agarie, Sakae

    2004-10-01

    An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process. PMID:15361538

  13. Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne.

    Science.gov (United States)

    Hong, Hoang Thi Kim; Nose, Akihiro; Agarie, Sakae

    2004-10-01

    An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.

  14. A connecting hinge represses the activity of endothelial nitric oxide synthase

    OpenAIRE

    Haque, Mohammad Mahfuzul; Panda, Koustubh; Tejero, Jesús; Aulak, Kulwant S.; Fadlalla, Mohammed Adam; Mustovich, Anthony T.; Stuehr, Dennis J

    2007-01-01

    In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that e...

  15. 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

  16. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  17. A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity.

    Science.gov (United States)

    Harada, Emiko; von Roepenack-Lahaye, Edda; Clemens, Stephan

    2004-12-01

    Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

  18. [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

  19. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

    Science.gov (United States)

    Leirós, C P; Rosignoli, F; Genaro, A M; Sales, M E; Sterin-Borda, L; Santiago BordaE

    2000-03-15

    Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

  20. Downregulation of lncRNA-MALAT1 Affects Proliferation and the Expression of Stemness Markers in Glioma Stem Cell Line SHG139S.

    Science.gov (United States)

    Han, Yong; Zhou, Liang; Wu, Tingfeng; Huang, Yulun; Cheng, Zhe; Li, Xuetao; Sun, Ting; Zhou, Youxin; Du, Ziwei

    2016-10-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is among the most abundant and highly conserved lncRNAs, which has been detected in a wide variety of human tumors, including gastric cancer, gallbladder cancer, and so on. Previous research has showed that MALAT1 can activate LTBP3 gene in mesenchymal stem cells. However, the specific roles of MALAT1 in glioma stem cells (GSCs) remain unclear. In this study, we aimed to identify the effects of MALAT1 on proliferation and the expression of stemness markers on glioma stem cell line SHG139S. Our results showed that downregulation of MALAT1 suppressed the expression of Sox2 and Nestin which are related to stemness, while downregulation of MALAT1 promoted the proliferation in SHG139S. Further research on the underlying mechanism showed that the effects of MALAT1 downregulation on SHG139S were through regulating ERK/MAPk signaling activity. And we also found that downregulation of MALAT1 could activate ERK/MAPK signaling and promoted proliferation in SHG139 cells. These findings show that MALAT1 plays an important role in regulating the expression of stemness markers and proliferation of SHG139S, and provide a new research direction to target the progression of GSCs. PMID:26649728

  1. 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.

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Chih-Jung Kuo

    2008-01-01

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

  4. 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...

  5. The metabolism of malate by cultured rat brain astrocytes

    International Nuclear Information System (INIS)

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

  6. 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.

  7. 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.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    Silva José Carlos da

    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%.

  10. Extract of Meretrix meretrix Linnaeus induces angiogenesis in vitro and activates endothelial nitric oxide synthase

    Science.gov (United States)

    Liu, Ming; Wei, Jianteng; Wang, Hui; Ding, Lili; Zhang, Yuyan; Lin, Xiukun

    2012-09-01

    Meretrix meretrix Linnaeus has long been used as traditional Chinese medicine in oriental medicine. The angiogentic activity of the extract of M. meretrix was investigated in this study, using human umbilical vein endothelial cells (HUVECs). Extract of M. meretrix Linnaeus (AFG-25) was prepared with acetone and ethanol precipitation, and further separated by Sephadex G-25 column. The results show that AFG-25 promoted proliferation, migration, and capillary-like tube formation in HUVECs, and in the presence of eNOS inhibitor NMA, the tube formation induced by AFG-25 is inhibited significantly. Moreover, AFG-25 could also promote the activation of endothelial nitric oxide synthase (eNOS) and the resultant elevation of nitric oxide (NO) production. The results suggested that M. meretrix contains active ingredients with angiogentic activity and eNOS/NO signal pathway is in part involved in the proangiogenesis effect induced by AFG-25.

  11. Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Shuai Yang

    Full Text Available To study the role of long non-coding RNA (lncRNA MALAT1 in transforming growth factor beta 1 (TGF-β1-induced epithelial-mesenchymal transition (EMT of retinal pigment epithelial (RPE cells.ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1 at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA. The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR vitreous samples.The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.

  12. 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.

  13. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

    Science.gov (United States)

    Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E; Khosla, Chaitan

    2016-08-16

    Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.

  14. Characterization of a bifunctional enzyme with (p)ppGpp-hydrolase/synthase activity in Leptospira interrogans.

    Science.gov (United States)

    He, Ping; Deng, Cong; Liu, Boyu; Zeng, LingBing; Zhao, Wei; Zhang, Yan; Jiang, XuCheng; Guo, XiaoKui; Qin, JinHong

    2013-11-01

    Alarmone Guanosine 5'-diphosphate (or 5'-triphosphate) 3'-diphosphate [(p)ppGpp] is the key component that globally regulates stringent control in bacteria. There are two homologous enzymes, RelA and SpoT in Escherichia coli, which are responsible for fluctuations in (p)ppGpp concentration inside the cell, whereas there exists only a single RelA/SpoT enzyme in Gram-positive bacteria. We have identified a bifunctional enzyme with (p)ppGpp-hydrolase/synthase activity in Leptospira interrogans. We show that the relLin gene (LA_3085) encodes a protein that fully complements the relA/spoT double mutants in E. coli. The protein functions as a (p)ppGpp degradase as well as a (p)ppGpp synthase when the cells encounter amino acid stress and deprivation of carbon sources. N-terminus HD and RSD domains of relLin (relLinN ) were observed to restore growth of double mutants of E. coli. Finally, We demonstrate that purified RelLin and RelLinN show high (p)ppGpp synthesis activity in vitro. Taken together, our results suggest that L. interrogans contain a single Rel-like bifunctional protein, RelLin , which plays an important role in maintaining the basal level of (p)ppGpp in the cell potentially contributing to the regulation of bacterial stress response.

  15. 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 ...

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. Regulation of sucrose synthase activity and sugar yield by nitrogen in sugar beet

    Institute of Scientific and Technical Information of China (English)

    LI Caifeng; MA Fengming; LI Wenhua; WANG Rui; CHEN Shengyong; LUO Yu

    2007-01-01

    The content of sugar is influenced by sucrose synthase (SS) activity in roots. The effects of nitrogen level in the aminonitrate ratio on SS activity of leaves and roots, roots yield and sugar content in sugar beet were studied in the field experiment by nutrient solution culture. The results showed that SS activity in leaves was lower than that in roots. With nitrogen level increasing,SS decomposition activity enhanced, and synthesis activity reduced. SS activity was regulated by different nitrogen forms and the ratio of NO3- and NH4+. SS synthesis activity was enhanced as NH4+ increasing when NO3-: NH4+≥ 1, and it decreased as increasing NH4+ when NO3-: NH4+≤1, and it was the highest when NO3-: NH4+=1. SS decomposition activity was enhanced as NO3- increasing.Sucrose content in root was lowed as nitrogen level increasing, but it was enhanced as NH4+ increasing in the same nitrogen level.Root and sugar yield were the highest in the medium nitrogen level and NO3-: NH4+=1. The result in field experiment corresponded with that in the nutrient fluid culture. It provides a basis for using reasonably nitrogen fertilizer in sugar beet production.

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

    Science.gov (United States)

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

    2002-11-01

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

  2. 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.

  3. ‘Dopamine-first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

    Science.gov (United States)

    Lichman, Benjamin R; Gershater, Markus C; Lamming, Eleanor D; Pesnot, Thomas; Sula, Altin; Keep, Nicholas H; Hailes, Helen C; Ward, John 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 proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity. PMID:25620686

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

    Science.gov (United States)

    Lichman, Benjamin R; Gershater, Markus C; Lamming, Eleanor D; Pesnot, Thomas; Sula, Altin; Keep, Nicholas H; Hailes, Helen C; Ward, John M

    2015-03-01

    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 proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the 'dopamine-first' mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity. PMID:25620686

  5. A connecting hinge represses the activity of endothelial nitric oxide synthase.

    Science.gov (United States)

    Haque, Mohammad Mahfuzul; Panda, Koustubh; Tejero, Jesús; Aulak, Kulwant S; Fadlalla, Mohammed Adam; Mustovich, Anthony T; Stuehr, Dennis J

    2007-05-29

    In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K(m)O(2) for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.

  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

    labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either...

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

    Directory of Open Access Journals (Sweden)

    Vijay Venugopal

    2010-12-01

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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Yan-Li Yao

    2012-07-01

    Full Text Available 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.

  10. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  11. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption.

  12. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption. PMID:25900579

  13. 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.

  14. NMR crystallography of enzyme active sites: probing chemically detailed, three-dimensional structure in tryptophan synthase.

    Science.gov (United States)

    Mueller, Leonard J; Dunn, Michael F

    2013-09-17

    NMR crystallography applied to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation.

  15. The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

    Science.gov (United States)

    Lee, Miyoung; Choi, Yongwook; Burla, Bo; Kim, Yu-Young; Jeon, Byeongwook; Maeshima, Masayoshi; Yoo, Joo-Yeon; Martinoia, Enrico; Lee, Youngsook

    2008-10-01

    Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

  16. Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2008-03-01

    Full Text Available Abstract Background β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha. Results The P. chrysogenum pcbC gene encoding isopenicillin N synthase (IPNS was successfully expressed in H. polymorpha, but the protein produced was unstable and inactive when the host was grown at its optimal growth temperature (37°C. Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25°C or 30°C. Furthermore, IPNS produced at these lower cultivation temperatures was functionally active. Localization experiments demonstrated that, like in P. chrysogenum, in H. polymorpha IPNS is located in the cytosol. Conclusion In P. chrysogenum, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies. In this study, we focus on the cytosolic enzyme IPNS. Our data show that high amounts of functionally active IPNS enzyme can be produced in the heterologous host during cultivation at 25°C, the optimal growth temperature for P. chrysogenum. This is a new step forward in the metabolic reprogramming of H. polymorpha to produce penicillin.

  17. Elevation in sphingomyelin synthase activity is associated with increases in amyloid-beta peptide generation.

    Directory of Open Access Journals (Sweden)

    Jen-Hsiang T Hsiao

    Full Text Available A pathological hallmark of Alzheimer's disease (AD is the presence of amyloid-beta peptide (Aβ plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP, a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose- and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.

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

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    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.

  19. 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...

  20. 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...

  1. Malolactic Fermentation : Electrogenic Malate Uptake and Malate/Lactate Antiport Generate Metabolic Energy

    NARCIS (Netherlands)

    Poolman, Bert; Molenaar, Douwe; Smid, Eddy J.; Ubbink, Trees; Abee, Tjakko; Renault, Pierre P.; Konings, Wil N.

    1991-01-01

    The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generate

  2. 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, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

  3. 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

  4. 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

  5. [DYNAMICS OF GLUTAMINE SYNTHASE ACTIVITY IN RAT BRAIN IN PRENATAL HYPOXIA MODEL].

    Science.gov (United States)

    Khairova, V R; Safarov, M I

    2015-01-01

    Prenatal ontogenesis is a period of high sensitivity to stressful impact, so any stressor can lead to changes of physiological, biochemical indicators, behavioral and cognitive functions. The most common and clinically significant stress factor, which the embryo may be exposed during embryonic development, is hypoxia. In this case pathological changes in the central nervous system depend on the duration and severity of hypoxic exposure, individual tolerance and the stage of prenatal development, at each of which in the brain take place the basic histogenetic processes. By activating energetically disadvantageous anaerobic glycolysis hypoxia leads to excess of glutamate emission and cell apoptosis. Glutamine synthase is a basic enzyme that regulates metabolism of glutamate, catalyzing conversion of glutamate to glutamine with ammonia detoxification. The aim of the presented work was to reveal changes in the activity of one of the key enzyme of glutamate metabolism- glutamine synthetase in the brain of offspring of white rats undergone to hypoxia at different stages of prenatal ontogenesis. Hypoxia was created by placing female rats at stages of the pregnancy, corresponding to progestation period of organogenesis and fetal period of prenatal development, in the hypobaric chamber with exposure to 5% oxygen and 95% nitrogen gas mixture during 30 minutes per day. The offspring obtained from females of control and experimental groups were used for biochemical determinations in the age of 1 and 3 month. It has been established that hypoxia exposed to pregnant females during embryonic organogenesis causes significant changes in enzyme activity, particularly pronounced in the cerebral cortex and cerebellum, as compared with progestational and fetal hypoxia. Enzyme activity decreased in a greater degree in one-month-old rats undergone to prenatal hypoxia, than three- month-old animals. Thus, stress during intensive processes of proliferation and migration of cells of the

  6. 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.

  7. 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.

  8. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    Science.gov (United States)

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

    2012-11-28

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

  9. Nitration of tyrosyl residues in human alpha-lactalbumin. Effect on lactose synthase specifier activity.

    Science.gov (United States)

    Prieels, J P; Dolmans, M; Leonis, J; Brew, K

    1975-12-15

    Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives. PMID:812700

  10. 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

  11. Occurrence of the malate-aspartate shuttle in various tumor types.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1976-04-01

    The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm. PMID:177206

  12. Exploring geometric properties of gold nanoparticles using TEM images to explain their chaperone like activity for citrate synthase

    OpenAIRE

    Kaushik, Vikas; Lahiri, Tapobrata; Singha, Shantiswaroop; Dasgupta, Anjan Kumar; Mishra, Hrishikesh; Kumar, Upendra; Kumar, Rajeev

    2011-01-01

    Study on geometric properties of nanoparticles and their relation with biomolecular activities, especially protein is quite a new field to explore. This work was carried out towards this direction where images of gold nanoparticles obtained from transmission electron microscopy were processed to extract their size and area profile at different experimental conditions including and excluding a protein, citrate synthase. Since the images were ill-posed, texture of a context-window for each pixe...

  13. 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 ...

  14. A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs.

    Science.gov (United States)

    Behm-Ansmant, Isabelle; Massenet, Séverine; Immel, Françoise; Patton, Jeffrey R; Motorin, Yuri; Branlant, Christiane

    2006-08-01

    Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.

  15. 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

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

    Science.gov (United States)

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

    2012-01-01

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

  17. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    Science.gov (United States)

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  18. 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

  19. 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

    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...

  20. 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...

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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...

  6. l-Malate Production by Metabolically Engineered Escherichia coli▿ †

    OpenAIRE

    Zhang, X; Wang, X.; Shanmugam, K. T.; Ingram, L O

    2010-01-01

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

  7. Activated platelets from diabetic rats cause endothelial dysfunction by decreasing Akt/endothelial NO synthase signaling pathway.

    Directory of Open Access Journals (Sweden)

    Keiko Ishida

    Full Text Available Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. The purpose of this study was to characterize circulating platelets in diabetes and clarify their effects on endothelial function. Male Wistar rats were injected with streptozotocin (STZ to induce diabetes. Each experiment was performed by incubating carotid arterial rings with platelets (1.65×10(7 cells/mL; 30 min isolated from STZ or control rats. Thereafter, the vascular function was characterized in isolated carotid arterial rings in organ bath chambers, and each expression and activation of enzymes involved in nitric oxide and oxidative stress levels were analyzed. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in carotid arteries treated with platelets isolated from STZ rats. Similarly, treatment with platelets isolated from STZ rats significantly reduced ACh-induced Akt/endothelial NO synthase signaling/NO production and enhanced TXB2 (metabolite of TXA2, while CD61 (platelet marker and CD62P (activated platelet marker were increased in carotid arteries treated with platelets isolated from STZ rats. Furthermore, the platelets isolated from STZ rats decreased total eNOS protein and eNOS dimerization, and increased oxidative stress. These data provide direct evidence that circulating platelets isolated from diabetic rats cause dysfunction of the endothelium by decreasing NO production (via Akt/endothelial NO synthase signaling pathway and increasing TXA2. Moreover, activated platelets disrupt the carotid artery by increasing oxidative stress.

  8. Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

    Directory of Open Access Journals (Sweden)

    Olga N. Rozova

    2015-02-01

    Full Text Available We have expressed the l-malate dehydrogenase (MDH genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa enzyme displaying nearly equal reductive (malate formation and oxidative (oxaloacetate formation activities and higher affinity to malate (Km = 0.11 mM than to oxaloacetate (Km = 0.34 mM. The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa, two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM than to malate (Km = 1.28 mM. The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.

  9. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  10. 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

  11. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

    Science.gov (United States)

    Tesfaye, M; Temple, S J; Allan, D L; Vance, C P; Samac, D A

    2001-12-01

    Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity. PMID:11743127

  12. 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.

  13. 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

  14. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles

    DEFF Research Database (Denmark)

    Kolnes, Anders J; Birk, Jesper Bratz; Eilertsen, Einar;

    2015-01-01

    Adrenaline increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated...... in condition with decreased GS activation. Saline or adrenaline (0.02mg/100g rat) was injected subcutaneously in Wistar rats (~130 g) with low (24 h fasted), normal (normal diet) and high glycogen content (fasted-refed) and epitrochlearis muscles were removed after 3 h and incubated ex vivo eliminating...... adrenaline action. Adrenaline injection reduced glycogen content in epitrochlearis muscles with high (120.7±17.8 vs 204.6±14.5 mmol•kg(-1); p

  15. 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.

  16. Effects of glucocorticoid dexamethasone on serum nitric oxide synthase activity and nitric oxide levels in a rat model of lung disease-induced brain injury

    Institute of Scientific and Technical Information of China (English)

    Huajun Li; Ligang Jiang; Meng Xia; Haiping Li; Fanhua Meng; Wei Li; Lifeng Liu; Zhaohui Wang

    2011-01-01

    In this study, we investigated the effects of dexamethasone, pertussis toxin (a Gi protein inhibitor), and actinomycin (a transcription inhibitor) on serum nitric oxide synthase activity and nitric oxide content in a rat model of lung disease-induced brain injury. High-dose dexamethasone (13 mg/kg) and dexamethasone + actinomycin reduced lung water content, increased serum nitric oxide synthase activity and nitric oxide content, diminished inflammatory cell infiltration in pulmonary alveolar interstitium, attenuated meningeal vascular hyperemia, reduced glial cell infiltration, and decreased cerebral edema. These results demonstrate that high-dose glucocorticoid treatment can reduce the severity of lung disease-induced brain injury by increasing nitric oxide synthase activity and nitric oxide levels.

  17. 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)

  18. The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian B; Nielsen, Jakob N.; Birk, Jesper Bratz;

    2004-01-01

    The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). ...

  19. Exploring geometric properties of gold nanoparticles using TEM images to explain their chaperone like activity for citrate synthase

    Science.gov (United States)

    Kaushik, Vikas; Lahiri, Tapobrata; Singha, Shantiswaroop; Dasgupta, Anjan Kumar; Mishra, Hrishikesh; Kumar, Upendra; Kumar, Rajeev

    2011-01-01

    Study on geometric properties of nanoparticles and their relation with biomolecular activities, especially protein is quite a new field to explore. This work was carried out towards this direction where images of gold nanoparticles obtained from transmission electron microscopy were processed to extract their size and area profile at different experimental conditions including and excluding a protein, citrate synthase. Since the images were ill-posed, texture of a context-window for each pixel was used as input to a back-propagation network architecture to obtain decision on its membership as nanoparticle. The segmented images were further analysed by k-means clustering to derive geometric properties of individual nanoparticles even from their assembled form. The extracted geometric information was found to be crucial to give a model featuring porous cage like configuration of nanoparticle assembly using which the chaperone like activity of gold nanoparticles can be explained. PMID:22355230

  20. 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.

  1. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    Science.gov (United States)

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.

  2. Genetic Control of Malate Dehydrogenase Isozymes in Maize

    OpenAIRE

    Goodman, M. M.; Stuber, C. W.; Lee, C N; Johnson, F. M.

    1980-01-01

    At least six nuclear loci are responsible for the genetic control of malate dehydrogenase (L-malate: NAD oxidoreductase; EC 1.1.1.37; MDH) in coleoptiles of maize. Three independently segregating loci (Mdh1, Mdh2, Mdh3) govern the production of MDH isozymes resistant to inactivation by ascorbic acid and found largely or solely in the mitochondria. A rare recessive allele found at a fourth nuclear locus (mmm) causes increased electrophoretic mobility of the MDH isozymes governed by the Mdh1, M...

  3. 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

  4. Doubling the CO{sub 2} concentration enhanced the activity of carbohydrate-metabolism enzymes, source carbohydrate production, photoassimilate transport, and sink strength for Opuntia ficus-indica

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ning; Nobel, P.S. [Univ. of California, Los Angeles, CA (United States)

    1996-03-01

    After exposure to a doubled CO{sub 2} concentration of 750 {mu}mol mol{sup -1} air for about 3 months, glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO{sub 2} concentration of 370 {mu}mol mol{sup -1}, but sucrose content was virtually unaffected. Doubling the CO{sub 2} concentration increased the noncturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32% soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO{sub 2} accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO{sub 2} increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO{sub 2} concentrations occurs for O. ficus-indica, consistent with its higher source capacity and sink strength than under current CO{sub 2}. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops.

  5. 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.

  6. 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

  7. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    Science.gov (United States)

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

  8. Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

    Science.gov (United States)

    Yilmaz, O; Całka, J; Bukowski, R; Zalecki, M; Wasowicz, K; Jaroszewski, J J; Markiewicz, W; Bulbul, A; Ucar, M

    2012-04-15

    The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from

  9. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    Science.gov (United States)

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  10. 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.

  11. 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.

  12. 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

  13. Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carbosylation, oxaloacetate reduction and malate export

    NARCIS (Netherlands)

    Zelle, R.M.; Hulster, de E.; Winden, van W.A.; Waard, de P.; Dijkema, C.; Winkler, A.A.; Geertman, J.M.A.

    2008-01-01

    Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production

  14. Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export

    NARCIS (Netherlands)

    Zelle, R.M.; De Hulster, E.; Van Winden, W.A.; De Waard, P.; Dijkema, C.; Winkler, A.A.; Geertman, J.M.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.

    2008-01-01

    Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production

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

    Science.gov (United States)

    Piłsyk, Sebastian; Perlińska-Lenart, Urszula; Górka-Nieć, Wioletta; Graczyk, Sebastian; Antosiewicz, Beata; Zembek, Patrycja; Palamarczyk, Grażyna; Kruszewska, Joanna S

    2014-07-10

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

  16. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A;

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  17. Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

    DEFF Research Database (Denmark)

    Mazumdar, Tuhina; Gorgun, F Murat; Sha, Youbao;

    2010-01-01

    activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn...

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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...

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

    Science.gov (United States)

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

    2013-08-01

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

  5. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    Science.gov (United States)

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

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

  6. 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.

  7. 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

  8. 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.

  9. 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

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

    Science.gov (United States)

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

    2011-11-01

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

  11. Changes of Nitric Oxide Synthase Activity in Penumbral and Core Area during Focal Cerebral Ischemia and Reperfusion in Rats

    Institute of Scientific and Technical Information of China (English)

    GUZhen; ZHOUJian-ping; WUWen-zhong; ZHANGYong-jie; HANQun-ying; WANGHe-ming

    2004-01-01

    Objecivee: To study the changes of nitric oxide synthase (NOS) activity in penumbral and core area during focal cerebral ischemia and reperfusion, and to explore the therapeutic window of focal cerebral ischemia. Methods:The middle cerebral artery of rats was occluded for 15, 30,60,90 and 120 min by an inraluminal filament respectively,and recirculation was instituted for 24 h. The changes of NOS activity in ischemic core area(parietal cortex and caudoputamen) and penumbral area ( frontal cortex)were examined after focal cerebral ischemla and reperfusion using NADPH-d histochemistry, technique. Results. The NOS activity of the ischemic penumbral area peaked at 60 min while the ischemic core area peaked at 30 min then declined at 90-120 rain sharply. Conclusion: NOS takes part in cerebral ischemic damage during focal cerebral ischemia and reperfusion. The NOS activity of the ischemic penmnbral area is different from the ischemic core area. The peak time of the penumbral area is delayed comparing with the core area. The data suggest that the best time to apply NOS inhibitor is within 30 min in ischemic core area, and 60 rain in penumbral area.

  12. Steroid receptor RNA activator (SRA modification by the human pseudouridine synthase 1 (hPus1p: RNA binding, activity, and atomic model.

    Directory of Open Access Journals (Sweden)

    Tiphaine Huet

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

  13. Steroid receptor RNA activator (SRA) modification by the human pseudouridine synthase 1 (hPus1p): RNA binding, activity, and atomic model.

    Science.gov (United States)

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

    2014-01-01

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

  14. Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

    Institute of Scientific and Technical Information of China (English)

    Tóshiko TAKAHASHI-ÍÑIGUEZ; Nelly ABURTO-RODRÍGUEZ; Ana Laura VILCHIS-GONZÁLEZ; María Elena FLORES

    2016-01-01

    题目:微生物苹果酸脱氢酶的功能、动力学特征、晶体结构以及调控概苹果酸脱氢酶(MDH)广泛存在于动物、植物以及微生物体内,是生物体进行糖代谢的关键酶之一。在辅酶I(NAD+)或辅酶II(NADP+)的作用下,能够催化草酰乙酸和苹果酸之间相互转化。虽然目前真核微生物中MDH已被广泛研究,但是对原核生物中的这种酶却鲜有报道。因此,有必要对MDH的相关研究信息进行综述,以期更好地了解这种酶的功能。本文综述了细菌相关研究的各种数据信息,进一步挖掘MDH的分子多样性,包括分子量、低聚态、辅因子与底物的结合力,以及酶反应方向的差异等。通过对不同细菌来源的MDH的晶体结构的分析,可鉴别底物与辅因子结合的部位以及形成二聚体的重要残基。对这些结构信息的了解将有利于指导研究人员对酶的结构进行修饰从而提高其催化能力,比如增加酶的活性、辅助因子的结合能力、底物特异性和热稳定性等。另外,本文通过分析比较MDH 系统发生树的重建,将其蛋白超家族分成两个主分支,同时在古生菌、细菌和真核微生物等不同细胞的MDH之间建立联系。%Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as wel as differ-ences in the direction

  15. Inhibition of glycogen synthase kinase 3β activity with lithium prevents and attenuates paclitaxel-induced neuropathic pain.

    Science.gov (United States)

    Gao, M; Yan, X; Weng, H-R

    2013-12-19

    Paclitaxel (taxol) is a first-line chemotherapy-drug used to treat many types of cancers. Neuropathic pain and sensory dysfunction are the major toxicities, which are dose-limiting and significantly reduce the quality of life in patients. Two known critical spinal mechanisms underlying taxol-induced neuropathic pain are an increased production of pro-inflammatory cytokines including interleukin-1β (IL-1β) and suppressed glial glutamate transporter activities. In this study, we uncovered that increased activation of glycogen synthase kinase 3beta (GSK3β) in the spinal dorsal horn was concurrently associated with increased protein expressions of GFAP, IL-1β and a decreased protein expression of glial glutamate transporter 1 (GLT-1), as well as the development and maintenance of taxol-induced neuropathic pain. The enhanced GSK3β activities were supported by the concurrently decreased AKT and mTOR activities. The changes of all these biomarkers were basically prevented when animals received pre-emptive lithium (a GSK3β inhibitor) treatment, which also prevented the development of taxol-induced neuropathic pain. Further, chronic lithium treatment, which began on day 11 after the first taxol injection, reversed the existing mechanical and thermal allodynia induced by taxol. The taxol-induced increased GSK3β activities and decreased AKT and mTOR activities in the spinal dorsal horn were also reversed by lithium. Meanwhile, protein expressions of GLT-1, GFAP and IL-1β in the spinal dorsal horn were improved. Hence, suppression of spinal GSK3β activities is a key mechanism used by lithium to reduce taxol-induced neuropathic pain, and targeting spinal GSK3β is an effective approach to ameliorate GLT-1 expression and suppress the activation of astrocytes and IL-1β over-production in the spinal dorsal horn.

  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. The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

    Science.gov (United States)

    Singh, Dhirendra; Sielaff, Hendrik; Sundararaman, Lavanya; Bhushan, Shashi; Grüber, Gerhard

    2016-02-01

    A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.

  19. By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, stichoposide D inhibits growth of leukemia xenografts.

    Science.gov (United States)

    Yun, Seong-Hoon; Park, Eun-Seon; Shin, Sung-Won; Ju, Mi-Ha; Han, Jin-Yeong; Jeong, Jin-Sook; Kim, Sung-Hyun; Stonik, Valentin A; Kwak, Jong-Young; Park, Joo-In

    2015-09-29

    Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-β-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia.

  20. 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

  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. 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

  3. Methylene bridge regulated geometrical preferences of ligands in cobalt(III) coordination chemistry and phenoxazinone synthase mimicking activity.

    Science.gov (United States)

    Panja, Anangamohan; Shyamal, Milan; Saha, Amrita; Mandal, Tarun Kanti

    2014-04-14

    Two new azide bound cobalt(III) complexes, [Co(L(1))(N3)3] (fac-1) and [Co(L(2))(N3)3] (mer-2), where L(1) is bis(2-pyridylmethyl)amine and L(2) is (2-pyridylmethyl)(2-pyridylethyl)amine, derived from tridentate reduced Schiff-base ligands have been reported. Interestingly, a methylene bridge regulated preferential coordination mode of ligands is noticed in their crystal structures: it is found in a facial arrangement in fac-1 and has a meridional disposition in mer-2. Both complexes show phenoxazinone synthase-like activity and the role of the structural factor on the catalytic activity is also explored. Moreover, the easily reducible cobalt(III) center in mer-2 favors the oxidation of o-aminophenol. The ESI-MS positive spectra together with UV-vis spectroscopy clearly suggest the formation of a catalyst-substrate adduct by substitution of the coordinated azide ions in the catalytic cycle.

  4. 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

  5. Ligands of Peroxisome Proliferator-activated Receptor Inhibit Homocysteineinduced DNA Methylation of Inducible Nitric Oxide Synthase Gene

    Institute of Scientific and Technical Information of China (English)

    Yideng JIANG; Jianzhong ZHANG; Jiantuan XIONG; Jun CAO; Guizhong LI; Shuren WANG

    2007-01-01

    Homocysteine (Hcy) is a risk factor for atherosclerosis. It is generally accepted that inducible nitric oxide synthase (iNOS) is a key enzyme in the regulation of vascular disease. The aim of the present study is to investigate the effects of peroxisome proliferator-activated receptor ligands on iNOS in the presence of Hcy in human monocytes. Foam cells, induced by oxidize low density lipoprotein (ox-LDL) and phorbol myristate acetate (PMA) in the presence of different concentrations of Hcy, clofibrate and pioglitazone in human monocytes for 4 d, were examined by oil red O staining. The activity of iNOS was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. The capability of DNA methylation was measured by assaying endogenous C5 DNA methyltransferase (C5MTase)activity, and the iNOS promoter methylation level was determined by quantitative MethyLight assays. The results indicated that Hcy increased the activity of C5MTase and the level of iNOS gene DNA methylation,resulting in a decrease of iNOS expression. Clofibrate and pioglitazone could antagonize the Hcy effect on iNOS expression through DNA methylation, resulting in attenuation of iNOS transcription. These findings suggested that Hcy decreased the expression of iNOS by elevating iNOS DNA methylation levels, which can repress the transcription of some genes. Peroxisome proliferator-activated receptor α/γ ligands can down-regulate iNOS DNA methylation, and could be useful for preventing Hcy-induced atherosclerosis by repressing iNOS expression.

  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. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

    Science.gov (United States)

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  8. A novel malate dehydrogenase from Ceratonia siliqua L. seeds with potential biotechnological applications.

    Science.gov (United States)

    Muccio, Clelia; Guida, Vincenzo; Di Petrillo, Amalia; Severino, Valeria; Di Maro, Antimo

    2012-12-01

    A novel malate dehydrogenase (MDH; EC 3.1.1.1.37), hereafter MDHCs, from Ceratonia siliqua seeds, commonly known as Carob tree, was purified by using ammonium sulphate precipitation, ion exchange chromatography on SteamLine SP and gel-filtration. The molecular mass of the native protein, obtained by analytical gel-filtration, was about 65 kDa, whereas, by using SDS-PAGE analysis, with and without reducing agent, was 34 kDa. The specific activity of purified MDHCs (0.25 mg/100 g seeds) was estimated to be 188 U/mg. The optimum activity of the enzyme is at pH 8.5, showing a decrease in the presence of Ca(2+), Mg(2+) and NaCl. The N-terminal sequence of the first 20 amino acids of MDHCs revealed 95 % identity with malate dehydrogenase from Medicago sativa L. Finally, the enzymatic activity of MDHCs was preserved even after absorption onto a PVDF membrane. To our knowledge, this is the first contribution to the characterization of an enzyme from Carob tree sources.

  9. 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

  10. 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.

  11. 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

  12. 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.

  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. 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.

  16. Role of Nitric Oxide Dependence on Nitric Oxide Synthase-like Activity in the Water Stress Signaling of Maize Seedling

    Institute of Scientific and Technical Information of China (English)

    Gang-Ping Hao; Yu Xing; Jian-Hua Zhang

    2008-01-01

    Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-lika (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings. NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramathylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation Inhibited by NOS inhibitor L-NAME In dehydration-treated maize seedlings Indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-trested maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutsse (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity

  17. Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Dahari, Dhiana Efani; Salleh, Raifana Mohamad; Mahmud, Fauze; Chin, Lee Ping; Embi, Noor; Sidek, Hasidah Mohd

    2016-08-01

    Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition

  18. 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

  19. 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

  20. 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...

  1. 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...

  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. 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...

  4. Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg(2+) Ions and Severe Inhibitory Effects of Divalent Cations.

    Science.gov (United States)

    Tlapak-Simmons, Valarie L; Medina, Andria P; Baggenstoss, Bruce A; Nguyen, Long; Baron, Christina A; Weigel, Paul H

    2011-11-15

    Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). Streptococcus equisimilis HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg(2+) for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg(2+) with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg(2+) dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg(2+). In the presence of Mg(2+), other divalent cations inhibited SeHAS with different potencies (Cu(2+)~Zn(2+) >Co(2+) >Ni(2+) >Mn(2+) >Ba(2+) Sr(2+) Ca(2+)). Some divalent metal ions likely inhibit by displacement of Mg(2+)-UDP-Sugar complexes (e.g. Ca(2+), Sr(2+) and Ba(2+) had apparent Ki values of 2-5 mM). In contrast, Zn(2+) and Cu(2+) inhibited more potently (apparent Ki ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu(2+), but not Zn(2+), was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn(2+) or Cu(2+). Wildtype SeHAS allowed to make HA prior to exposure to Zn(2+) or Cu(2+) was protected from inhibition, indicating that access of metal ions to sensitive functional groups was hindered in processively acting HA•HAS complexes. We conclude that clustered Cys residues mediate cooperative interactions with Mg(2+) and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.

  5. 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

  6. 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

  7. 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.

  8. Dihydromyricetin protects neurons in an MPTP-induced model of Parkinson's disease by suppressing glycogen synthase kinase-3 beta activity

    Science.gov (United States)

    Ren, Zhao-xiang; Zhao, Ya-fei; Cao, Ting; Zhen, Xue-chu

    2016-01-01

    Aim: It is general believed that mitochondrial dysfunction and oxidative stress play critical roles in the pathology of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, has recently been found to elicit potent anti-oxidative effects. In the present study, we explored the role of DHM in protecting dopaminergic neurons. Methods: Male C57BL/6 mice were intraperitoneally injected with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 d to induce PD. Additionally, mice were treated with either 5 or 10 mg/kg DHM for a total of 13 d (3 d before the start of MPTP, during MPTP administration (7 d) and 3 d after the end of MPTP). For the saline or DHM alone treatment groups, mice were injected with saline or DHM for 13 d. On d 14, behavioral tests (locomotor activity, the rotarod test and the pole test) were administered. After the behavioral tests, the mice were sacrificed, and brain tissue was collected for immunofluorescence staining and Western blotting. In addition, MES23.5 cells were treated with MPP+ and DHM, and evaluated using cell viability assays, reactive oxygen species (ROS) measurements, apoptosis analysis and Western blotting. Results: DHM significantly attenuated MPTP-induced mouse behavioral impairments and dopaminergic neuron loss. In the MES23.5 cells, DHM attenuated MPP+-induced cell injury and ROS production in a dose-dependent manner. In addition, DHM increased glycogen synthase kinase-3 beta phosphorylation in a dose- and time-dependent manner, which may be associated with DHM-induced dopaminergic neuronal protection. Conclusion: The present study demonstrated that DHM is a potent neuroprotective agent for DA neurons by modulating the Akt/GSK-3β pathway, which suggests that DHM may be a promising therapeutic candidate for PD. PMID:27374489

  9. 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.

  10. 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.

  11. 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

  12. Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA.

    Science.gov (United States)

    Patton, Jeffrey R; Padgett, Richard W

    2003-06-01

    The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.

  13. Propofol improves cardiac functional recovery after ischemia-reperfusion by upregulating nitric oxide synthase activity in the isolated rat hearts

    Institute of Scientific and Technical Information of China (English)

    SUN Hai-yan; XUE Fu-shan; XU Ya-chao; LI Cheng-wen; XIONG Jun; LIAO Xu; ZHANG Yan-ming

    2009-01-01

    Background There are few studies to assess whether propofol attenuates myocardial ischemia-reperfusion injury via a mechanism related to nitric oxide (NO) route, so we designed this randomized blinded experiment to observe the changes of NO contents, nitric oxide synthase (NOS) activity, NOS contents in the myocardium, and cardiac function in ischemic reperfused isolated rat hearts, and to assess the relation between myocardial NO system and cardioprotection of propofol.Methods The hearts of 30 Sprague-Dawley male rats were removed, mounted on a Langendorff apparatus, and randomly assigned to one of three groups (n=10 each group) to be treated with the following treatments in a blinded manner: Group 1, control group, after perfusion with pure Krebs Henseleit bicarbonate (K-HBB) buffer solution for 15 minutes, hearts were subjected to 20 minutes global ischemia followed by 60 minutes reperfusion with pure K-HBB buffer; Group 2, after perfusion with K-HBB buffer solution containing propofol (10 μg/ml) for 15 minutes, the hearts underwent 20 minutes global ischemia followed by 60 minutes reperfusion with the same K-HBB buffer solution; Group 3, after perfusion with K-HBB buffer solution containing propofol (10 μg/ml) and L-NAME (100 μmol/L) for 15 minutes, the hearts underwent 20 minutes global ischemia followed by 60 minutes reperfusion with the same K-HBB buffer solution. The cardiac function was continuously monitored throughout the experiment.The coronary flow was also measured. An ISO-NO electrode was placed into the right atrium close to the coronary sinus to continuously measure NO concentration in the coronary effluent. The tissue samples from apex of hearts in Groups 1 and 2 were obtained to measure the NOS activity by spectrophotometry and the NOS contents by immunohistochemistry, respectively.Results The cardiac function was significantly inhibited after ischemia and then gradually improved with reperfusion in all three groups. As compared with Group 1

  14. Purification and activity evaluation of methionine synthase%蛋氨酸合酶活性筛选体系的建立

    Institute of Scientific and Technical Information of China (English)

    郭莹; 李超; 张志丽; 田超; 王孝伟; 刘俊义

    2012-01-01

    钴胺素依赖的蛋氨酸合酶催化N5-甲基四氢叶酸转移甲基至同型半胱氨酸生成蛋氨酸和四氢叶酸,直接参与蛋氨酸循环、叶酸循环及含硫氨基酸代谢,与DNA、蛋白质合成及生物甲基化有密切关系.本研究采用蛋白层析技术,将大鼠肝匀浆经超声破碎和高速离心处理后,依次经过DE-52批处理、Q Sepharose Fast Flow离子交换层析和CHT陶瓷羟基磷灰石吸附柱层析进行纯化,并对纯化产物进行了SDS-PAGE和Western blotting 鉴定.采用分光光度法测定蛋氨酸合酶的活性,对纯化酶的酶促反应动力学进行了研究,确定了最佳反应条件,动力学结果显示蛋氨酸合酶的双底物酶促反应的机制为乒乓机制.研究表明,采用层析技术纯化得到的蛋氨酸合酶适用于以其为靶点的化合物高通量筛选.%Methionine synthase (MS, EC2.1.1.13), a key enzyme in the folate metabolism area catalyzing methyl transfer from N5-methyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine, takes a core position in folate cycle, one-carbon-unit transfer and sculpture amino acid pathways. Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme was purified 609-fold to near homogeneity by batch chromatography on DE-52, anion-exchange chromatography on Q Sepharose Fast Flow and CHT-I hydroxyapatite column and was identified by SDS-PAGE and Western blotting. The enzyme activity was determined by spectrophotometric assay. In addition, the influencing factor and optimal reaction condition were performed. The steady state kinetic of rat liver methionine synthase was similar to that of other mammalian cobalamin-dependent methionine synthase which employed a Ping-Pong mechanism. The result indicated that cobalamin-dependent methionine synthase purified from rat liver is suitable for screening and studying methionine synthase specific inhibitors.

  15. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    1988-08-25

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.

  16. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    International Nuclear Information System (INIS)

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15

  17. Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice.

    Directory of Open Access Journals (Sweden)

    Tim Peters

    Full Text Available Long non-coding RNAs (lncRNAs are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1 is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC in mice.Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001 but to a similar extend also in Malat-1 knockout (KO mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01 with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01 but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05. Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-15

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

  19. 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.

  20. Safety Assessment of Dialkyl Malates as Used in Cosmetics.

    Science.gov (United States)

    Becker, Lillian C; Bergfeld, Wilma F; Belsito, Donald V; Hill, Ronald A; Klaassen, Curtis D; Liebler, Daniel C; Marks, James G; Shank, Ronald C; Slaga, Thomas J; Snyder, Paul W; Andersen, F Alan

    2015-01-01

    The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of 6 dialkyl malate compounds used in cosmetics. These ingredients function mostly as skin-conditioning agents-emollients. The Panel reviewed relevant animal and human data related to the ingredients along with a previous safety assessment of malic acid. The similar structure, properties, functions, and uses of these ingredients enabled grouping them and using the available toxicological data to assess the safety of the entire group. The Panel concluded that these dialkyl maleate compounds are safe in the present practices of use and concentration as given in this safety assessment.

  1. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

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

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  2. 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...

  3. 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...

  4. Independent fluctuations of malate and citrate in the CAM species Clusia hilariana Schltdl. under low light and high light in relation to photoprotection.

    Science.gov (United States)

    Miszalski, Zbigniew; Kornas, Andrzej; Rozpądek, Piotr; Fischer-Schliebs, Elke; Lüttge, Ulrich

    2013-03-15

    Clusia hilariana Schltdl. is described in literature as an obligate Crassulacean acid metabolism (CAM) species. In the present study we assessed the effect of irradiance with low light (LL, 200μmolm(-2)s(-1)) and high light (HL, 650-740μmolm(-2)s(-1)), on the interdependency of citrate and malate diurnal fluctuations. In plants grown at HL CAM-type oscillations of concentration of citrate and malate were obvious. However, at LL daily courses of both acids do not seem to indicate efficient utilization of these compounds as CO2 and NADPH sources. One week after transferring plants from LL to HL decarboxylation of malate was accelerated. Thus, in the CAM plant C. hilariana two independent rhythms of accumulation and decarboxylation of malate and citrate take place, which appear to be related to photosynthesis and respiration, respectively. Non photochemical quenching (NPQ) of photosystem II, especially well expressed during the evening hours was enhanced. Exposure to HL for 7 d activated oxidative stress protection mechanisms such as the interconversion of violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) (epoxydation/de-epoxydation) measured as epoxydation state (EPS). This was accompanied by a slight increase in the total amount of these pigments. However, all these changes were not observed in plants exposed to HL for only 2 d. Besides violaxanthin cycle components also lutein, which shows a small, but not significant increase, may be involved in dissipating excess light energy in C. hilariana.

  5. 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 PCOS patients (P

  6. Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Vind, B F; Birk, Jesper Bratz; Vienberg, Sara Gry;

    2012-01-01

    AIMS/HYPOTHESIS: In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved...... insulin signalling to glycogen synthesis remains to be clarified. METHODS: Whole-body glucose metabolism was studied in 12 patients with type 2 diabetes, and 10 lean and 10 obese non-diabetic controls by means of indirect calorimetry and tracers during a euglycaemic-hyperinsulinaemic clamp. The diabetic...

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

    Science.gov (United States)

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

    2004-01-01

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

  8. Fluvoxamine alters the activity of energy metabolism enzymes in the brain

    Directory of Open Access Journals (Sweden)

    Gabriela K. Ferreira

    2014-09-01

    Full Text Available Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and creatine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

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

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes; Maretty, Lasse;

    2014-01-01

    Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS...... increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion...

  10. 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...

  11. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

    Science.gov (United States)

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

    2016-03-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. PMID:26637549

  12. 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.

  13. Organic acid metabolism and root excretion of malate in wheat cultivars under aluminium stress.

    Science.gov (United States)

    de Andrade, Leide Rovênia Miranda; Ikeda, Motoki; do Amaral, Lourdes Isabel Velho; Ishizuka, Junji

    2011-01-01

    The effects of aluminium (Al) on the metabolism of organic acids synthesised via nonphotosynthetic carbon fixation in the roots and on malate exudation were investigated in Al-tolerant Shirosanjyaku (SH) and Al-sensitive Chikushikomugi (CK) wheat cultivars labelled with bicarbonate-(14)C. Aluminum triggered the excretion of (14)C into the solution, especially in the SH that excreted 2.5 times more (14)C than the CK. The loss of radioactivity (about 10%) into the solution represented a small drain in the (14)C reserve found in the roots. In the organic acid fraction within the roots, malate contained the greatest amount of (14)C, and this amount decreased rapidly with time in both cultivars. The disappearance of radioactivity in the malate resulted from metabolism and translocation rather than to root efflux. Aluminium decreased the malate concentrations in roots of both cultivars. The Al-sensitive cultivar had higher concentrations of malate regardless of the presence of Al. It was therefore assumed that the decrease of malate concentration in roots under Al stress did not result from the decline in malate synthesis but due to an increase in malate decomposition. This response was interpreted as the result of the Al-induced stress and not as the cause of a differential Al-tolerance between the wheat cultivars. An important component of the differential Al tolerance between SH and CK is the greater ability of the Al-tolerant cultivar to excrete malate from the roots, which is independent of its internal concentration in the roots.

  14. 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;

    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......-related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. MATERIALS AND METHODS: The femoral vein was catheterised in rats and GTN was infused (4 µg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP and Western blot for measuring n......NOS protein expression. The effect of olcegepant, L-nitro-arginine methyl ester (L-NAME) and neurokinin (NK)-1 receptor antagonist L-733060 were analysed on Fos activation. RESULTS: GTN-treated rats showed a significant increase of nNOS and CGRP in dura mater and CGRP in the trigeminal nucleus caudalis (TNC...

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. The prostaglandin F synthase activity of the human aldose reductase AKR1B1 brings new lenses to look at pathologic conditions.

    Directory of Open Access Journals (Sweden)

    Eva eBresson

    2012-05-01

    Full Text Available Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs, lead us to the discovery that AKR1B5 and later AKR1B1 were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2 and PGF2α may constitute a functional dyad with physiological relevance at least as important as the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231 also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region (MSRR containing two putative antioxidant response elements (ARE adjacent to TonE and AP1.We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors (ARIs like alrestatin, statil (ponalrestat and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human

  20. Sevoflurane and nitric oxide synthase expression in rat cochlea

    Institute of Scientific and Technical Information of China (English)

    Yuantao Li; Qingzhong Hou; Mingguang Wu; Xiaolei Huang; Jun Cao; Yin Gu; Xiaofei Qi; Yawen Li

    2010-01-01

    Sevoflurane exhibits anesthetic action by inhibiting the auditory cortex,brain stem nitric oxide synthase activity,and reducing nitric oxide(NO),thereby interfering with the hearing process.However,the influence of sevoflurane on peripheric receptor(cochlea)NO remains poorly understood.Results from the present study showed that sevoflurane downregulated cochlear inducible NO synthase,endothelial NO synthase and neuronal NO synthase expression in a dose dependent manner.This suggests that sevoflurane can decrease cochlear NO synthase expression in a dose dependent manner.

  1. 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.

  2. Involvement of salicylic acid on antioxidant and anticancer properties, anthocyanin production and chalcone synthase activity in ginger (Zingiber officinale Roscoe) varieties.

    Science.gov (United States)

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

    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 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 flavonoids in ginger can be increased

  3. 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

  4. 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.

  5. Processes of malate catabolism during the anaerobic metabolism of grape berries

    International Nuclear Information System (INIS)

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

  6. Leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of prostaglandin and constitutive nitric oxide synthase pathways.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2008-04-01

    Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE2 generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE2. The leptin-induced changes in arachidonic acid release and PGE2 generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

  7. The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells

    Science.gov (United States)

    Menendez, Javier A.; Vellon, Luciano; Espinoza, Ingrid; Lupu, Ruth

    2016-01-01

    The angiogenic inducer CCN1 (Cysteine-rich 61, CYR61) is differentially activated in metastatic breast carcinomas. However, little is known about the precise mechanisms that underlie the pro-metastatic actions of CCN1. Here, we investigated the impact of CCN1 expression on fatty acid synthase (FASN), a metabolic oncogene thought to provide cancer cells with proliferative and survival advantages. Forced expression of CCN1 in MCF-7 cells robustly up-regulated FASN protein expression and also significantly increased FASN gene promoter activity 2- to 3-fold, whereas deletion of the sterol response element-binding protein (SREBP) binding site in the FASN promoter completely abrogated CCN1-driven transcriptional activation. Pharmacological blockade of MAPK or PI-3'K activation similarly prevented the ability of CCN1 to induce FASN gene activation. Pharmacological inhibition of FASN activity with the mycotoxin cerulenin or the small compound C75 reversed CCN1-induced acquisition of estrogen independence and resistance to hormone therapies such as tamoxifen and fulvestrant in anchorage-independent growth assays. This study uncovers FASNdependent endogenous lipogenesis as a new mechanism controlling the metastatic phenotype promoted by CCN1. Because estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer, this previously unrecognized CCN1-driven lipogenic phenotype represents a novel metabolic target to clinically manage metastatic disease progression.

  8. 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

    2016-01-01

    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.

  9. 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.

  10. 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 ...

  11. 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.

  12. Purification and characterization of the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana.

    Science.gov (United States)

    An, Yan; Cao, Youzhi; Xu, Yingwu

    2016-07-01

    Malate dehydrogenase (MDH) ubiquitously exists in living organisms and has many isoforms in a single species. MDHs from some classes have been characterized for their catalytic properties, which show significant variations despite that they share high sequence identity for the active sites. One class MDH, the plastid-localized NAD-dependent MDH (plNAD-MDH) is known to be important for plant survival in a dark environment, but its biochemical and enzymatic properties have not been well characterized. This study attempts to fill the gap. plNAD-MDH was expressed in an Escherichia coli system and purified using nickel-affinity chromatography followed by size exclusion chromatography. The N-terminal fusion his-tag was removed by protease cleavage. The gel filtration assay and glutaraldehyde cross-linking results showed that the active enzyme was a homodimer in solution. Further assay indicated that plNAD-MDH is most active at a neutral pH value. The Km values for oxaloacetate and NADH are found in the submillimolar order, a median range for most MDHs. The maximum reaction rate values, however, are dramatically different from other plant MDHs, indicating that plNAD-MDH has different substrate specificity. Moreover, we obtained crystals for this enzyme, which laid the groundwork for further analysis of the enzymatic mechanism from structural stand point. PMID:26095832

  13. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration.

    Science.gov (United States)

    Ban, Hyun Seung; Xu, Xuezhen; Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  14. 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

  15. 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.

  16. A Review on Molecular Physiology of Malate and Lactate Dehydrogenases in Fishes

    Institute of Scientific and Technical Information of China (English)

    G.TRIPATHI

    1993-01-01

    The malate(EC1.1.1.37)and lactate(EC1.1.1.27)dehydrogenases are the metaboliic enzymes directly or indirectly involved in energy production,gluconeogenesis and lipogenesis.Malate dehydrogenase(MDH)exists in two isoenzymic forms,cytoplasmic(cMDH)and mitochondrial(mMDH),composed of A and /or B subunits(dimeric molecule:MW 40,000-120,000).Lactate dehydrogenase(LDH)has tetrameric(W 35,000-110,000)structure made up of either A and/or B,or C(,C,E,F)subunits,They catalyze an ordered bisubstrate(substrate and coenzyme) reaction in cytosol(cMDH and LDH)and mitochondrion(mMDH)for specific purposes.The cMDH,mMDHand LDH generally exhibit the maxium velocity(Vmax)of 50-500,0.5-15,and 80-400 units/g wet wt.respectively at an optimum pH(6.5-8.0)and temperature(20-30℃).The several folds higher activity of cMDH as compared to mMDH is to carry out three different(energy production,gluconeogenesis,lipogenesis)metabolic functions rather catalysin only Krebs cycle as mMDH does.Kinetic constant(Km)of cMDH,mMDH and LDH for substrate and coenzyme varies within the rage of 0.06-0.30,0.04-0.20,and 0.04-0.50 mmol·L-1 respectively reflecting their affinities for the substrates.Activiities of these enzymes are inhibited by substrates and coenzymes both.A number of environmental and physiological signals considerably influence the enzyme activities.The extreme pH of the medium decreases the activities of cMDH,mMDH and LDH.Seasonal changes in environmental factors(temperature,photoperiod,rainfall,food availability etc.)alter enzyme activities and may affect the expression of subunits.Thermal acclimation exerts tissue and species-spectific changes in Km,activity and subunit expression of cMDH,mMDH and LDH.Activities of these enzymes substantially deline duuring starvation periods.Enzyme scaling shows a decrease in cMDH and an increase in mMDH and LDH activities as a function of increasing body-size.Metabolic hormones may either decrease or increase the activities of enzymes or they do not

  17. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    Science.gov (United States)

    Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

    2002-01-01

    α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

  18. The non-coding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells

    OpenAIRE

    Gutschner, Tony; Hämmerle, Monika; Eißmann, Moritz; Hsu, Jeff; Kim, Youngsoo; Hung, Gene; Revenko, Alexey; Arun, Gayatri; Stentrup, Marion; Groß, Matthias; Zörnig, Martin; MacLeod, A. Robert; Spector, David L.; Diederichs, Sven

    2012-01-01

    The long non-coding RNA MALAT1, also known as MALAT-1 or NEAT2, is a highly conserved nuclear ncRNA and a predictive marker for metastasis development in lung cancer. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements using Zinc Finger Nucleases. The achieved 1000-fold MALAT1 silencing provides a unique loss-of-function model. Proposed mechanisms of action include regulation of splicing or ...

  19. Evaluation of Tear Malate Dehydrogenase 2 in Mild Dry Eye Disease

    Institute of Scientific and Technical Information of China (English)

    Qing Guo; Houbin Huang; Yuli Pi; Hancheng Zhang

    2014-01-01

    Purpose: To evaluate the effect of tear malate dehydrogenase 2 on monitoring ocular surface injury in mild dry eye (DE) disease. Methods: A total of 15 DE patients (30 eyes) with mild sub-jective symptoms but no ocular surface fluorescein staining signs were enrolled in this study. (DE group)..The control group was 15 healthy age- and sex-matched volunteers (30 eyes)..All subjects were asked to fill out a DE symptoms questionnaire and take different tests including tear MDH and MDH2 activities evaluation,..tear breakup time. (TBUT), Schirmer I,.and slit-lamp examination of the ocular surface. We investigated different changes in tear MDH and MDH2 ac-tivities in the DE group and control group,.discussed the as-sociation between tear MDH2 activity and DE symptoms, and the relationship between tear MDH2 activity and diagnostic tests (Schirmer I and TBUT). We also analyzed the changes in tear MDH2 activities after the treatment with artificial tears. Results:.Tear MDH activities in the DE group and control group were 288 ±102 U/L and 259 ±112 U/L,.respectively, and this difference was not statistically significant (P>0.05). The tear MDH2 activities in DE group were significantly in-creased compared with control group. Tear MDH2 was signif-icantly and negatively correlated with the Schirmer’s value (r=-0.733,P Conclusion: Tear MDH2 activity can indicate ocular surface injury in mild DE patients and may be used to monitor the re-sponse to therapy.

  20. 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.

  1. Activation of nuclear factor Κb and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans

    Institute of Scientific and Technical Information of China (English)

    曾焱华; 吴移谋; 张文波; 余敏君; 朱翠明; 谭立志

    2004-01-01

    Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression.Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3β in a hamster model of oral oncogenesis.

    Science.gov (United States)

    Sophia, Josephraj; Kiran Kishore T, Kranthi; Kowshik, Jaganathan; Mishra, Rajakishore; Nagini, Siddavaram

    2016-02-23

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1(Thr286) and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment.

  7. Aronia melanocarpa juice, a rich source of polyphenols, induces endothelium-dependent relaxations in porcine coronary arteries via the redox-sensitive activation of endothelial nitric oxide synthase.

    Science.gov (United States)

    Kim, Jong Hun; Auger, Cyril; Kurita, Ikuko; Anselm, Eric; Rivoarilala, Lalainasoa Odile; Lee, Hyong Joo; Lee, Ki Won; Schini-Kerth, Valérie B

    2013-11-30

    This study examined the ability of Aronia melanocarpa (chokeberry) juice, a rich source of polyphenols, to cause NO-mediated endothelium-dependent relaxations of isolated coronary arteries and, if so, to determine the underlying mechanism and the active polyphenols. A. melanocarpa juice caused potent endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to A. melanocarpa juice were minimally affected by inhibition of the formation of vasoactive prostanoids and endothelium-derived hyperpolarizing factor-mediated responses, and markedly reduced by N(ω)-nitro-l-arginine (endothelial NO synthase (eNOS) inhibitor), membrane permeant analogs of superoxide dismutase and catalase, PP2 (Src kinase inhibitor), and wortmannin (PI3-kinase inhibitor). In cultured endothelial cells, A. melanocarpa juice increased the formation of NO as assessed by electron paramagnetic resonance spectroscopy using the spin trap iron(II)diethyldithiocarbamate, and reactive oxygen species using dihydroethidium. These responses were associated with the redox-sensitive phosphorylation of Src, Akt and eNOS. A. melanocarpa juice-derived fractions containing conjugated cyanidins and chlorogenic acids induced the phosphorylation of Akt and eNOS. The present findings indicate that A. melanocarpa juice is a potent stimulator of the endothelial formation of NO in coronary arteries; this effect involves the phosphorylation of eNOS via the redox-sensitive activation of the Src/PI3-kinase/Akt pathway mostly by conjugated cyanidins and chlorogenic acids.

  8. Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations

    Directory of Open Access Journals (Sweden)

    Anna Dowierciał

    2014-01-01

    Full Text Available The crystal structure of mouse thymidylate synthase (mTS in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB and thus supporting tighter binding of ligands, and the other being more open (dimer CD and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.

  9. 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

  10. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression.

    Directory of Open Access Journals (Sweden)

    Alessio Ligabue

    Full Text Available BACKGROUND: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS. Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G(1 phase and hTS is localized in the nuclei during S and G(2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. CONCLUSIONS/SIGNIFICANCE: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5

  11. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Fourmann

    Full Text Available Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.

  12. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    Science.gov (United States)

    Fourmann, Jean-Baptiste; Tillault, Anne-Sophie; Blaud, Magali; Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.

  13. CCR5-Dependent Activation of mTORC1 Regulates Translation of Inducible NO Synthase and COX-2 during Encephalomyocarditis Virus Infection.

    Science.gov (United States)

    Shaheen, Zachary R; Naatz, Aaron; Corbett, John A

    2015-11-01

    Encephalomyocarditis virus (EMCV) infection of macrophages results in the expression of a number of inflammatory and antiviral genes, including inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. EMCV-induced macrophage activation has been shown to require the presence of CCR5 and the activation of PI3K-dependent signaling cascades. The purpose of this study was to determine the role of PI3K in regulating the macrophage responses to EMCV. We show that PI3K regulates EMCV-stimulated iNOS and COX-2 expression by two independent mechanisms. In response to EMCV infection, Akt is activated and regulates the translation of iNOS and COX-2 through the mammalian target of rapamycin complex (mTORC)1. The activation of mTORC1 during EMCV infection is CCR5-dependent and appears to function in a manner that promotes the translation of iNOS and COX-2. CCR5-dependent mTORC1 activation functions as an antiviral response, as mTORC1 inhibition increases the expression of EMCV polymerase. PI3K also regulates the transcriptional induction of iNOS and COX-2 in response to EMCV infection by a mechanism that is independent of Akt and mTORC1 regulation. These findings indicate that macrophage expression of the inflammatory genes iNOS and COX-2 occurs via PI3K- and Akt-dependent translational control of mTORC1 and PI3K-dependent, Akt-independent transcriptional control.

  14. 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.

  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...

  17. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  18. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

    DEFF Research Database (Denmark)

    Maile, C A; Hingst, Janne Rasmuss; Mahalingan, K K;

    2016-01-01

    and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle...

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

    Science.gov (United States)

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-10-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

  4. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  5. 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

  6. Development and validation of HPTLC method for the estimation of almotriptan malate in tablet dosage form

    Directory of Open Access Journals (Sweden)

    Suneetha A

    2010-01-01

    Full Text Available A new, simple, precise and accurate high performance thin layer chromatographic method has been proposed for the determination of almotriptan malate in a tablet dosage form. The drug was separated on aluminum plates precoated with silica gel 60 GF 254 with butanol:acetic acid:water (3:1:1 was used as mobilephase. Quantitative analysis was performed by densitometric scanning at 300 nm. The method was validated for linearity, accuracy, precision and robustness. The calibration plot was linear over the range of 100-700 ng/band for almotriptan malate. The method was successfully applied to the analysis of drug in a pharmaceutical dosage form.

  7. 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.

  8. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.

    Science.gov (United States)

    Schleicher, Ulrike; Paduch, Katrin; Debus, Andrea; Obermeyer, Stephanie; König, Till; Kling, Jessica C; Ribechini, Eliana; Dudziak, Diana; Mougiakakos, Dimitrios; Murray, Peter J; Ostuni, Renato; Körner, Heinrich; Bogdan, Christian

    2016-05-01

    Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  9. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection

    Directory of Open Access Journals (Sweden)

    Ulrike Schleicher

    2016-05-01

    Full Text Available Neutralization or deletion of tumor necrosis factor (TNF causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1 expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO synthase (NOS2 was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  10. 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.

  11. 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

  12. 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

  13. Identification of 2-aminothiazole-4-carboxylate derivatives active against Mycobacterium tuberculosis H37Rv and the beta-ketoacyl-ACP synthase mtFabH.

    Directory of Open Access Journals (Sweden)

    Qosay Al-Balas

    Full Text Available BACKGROUND: Tuberculosis (TB is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. METHODOLOGY/PRINCIPAL FINDINGS: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H(37R(v and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H(37R(v with an MIC of 0.06 microg/ml (240 nM, but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido-5-(3-chlorophenylthiazole-4-carboxylate inhibited mtFabH with an IC(50 of 0.95+/-0.05 microg/ml (2.43+/-0.13 microM but was not active against the whole cell organism. CONCLUSIONS/SIGNIFICANCE: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.

  14. 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

  15. 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

  16. 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.

  17. 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

    and 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.......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...... and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P values of CS and V.O2max did not correlate (r =- 0.07, n = 148, P = 0...

  18. L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

    Science.gov (United States)

    Zeng, X; Wu, J; Wu, Q; Zhang, J

    2015-01-01

    Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.

  19. 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...

  20. 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 ...

  1. 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

    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...

  2. Glycogen synthase kinase 3 β activity is required for hBora/Aurora A-mediated mitotic entry.

    Science.gov (United States)

    Lee, Yu-Cheng; Liao, Po-Chi; Liou, Yih-Cherng; Hsiao, Michael; Huang, Chi-Ying; Lu, Pei-Jung

    2013-03-15

    The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G 2 phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3β can interact with hBora and phosphorylate hBora at Ser274 and Ser278. Pharmacological inhibitors of GSK3β reduced the retarded migrating band of hBora in cells and diminished the phosphorylation of hBora by in vitro kinase assay. Moreover, as well as in GSK3β activity-inhibited cells, specific knockdown of GSK3β by shRNA and S274A/S278 hBora mutant-expressing cells also exhibited the reduced Plk1 activation and a delay in mitotic entry. It suggests that GSK3β activity is required for hBora-mediated mitotic entry through Ser274 and Ser278 phosphorylation.

  3. The Pb-hyperaccumulator aquatic fern Salvinia minima Baker, responds to Pb(2+) by increasing phytochelatins via changes in SmPCS expression and in phytochelatin synthase activity.

    Science.gov (United States)

    Estrella-Gómez, N; Mendoza-Cózatl, D; Moreno-Sánchez, R; González-Mendoza, D; Zapata-Pérez, O; Martínez-Hernández, A; Santamaría, J M

    2009-03-01

    The relationship between accumulation of Pb(2+) and the activation of chelation and metal sequestration mechanisms mediated by phytochelatins (PC) was analyzed in the Pb(2+) hyperaccumulator aquatic fern Salvinia minima, after exposure to 40microM Pb(NO(3))(2). The tissue accumulation pattern of lead and the phytochelatin biosynthesis responses were analyzed in both, S. minima submerged root-like modified fronds (here named "roots"), and in its aerial leaf-like fronds ("leaves"). S. minima roots accumulated a significantly higher concentrations of Pb(+2) than leaves did. Accumulation of Pb(2+) in roots was bi-phasic with a first uptake phase reached after 3h exposure and a second higher uptake phase reached after 24h exposure. In leaves, a single delayed, smaller uptake phase was attained only after 9h of exposure. In roots lead accumulation correlated with an increased phytochelatin synthase (PCS) activity and an enhanced PC production. A higher proportion of polymerized PC(4) was observed in both tissues of exposed S. minima plants relative to unexposed ones, although a higher concentration of PC(4) was found in roots than in leaves. PCS activity and Pb(2+) accumulation was also higher in roots than in leaves. The expression levels of the S. minima PCS gene (SmPCS), in response to Pb(2+) treatment, were also evaluated. In S. minima leaves, the accumulation of Pb(2+) correlated with a marked increase in expression of SmPCS, suggesting a transcriptional regulation in the PCS activation and PC accumulation in this S. minima tissue. However, in roots, the basal expression of SmPCS was down-regulated after Pb(2+) treatment. This fact did not correlate with the later but strong increase in both, PCS activity and PC production; suggesting that the PC biosynthesis activation in S. minima roots occurs only by post-translational activation of PCS. Taken together, our data suggest that the accumulation of PC in S. minima is a direct response to Pb(2+) accumulation, and

  4. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  5. 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...

  6. 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

  7. The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit.

    Science.gov (United States)

    Starrett, D A; Laties, G G

    1991-03-01

    When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A(+)) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.

  8. 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

  9. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  10. Homochiral Cu(II) and Ni(II) malates with tunable structural features

    Science.gov (United States)

    Zavakhina, Marina S.; Samsonenko, Denis G.; Virovets, Alexander V.; Dybtsev, Danil N.; Fedin, Vladimir P.

    2014-02-01

    Four new homochiral metal-organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H2O (1), [Cu(mal)(bpe)]·2H2O (2), [Ni(mal)(bpy)]·1.3CH3OH (3) and [Ni(mal)(bpe)]·4H2O (4) (mal=S-malate, bpy=4,4‧-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1-3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral {Ni(mal)} motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %.

  11. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

    NARCIS (Netherlands)

    Klei, I.J. van der; Faber, K.N.; Keizer-Gunnink, I.; Gietl, C.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstr

  12. The intracellular localization of malate dehydrogenase isoenzymes in Pisum arvense roots

    Directory of Open Access Journals (Sweden)

    Genowefa Kubik-Dorosz

    2014-02-01

    Full Text Available Mitochondria and plastids were isolated from Pisum arvense root cells by sucrose density gradient centrifugation. The individual subcellular fractions so obtained were subjected to isoelectric focusing on cellulose acetate strips. Mitochondria and plastids each contained one NAD -malate dehydrogenase, while three isoenzymes were associated with the supernatant.

  13. Homochiral Cu(II) and Ni(II) malates with tunable structural features

    International Nuclear Information System (INIS)

    Four new homochiral metal–organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H2O (1), [Cu(mal)(bpe)]·2H2O (2), [Ni(mal)(bpy)]·1.3CH3OH (3) and [Ni(mal)(bpe)]·4H2O (4) (mal=S-malate, bpy=4,4′-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1–3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral (Ni(mal)) motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %. - Graphical abstract: Four new homochiral metal–organic frameworks are built from Ni2+ or Cu2+ cations, S-malate anions and N-donor linkers of different length, which controls the size of pores and guest accessible volume of the homochiral structure. Display Omitted - Highlights: • Four new homohiral metal–organic frameworks based on Ni2+ and Cu2+. • Cu(II)–malate layers and Ni(II)–malate chains are connected by N-donor linkers. • N-donor linkers of different length control the size of pores

  14. 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

  15. 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.

  16. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  17. 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.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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

  20. 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

  1. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  2. Clinical Significance of Long Non-coding RNA MALAT1 Expression in Tissue and Serum of Breast Cancer.

    Science.gov (United States)

    Miao, Yufeng; Fan, Rengen; Chen, Lige; Qian, Haixin

    2016-07-01

    Long non-coding RNAs (lncRNAs) have been proven to serve a critical role in cancer development and progression. The aim of this study was to elucidate clinical significance of lncRNA MALAT1 expression in breast cancer (BC). A total of 78 BC patients treated with radical resection were enrolled in this study. Quantitative reverse transcription-polymerase chain reaction was used to detect MALAT1 expression in tissues and serum samples. The receiver operating characteristics (ROC) curve was constructed to describe diagnostic specificity and sensitivity. Lentivirus-mediated RNA interference was used to knockdown MALAT1 in the MDA-MB-231 cell line, and then cell proliferation and invasion were explored. Results showed that MALAT1 expression was significantly up-regulated in 85.9% (67/78) of cancerous tissues compared with normal counterparts (P<0.01). Further, an elevated MALAT1 expression in BC tissue was significantly associated with lymph metastasis (P=0.037) and adverse 5-year disease-free survival (mean 48.5 months vs 62.7 months, P=0.012). Suppression of lncRNA MALAT1 significantly inhibited BC cells proliferation, migration and invasion, induced apoptosis and cell cycle G1 arrest. In addition, serum MALAT1 levels in BC patients were much higher than levels in patients with benign breast disease (P<0.001), its diagnostic efficacy was satisfactory, area under the curve (AUC) was 0.833. In conclusion, MALAT1 upregulation plays an important rolein BC development, and serum MALAT1 level may be a potential tumor marker for BC diagnosis.

  3. 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

    The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase...

  4. 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

  5. Prediction of MALAT1 Mediated Regulatory Network by Bioinformatics Analysis%生物信息学分析及预测长链非编码RNA MALAT1的分子调控网络

    Institute of Scientific and Technical Information of China (English)

    韩泽平; 何金花; 黎毓光; 杨晓燕; 李秋娴; 朱剑霞

    2013-01-01

    目的 运用在线数据库对长链非编码RNA MALAT1(metastasis-associated lung adenocarcinoma transcript 1,MALAT1)进行生物信息学分析,并进一步推测其分子调控网络.方法 运用多个在线数据库,预测并筛选MALAT1的上游转录因子、下游miRNA 及其靶基因,筛选出均与其相关的疾病,进一步研究在该疾病中MALAT1的核心调控网络.结果 通过预测及筛选,以乳腺癌为研究对象,MALAT1受上游转录因子snail、sox-5、FOXQ1和RUNX1的调控,同时又可调控下游hsa-miR-320a的表达;而hsa-miR-320a的预测靶基因为CCR7、PTEN和FADD,它们又可与MALAT1的转录因子相互作用,形成了一个调控环路.结论 对MALAT1分子调控网络生物信息学的分析可有助于理解其在乳腺癌发生与发展中的作用机制并为后续实验提供良好的指导依据.%Objective To analyze the bioinformatics of the long noncoding RNA MALAT1 ( metastasis-associated lung adenocarcinoma transcript 1 , MALAT1) through online database , and to predict the molecular control network of MALAT1. Methods Several online databases were used to predict and screen the upstream transcription factors , the downstream miRNAs , the target genes and the related diseases of MALAT1. The role of MALAT1-mediated regulatory network was further examined in the diseases. Results After prediction and screening , with the breast cancer as the object of study, it was found that MALAT1 was regulated by the upstream transcription factors snail , sox-5, FOXQ1 and RUNX1, and meanwhile, it could regulate the expression of its downstream miRNA has-miR-320a. The target genes of hsa-miR-320a were speculated to be CCR7, PTEN and FADD, which could interact with the transcription factors of MALAT 1 and form a control loop of MALAT1. Conclusion Analyzing the MALAT1 molecular regulation network by bioinformatics method helps understand the mechanism of occurrence and development of breast cancer , and provide valuable guideline

  6. 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

  7. 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.

  8. Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Xiao Ming Wang

    Full Text Available PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs of Mycobacterium tuberculosis (Mtb. In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.

  9. 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.

  10. 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

  11. 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 ...

  12. 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...

  13. Duplication of Locus Coding of Malate Dehydrogenase in Populus tomentosa Carr.

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Carr. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-1 were found. Three allozyme bands at locus Mdh-1 were 9:6:1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-1 were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase (MDH) zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-1 coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P. tomentosa was discovered and reported for the first time.

  14. 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 ...

  15. 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...

  16. Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

    Science.gov (United States)

    Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba

    2014-09-15

    Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

    Science.gov (United States)

    Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

    2016-09-01

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

  2. 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.

  3. 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...

  4. 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

  5. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  6. Long Non Coding RNA MALAT1 Promotes Tumor Growth and Metastasis by inducing Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Zhou, Xuan; Liu, Su; Cai, Guoshuai; Kong, Lingping; Zhang, Tingting; Ren, Yu; Wu, Yansheng; Mei, Mei; Zhang, Lun; Wang, Xudong

    2015-11-02

    The prognosis of advanced oral squamous cell carcinoma (OSCC) patients remains dismal, and a better understanding of the underlying mechanisms is critical for identifying effective targets with therapeutic potential to improve the survival of patients with OSCC. This study aims to clarify the clinical and biological significance of metastasis-associated long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OSCC. We found that MALAT1 is overexpressed in OSCC tissues compared to normal oral mucosa by real-time PCR. MALAT1 served as a new prognostic factor in OSCC patients. When knockdown by small interfering RNA (siRNA) in OSCC cell lines TSCCA and Tca8113, MALAT1 was shown to be required for maintaining epithelial-mesenchymal transition (EMT) mediated cell migration and invasion. Western blot and immunofluorescence staining showed that MALAT1 knockdown significantly suppressed N-cadherin and Vimentin expression but induced E-cadherin expression in vitro. Meanwhile, both nucleus and cytoplasm levels of β-catenin and NF-κB were attenuated, while elevated MALAT1 level triggered the expression of β-catenin and NF-κB. More importantly, targeting MALAT1 inhibited TSCCA cell-induced xenograft tumor growth in vivo. Therefore, these findings provide mechanistic insight into the role of MALAT1 in regulating OSCC metastasis, suggesting that MALAT1 is an important prognostic factor and therapeutic target for OSCC.

  7. 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...

  8. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

  9. 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.

  10. 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 ...

  11. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α 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. PMID:21602811

  12. 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

  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. NADP-malate Dehydrogenase Isoforms of Wheat Leaves under Drought: Their Localization, and Some physicochemical and Kinetic Properties

    Directory of Open Access Journals (Sweden)

    H.G. Babayev

    2015-09-01

    Full Text Available Changes in sub-cellular localization, isoenzyme spectrum and kinetic characteristics of NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.82 in Triticum durum Desf. genotypes with contrasting drought tolerance have been studied. In chloroplast and cytosol fractions of mesophyll cells of wheat flag leaves 70-75% and 25-30% of the total NADP-MDH activity were found to be localized, respectively. Three isoforms of NADP-MDH with molecular weights of 66, 74 and 86 kDa were revealed in the chloroplast fraction, whereas in the cytosolic fraction molecular weights of the isoenzymes were found to be 42, 66 and 74 kDa. Drought caused the formation of a new 90 kDa isoform of the enzyme in the chloroplast fraction in anthesis phase of ontogenesis. In the drought-tolerant genotype the appearance of the new isoform in the chloroplast fraction was accompanied by a more rapid increase in Km and Vmax contrary to the chloroplast fraction of the drought-sensitive genotype manifesting a slight decrease in these parameters, suggesting one of the adaptive traits in forming drought tolerance in C3 plants.

  15. 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

  16. Long non-coding RNA MALAT-1 is downregulated in preeclampsia and regulates proliferation, apoptosis, migration and invasion of JEG-3 trophoblast cells.

    Science.gov (United States)

    Chen, Haiying; Meng, Tao; Liu, Xuemin; Sun, Manni; Tong, Chunxiao; Liu, Jing; Wang, He; Du, Juan

    2015-01-01

    Long non-coding RNA (lncRNA), as a newly identified subset of the transcriptome, has been implicated in a variety of physiological and pathological processes. Metastasis associated lung adenocarcinoma transcript-1 (MALAT-1), a lncRNA that was initially detected in the metastatic lung cancer, was reported to be overexpressed in placenta previa increta/percreta (I/P), which is caused by excessive trophoblast invasion. However, the role of MALAT-1 in the regulation of trophoblast behavior is not fully understood. In this study, we first examined the expression of MALAT-1 in the placentas from the patients with preeclampsia, the pathology of which is associated with inadequate trophoblast invasion, and found that the expression of MALAT-1 was downregulated in the preeclamptic placentas as compared to the normal placentas. We further investigated the function of MALAT-1 in JEG-3 trophoblast cell line using short interfering RNA (siRNA) against MALAT-1 transcripts. Silencing of MALAT-1 in JEG-3 cells suppressed proliferation and induced cell cycle arrest at G0/G1 phase. Reduced expression of MALAT-1 by RNA interference resulted in enhanced apoptosis in JEG-3 cells, accompanied with elevated levels of the pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). Moreover, the migration rate and the invasiveness of JEG-3 cells were suppressed when MALAT-1 was downregulated. In summary, our results suggest that MALAT-1 may play an important role in the regulation of proliferation, cell cycle, apoptosis, migration and invasion of trophoblast cells, and under-expression of MALAT-1 during early placentation may be involved in the pathogenesis of preeclampsia.

  17. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    Science.gov (United States)

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  18. Pu-erh tea, green tea, and black tea suppresses hyperlipidemia, hyperleptinemia and fatty acid synthase through activating AMPK in rats fed a high-fructose diet.

    Science.gov (United States)

    Huang, Hsiu-Chen; Lin, Jen-Kun

    2012-02-01

    Although green tea extract has been reported to suppress hyperlipidemia, it is unclear how tea extracts prepared from green, oolong, black and pu-erh teas modulate fatty acid synthase expression in rats fed on a high-fructose diet. In this animal study, we evaluated the hypolipidemic and hypoleptinemia effect of these four different tea leaves fed to male Wistar rats for 12 weeks. The results showed that a fructose-rich diet significantly elevated serum triacylglycerols, cholesterol, insulin, and leptin concentrations, as compared with those in the control group. Interestingly, consuming tea leaves for 12 weeks almost normalized the serum triacylglycerols concentrations. Again, rats fed with fructose/green tea and fructose/pu-erh tea showed the greatest reduction in serum TG, cholesterol, insulin and leptin levels. In contrast, serum cholesterol and insulin concentrations of the fructose/oolong tea-fed rats did not normalize. The relative epididymal adipose tissue weight was lower in all rats supplemented with tea leaves than those fed with fructose alone. There was molecular evidence of improved lipid homeostasis according to fatty acid synthase (FAS) protein expression. Furthermore, supplementation of green, black, and pu-erh tea leaves significantly decreased hepatic FAS mRNA and protein levels, and increased AMPK phosphorylation, compared with those of rats fed with fructose only. These findings suggest that the intake of green, black, and pu-erh tea leaves ameliorated the fructose-induced hyperlipidemia and hyperleptinemia state in part through the suppression of FAS protein levels and increased AMPK phosphorylation.

  19. Linking pseudouridine synthases to growth, development and cell competition.

    Science.gov (United States)

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  20. Inhibition of ruminal microbial methane production by beta-cyclodextrin iodopropane, malate and their combination in vitro.

    Science.gov (United States)

    Mohammed, N; Lila, Z A; Ajisaka, N; Hara, K; Mikuni, K; Hara, K; Kanda, S; Itabashi, H

    2004-06-01

    The objective of this study was to evaluate the effects of different concentrations of l-malate (0, 5, 10 and 20 mm), 2-iodopropane-beta-cyclodextrin complex (CD-IP) (0, 0.1, 0.2 and 0.4 mm) and a combination of malate (10 and 20 mm) plus CD-IP (0.2 and 0.4 mm) on methane production from corn starch. Ruminal fluid was collected from dairy cows, mixed with phosphate buffer (1 : 2) and incubated (30 ml) anaerobically at 38 degrees C for 6 h with or without additives. Fermentation of corn starch in the presence of malate resulted in an increase (p hydrogen production was increased (p Hydrogen production was also decreased (p inhibit methane production as well as to improve rumen fermentation and animal performance.

  1. 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.

  2. 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

  3. The HIF-2α-MALAT1-miR-216b axis regulates multi-drug resistance of hepatocellular carcinoma cells via modulating autophagy.

    Science.gov (United States)

    Yuan, Peng; Cao, Weibin; Zang, Quanling; Li, Guixin; Guo, Xiangfei; Fan, Jianghe

    2016-09-23

    In this study, we firstly investigated the association among lncRNA MALAT1, HIF-1α and HIF-2α in hepatocellular carcinoma (HCC) cells. Then, we investigated the regulative effect of MALAT1 on multi-drug resistance (MDR) in HCC cells and the underlying mechanism. The results showed that MALAT1 was over two times higher in BEL-7402/5-FU cells than in BEL-7402 cells. It was HIF-2α, but not HIF-1α induced MALAT1 upregulation in HCC cells. Dual luciferase assay demonstrated that there were at least two binding sites of miR-26b in MALAT1. Therefore, we infer that there is a HIF-2α-MALAT1-miR-216b axis in HCC cells. Cell viability assay showed that both MALAT1 siRNA and miR-216b mimics reduced IC50 of 5-FU, ADR and MMC in BEL-7402/5-FU cells. MALAT1 siRNA and miR-216b mimics showed similar effect as 3-MA on reducing LC3-II levels, inhibiting p62 degradation and suppressing GFP-LC3 puncta formation in BEL-7402/5-FU cells. Flow cytometric analysis showed that 3-MA treatment, MALAT1 siRNA and miR-216b mimics all promoted 5-FU induced apoptosis in BEL-7402/5-FU cells. Therefore, this study firstly revealed that there is a HIF-2α-MALAT1-miR-216b axis regulating MDR of HCC cells via modulating autophagy. PMID:27524242

  4. Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.

    Science.gov (United States)

    Kusaka, Jin; Shuto, Satoshi; Imai, Yukiko; Ishikawa, Kazuki; Saito, Tomo; Natori, Kohei; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2016-04-01

    The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.

  5. Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

    Science.gov (United States)

    Yamamura, Y; Mizuguchi, Y; Taura, F; Kurosaki, F

    2014-10-01

    A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells. PMID:25027024

  6. Solid and Solution Structural Studies of Dimeric and Tetrameric Molybdenum(Ⅵ) Malate Complexes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Rong-Hua; ZHOU Zhao-Hui; WAN Hui-Lin

    2008-01-01

    Reactions of potassium molybdate with racemic malic acid (H3mal = C4H6O5) result in the isolation of two mesomeric molybdenum malate complexes Ks[(MoO2)2O(R-mal)2][(MoO2)2O(S- mal)2]·4H2O 1 and (Him)2K6[(MoO2)4O3(R-mal)2][(MoO2)4O3(S-mal)2]·8H2O 2. Complex 1 belongs to the monoclinic system, space group C2/c with a = 14.8637(3), b = 6.9544(1), c = 19.6783(5) A, β = 100.081(2)°, V= 2002.70(7)A3, Mr = 1452.88, Z= 2, F(000) = 1416, T= 173 K, Dc = 2.409 g/cm3, μ(MoKa) = 2.167, R = 0.0283 and wR = 0.0733.2 is of triclinic system, space group P1 with a = 8.7707(2), b = 9.3310(3), c = 17.9093(7) A, α = 83.781(3), β = 85.626(2), γ= 84.822(2)°, V = 1447.84(8) A3, Mr = 2160.68, Z = 1, F(000) = 1048, T= 173 K, Dc = 2.478 g/cm3, μ(MoKα) = 2.230, R = 0.0234 and wR = 0.0584.1 is the first isolated dinuclear molybdenum(Ⅵ) malato complex in 1:1 molar ratio. The molybdenum atoms in the two complexes are six-coordinated in an approximately octahedral geometry. Two malates coordinate tridentately with the Mo atom via their α-alkoxy, α-carboxy and α-carboxy groups in 1 and 2. β-Carboxy group in 2 further links with the other two Mo atoms to give a tetrameric unit. The solution 1H and 13C NMR spectra indicate that dimeric malate molybdenum in 1 dissociates partly in solution and exists in an equilibrium with tetrameric species, while 2 is stable and retains its tetrameric structure without any dissociation.

  7. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Science.gov (United States)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  8. Resveratrol inhibits invasion and metastasis of colorectal cancer cells via MALAT1 mediated Wnt/β-catenin signal pathway.

    Directory of Open Access Journals (Sweden)

    Qing Ji

    Full Text Available Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC, in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1 also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/β-catenin signaling, thus the expression of its target genes such as c-Myc, MMP-7, as well as the expression of MALAT1. In detail, resveratrol down-regulates MALAT1, resulting in decreased nuclear localization of β-catenin thus attenuated Wnt/β-catenin signaling, which leads to the inhibition of CRC invasion and metastasis. This finding of ours surely provides important pre-clinical evidence supporting future use of resveratrol in prevention and treatment of CRC.

  9. 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.

  10. Heat shock protein 70 protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus via inhibition of nuclear factor-κB activation-induced nitric oxide synthase II expression.

    Science.gov (United States)

    Chang, Chiung-Chih; Chen, Shang-Der; Lin, Tsu-Kung; Chang, Wen-Neng; Liou, Chia-Wei; Chang, Alice Y W; Chan, Samuel H H; Chuang, Yao-Chung

    2014-02-01

    Status epilepticus induces subcellular changes that may eventually lead to neuronal cell death in the hippocampus. Based on an animal model of status epilepticus, our laboratory showed previously that sustained hippocampal seizure activity activates nuclear factor-κB (NF-κB) and upregulates nitric oxide synthase (NOS) II gene expression, leading to apoptotic neuronal cell death in the hippocampus. The present study examined the potential modulatory role of heat shock protein 70 (HSP70) on NF-κB signaling in the hippocampus following experimental status epilepticus. In Sprague-Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Expression of HSP70 was elevated as early as 1h after the elicitation of sustained seizure activity, followed by a progressive elevation that peaked at 24h. Pretreatment with an antisense oligonucleotide against hsp70 decreased the HSP70 expression, and significantly augmented IκB kinase (IKK) activity and phosphorylation of IκBα, alongside enhanced nuclear translocation and DNA binding activity of NF-κB in the hippocampal CA3 neurons and glial cells. These cellular events were followed by enhanced upregulation of NOS II and peroxynitrite expression 3h after sustained seizure activity that led to an increase of caspase-3 and DNA fragmentation in the hippocampal CA3 neurons 7days after experimental status epilepticus. We concluded that HSP70 protects against apoptotic cell death induced by NF-κB activation and NOS II-peroxynitrite signaling cascade in the hippocampal CA3 and glial cells following experimental status epilepticus via suppression of IKK activity and deactivation of IκBα.

  11. 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

  12. 17β-Estradiol treatment inhibits breast cell proliferation, migration and invasion by decreasing MALAT-1 RNA level

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Ziyi [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China); Chen, Changjin [Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041 (China); Liu, Yu [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China); Wu, Chuanfang, E-mail: 879413966@qq.com [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610041 (China)

    2014-03-07

    Highlights: • E2 affects not only estrogen-receptor α positive breast cells but also negative ones. • 100 nM E2 treatment affects breast cells proliferation, migration. • 100 nM E2 treatment functions in an estrogen-receptor α-independent way. • E2 treatment decreases MALAT-1 RNA level by post-transcriptional regulation. - Abstract: Breast cancer cells, which express estrogen receptor α (ERα), respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. But breast cancer cells without ERα show no effect on low concentration of estrogen treatment. Proliferation, migration and invasion of MCF10a, MCF7 and MB231 cells treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) was performed. We identified the effects of E2 on these breast cell lines, and looked for the difference in the presence and absence of ERα. Specifically, we looked for the changes of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1), which is found extensively and highly expressed in several kinds of tumor cells, including breast carcinoma. It was observed that proliferation, migration and invasion of breast cells were greatly affected by high concentration E2 treatment and were not affected by low concentration E2 treatment in an ERα independent way. We found that the high concentration E2 treatment largely decreased MALAT-1 RNA level. Interestingly, MALAT-1 decreasing by knocking down showed similar effects on proliferation, migration and invasion. E2 treatment affects breast tumor or non-tumor cells proliferation, migration and invasion in an ERα -independent, but a dose-dependent way by decreasing the MALAT-1 RNA level.

  13. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

    Directory of Open Access Journals (Sweden)

    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  14. Acute Citrulline-Malate Supplementation and High-Intensity Cycling Performance.

    Science.gov (United States)

    Cunniffe, Brian; Papageorgiou, Maria; OʼBrien, Barbara; Davies, Nathan A; Grimble, George K; Cardinale, Marco

    2016-09-01

    Cunniffe, B, Papageorgiou, M, O'Brien, B, Davies, NA, Grimble, GK, and Cardinale, M. Acute citrulline-malate supplementation and high-intensity cycling performance. J Strength Cond Res 30(9): 2638-2647, 2016-Dietary L-citrulline-malate (CM) consumption has been suggested to improve skeletal muscle metabolism and contractile efficiency, which would be expected to predispose exercising individuals to greater fatigue resistance. The purpose of this study was to examine the effects of CM supplementation on acid-base balance and high-intensity exercise performance. In a double-blind, placebo-controlled, crossover study, 10 well-trained males consumed either 12 g of CM (in 400 ml) or lemon sugar-free cordial (placebo [PL]) 60 minutes before completion of 2 exercise trials. Each trial consisted of subjects performing 10 (×15 seconds) maximal cycle sprints (with 30-second rest intervals) followed by 5 minutes recovery before completing a cycle time-to-exhaustion test (TTE) at 100% of individual peak power (PP). Significant increases in plasma concentrations of citrulline (8.8-fold), ornithine (3.9-fold), and glutamine (1.3-fold) were observed 60 minutes after supplementation in the CM trial only (p ≤ 0.05) and none of the subjects experienced gastrointestinal side-effects during testing. Significantly higher exercise heart rates were observed in CM condition (vs. PL) although no between trial differences in performance related variables (TTE: [120 ± 61 seconds CM vs. 113 ± 50 seconds PL]), PP or mean power, ([power fatigue index: 36 ± 16% CM vs. 28 ± 18% PL]), subjective rating of perceived exertion or measures of acid-base balance (pH, lactate, bicarbonate, base-excess) were observed (p > 0.05). This study demonstrated that acute supplementation of 12 g CM does not provide acute ergogenic benefits using the protocol implemented in this study in well-trained males. PMID:26808848

  15. Evaluation of the Reproductive Toxicity, Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Zhang, Weijie; Zhao, Ting; Mao, Guanghua; Wang, Wei; Wu, Xueshan; Zhou, Zhaoxiang; Huang, Jing; Bao, Yongtuan; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the reproductive toxicity of chromium malate in Sprague-Dawley rats and then inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, and lipid metabolism. The results showed that no pathological, toxic feces and urine changes were observed in clinical signs of parental and fetal rats in chromium malate groups. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of chromium malate groups have no significant change compared with control group and chromium picolinate group. The serum and organ contents of Cr in chromium malate groups have no significant change when compared with control group. No measurable damage on liver, brain, kidney, and testis/uterus of chromium malate groups was found. No significant change in body mass, absolute and relative organ weights, and hematological and biochemical changes of rats were observed compared with the control and chromium picolinate groups. The results indicated that supplements with chromium malate does not cause obvious damage and has no obvious effect on glycometabolism, glycometabolism-related enzyme, and lipid metabolism on female and male rats. The results of this study suggested that chromium malate is safe for human consumption and has the potential for application as a functional food ingredient and dietary supplement. PMID:25876088

  16. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank;

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c...

  17. Metastasis-associated lung adenocarcinoma transcript 1 promotes the proliferation of chondro­sarcoma cell via activating Notch-1 signaling pathway

    Directory of Open Access Journals (Sweden)

    Xu FQ

    2016-04-01

    Full Text Available Fengqin Xu,1,* Zhi-qiang Zhang,2,* Yong-chao Fang,2 Xiao-lei Li,2 Yu Sun,2 Chuan-zhi Xiong,2 Lian-qi Yan,2 Qiang Wang2 1Department of Orthopaedics, Hongquan Hospital, 2Department of Orthopaedics, Subei People’s Hospital, Yangzhou, Jiangsu Province, People’s Republic of China *These authors contributed equally to this work Background: Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1 is identified to be overexpressed in several cancers. However, the role of MALAT-1 in chondrosarcoma is poorly understood.Methods: The expression of MALAT-1 and Notch-1 signaling pathway was detected in chondrosarcoma tissues and chondrosarcoma cells by quantitative real-time polymerase chain reaction (qRT-PCR and Western blot. 3-(4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay was performed to examine the cell viability of chondrosarcoma cells transfected with si-MALAT-1 or pcDNA-MALAT-1. Then the expression of Notch-1 signaling pathway was detected when MALAT-1 was upregulated or downregulated in chondrosarcoma cells. A subcutaneous chondrosarcoma cells xenograft model was used to confirm the effect of MALAT-1 on tumor growth in vivo.Results: We found the increased expression of MALAT-1 and Notch-1 signaling pathway in chondrosarcoma tissue and cells. MALAT-1 promoted the proliferation of chondrosarcoma cells. In addition, MALAT-1 activated the Notch-1 signaling pathway at posttranscriptional level in chondrosarcoma cells. Meanwhile, overexpression of Notch-1 reversed the effect of si-MALAT-1 on the proliferation of chondrosarcoma cells. Finally, we found that MALAT-1 promoted the tumor growth in a subcutaneous chondrosarcoma cells xenograft model, which confirmed the promoted effect of MALAT-1 on the tumor growth in vivo.Conclusion: Taken together, our study demonstrated that MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway. Keywords: MALAT-1, cell proliferation

  18. 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.

  19. 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

  20. Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study.

    Science.gov (United States)

    Jacques, Paul F; Bostom, Andrew G; Selhub, Jacob; Rich, Sharron; Ellison, R Curtis; Eckfeldt, John H; Gravel, Roy A; Rozen, Rima

    2003-01-01

    The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, methionine synthase and methionine synthase reductase, have also been identified. Methionine synthase catalyzes the remethylation of homocysteine to form methionine and methionine synthase reductase is required for the reductive activation of the cobalamin-dependent methionine synthase. The methionine synthase gene (MTR) mutation is an A to G substitution, 2756A-->G, which converts an aspartate to a glycine codon. The methionine synthase reductase gene (MTRR) mutation is an A to G substitution, 66A-->G, that converts an isoleucine to a methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a methionine load, a weak positive association was observed between change in homocysteine after a methionine load and the number of mutant MTR alleles (P-trend=0.04), but this association was not statistically significant according to the overall F-statistic (P=0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine. PMID:12482550

  1. 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

  2. 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;

    2013-01-01

    was positively associated with pAkt-T308 (P=0.01) and Akt2 activity (P=0.04), but not pAkt-S473 or IRS-1-PI3K activity. Furthermore, pAkt-T308 and Akt2 activity were negatively associated with NH(2)-terminal GS phosphorylation (P=0.001 for both), which in turn was negatively associated with insulin-stimulated GS...

  3. 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.

  4. 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.

  5. Intervertebral disk-like biphasic scaffold—demineralized bone matrix cylinder and poly(polycaprolactone triol malate)—for interbody spine fusion

    OpenAIRE

    Li Jin; Yuqing Wan; Shimer, Adam L.; Shen, Francis H.; Li, Xudong J

    2012-01-01

    Interbody fusion is an established procedure to preserve disk height and anterior fusion, but fusion with autografts, allografts, and metallic cages has its endogenous shortcomings. The objective of this study is to investigate whether a biphasic scaffold model, the native demineralized bone matrix cylinder in conjunction with degradable biomaterial poly(polycaprolactone triol malate), can be employed as a biological graft for interbody fusion. The poly(polycaprolactone triol malate) was synt...

  6. 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

  7. 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

  8. 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

  9. 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...

  10. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  11. 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

  12. Evolution and function of phytochelatin synthases.

    Science.gov (United States)

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  13. 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

  14. 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

    that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (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 contraction...... basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were...

  15. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    Science.gov (United States)

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  16. Kinetic simulation of malate-aspartate and citrate-pyruvate shuttles in association with Krebs cycle.

    Science.gov (United States)

    Korla, Kalyani; Vadlakonda, Lakshmipathi; Mitra, Chanchal K

    2015-01-01

    In the present work, we have kinetically simulated two mitochondrial shuttles, malate-aspartate shuttle (used for transferring reducing equivalents) and citrate-pyruvate shuttle (used for transferring carbon skeletons). However, the functions of these shuttles are not limited to the points mentioned above, and they can be used in different arrangements to meet different cellular requirements. Both the shuttles are intricately associated with Krebs cycle through the metabolites involved. The study of this system of shuttles and Krebs cycle explores the response of the system in different metabolic environments. Here, we have simulated these subsets individually and then combined them to study the interactions among them and to bring out the dynamics of these pathways in focus. Four antiports and a pyruvate pump were modelled along with the metabolic reactions on both sides of the inner mitochondrial membrane. Michaelis-Menten approach was extended for deriving rate equations of every component of the system. Kinetic simulation was carried out using ordinary differential equation solver in GNU Octave. It was observed that all the components attained steady state, sooner or later, depending on the system conditions. Progress curves and phase plots were plotted to understand the steady state behaviour of the metabolites involved. A comparative analysis between experimental and simulated data show fair agreement thus validating the usefulness and applicability of the model.

  17. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis.

    Science.gov (United States)

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  18. [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

  19. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  20. Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

    Science.gov (United States)

    Li, Guojing; Meng, Xiangzong; Wang, Ruigang; Mao, Guohong; Han, Ling; Liu, Yidong; Zhang, Shuqun

    2012-06-01

    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.

  1. Inhibition of lipopolysaccharide-inducible nitric oxide synthase and IL-1beta through suppression of NF-kappaB activation by 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin isolated from Ruta graveolens L.

    Science.gov (United States)

    Raghav, Sunil Kumar; Gupta, Bhawna; Shrivastava, Anju; Das, Hasi Rani

    2007-03-29

    The Ruta graveolens L. plant is used in traditional medicine to treat a large number of diseases. The methanol (50%) extract of the whole plant was observed to inhibit the expression of inducible nitric oxide synthase (iNOS) and the cycloxygenase-2 (COX-2) gene in lipopolysaccharide (LPS)-induced macrophage cells (J774A.1, [Raghav, S.K., Gupta, B., Agrawal, C., Goswami, K., Das, H.R., 2006b. Anti-inflammatory effect of Ruta graveolens L. in murine macrophage cells. J. Ethnopharmacol. 104, 234-239]). The effect of whole plant extract on the expression of other pro-inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12, interferon-gamma (IFN-gamma) and the activation of nuclear factor-kB (NF-kappaB) were investigated in LPS stimulated macrophage cells. An active compound was isolated from this methanol extract by further solvent fractionation and reverse phase high performance liquid chromatography (RP-HPLC). The purified compound was identified as 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin having IUPAC nomenclature of 6-hydroxy-7-methoxy-3-(2-methyl but-3-en-2yl)-2H-chromen-2-one by ESI-MS, MALDI, FT-IR and NMR. Effect of this purified compound was assessed on iNOS, COX-2 and various pro-inflammatory cytokine genes and was observed to inhibit both the protein and mRNA expression of iNOS and IL-1beta in LPS challenged macrophages. Electrophoretic mobility shift assay (EMSA) and Western blot analyses indicated that the plant extract and the isolated active compound blocked the LPS-induced activation of NF-kappaB through the prevention of inhibitor-kB (IkB) degradation. The purified compound also showed the anti-oxidant activity. The active compound at a dose of 40 mg/kg body weight was observed to inhibit the iNOS and IL-1beta gene expression significantly in endotoxin-induced inflammatory model of BALB/c mice. The low level of nitric oxide production was also observed in the sera of compound treated mice

  2. A new member of the chalcone synthase (CHS family in sugarcane

    Directory of Open Access Journals (Sweden)

    Contessotto Miriam G.G.

    2001-01-01

    Full Text Available Sequences from the sugarcane expressed sequence tag (SUCEST database were analyzed based on their identities to genes encoding chalcone-synthase-like enzymes. The sorghum (Sorghum bicolor chalcone-synthase (CHS, EC 2.3.1.74 protein sequence (gi|12229613 was used to search the SUCEST database for clusters of sequencing reads that were most similar to chalcone synthase. We found 121 reads with homology to sorghum chalcone synthase, which we were then able to sort into 14 clusters which themselves were divided into two groups (group 1 and group 2 based on the similarity of their deduced amino acid sequences. Clusters in group 1 were more similar to the sorghum enzyme than those in group 2, having the consensus sequence of the active site of chalcone and stilbene synthase. Analysis of gene expression (based on the number of reads from a specific library present in each group indicated that most of the group 1 reads were from sugarcane flower and root libraries. Group 2 clusters were more similar to the amino acid sequence of an uncharacterized pathogen-induced protein (PI1, gi|9855801 from the S. bicolor expressed sequence tag (EST database. The group 2 clusters sequences and PI1 proteins are 90% identical, having two amino acid changes at the chalcone and stilbene synthase consensi but conserving the cysteine residue at the active site. The PI1 EST has not been previously associated with chalcone synthase and has a different consensus sequence from the previously described chalcone synthase of sorghum. Most of the group 2 reads were from libraries prepared from sugarcane roots and plants infected with Herbaspirillum rubrisubalbicans and Gluconacetobacter diazotroficans. Our results indicate that we have identified a sugarcane chalcone synthase similar to the pathogen-induced PI1 protein found in the sorghum cDNA libraries, and it appears that both proteins represent new members of the chalcone and stilbene synthase super-family.

  3. 百合鳞茎蔗糖合成酶活性检测体系的建立%Establishment of Detection System for Sucrose Synthase Activity in Lily Bulb

    Institute of Scientific and Technical Information of China (English)

    孙红梅; 王微微; 何玲; 王春夏; 李天来

    2011-01-01

    为了建立富含多糖的百合鳞茎蔗糖合成酶(sucrose synthase,EC2.4.1.13,SuSy)活性检测体系,深入研究其蔗糖代谢机制,以兰州百合(Lilium davidii var.unicolor)鳞茎外层鳞片为试材,分别研究了提取缓冲液种类及pH值、反应温度、底物浓度以及缓冲液pH值对SuSy合成和分解方向活性的影响.结果表明:SuSy合成方向活性检测的最适提取缓冲液是pH值为7.8的TrisHCl,最适反应温度为50℃,底物果糖最适浓度为50 mmol· L-1,UDPG最适浓度为5 mmol·L-1,反应缓冲液Tris-HCl最适pH值为7.5;SuSy分解方向活性检测的最适提取缓冲液为pH值7.8的Hepes-NaOH,最适反应温度为40℃,底物蔗糖最适浓度为10mmol· L-1,UDP最适浓度为7 mmol·L-1,反应缓冲液Mes-NaOH最适pH值为4.5.%This investigation was designed to establish the detection system for sucrose synthase (EC 2.4.1.13, SuSy) activities in lily bulb enriched with polysaccharides, which provided a detection method for the further research on the mechanism of sucrose metabolism. The effects of extracting buffer types, pH, reaction temperature, substrate concentrations, pH of the reaction buffer on the SuSy activities in both synthesis and decomposition direction were respectively studied by using the exterior scales of Lilium davidii var. Unicolor at planting stage as materials. And the results showed that the optimum extraction buffer was Tris-HCl of pH7.8, the appropriate temperature was 50 ℃, the preferential substrate concentration of fructose was 50 mmol·L-1, the preferential substrate concentration of UDPG was 5 mmol·L-1, and the suitable pH for reaction buffer Tris-HCI was 7.5 in the detection of SuSy synthesis activities. In the detection of SuSy decomposition activities, the optimum extraction buffer was Hepes-NaOH of pH7.8, the appropriate temperature was 40 X!, the adequate substrate concentration of sucrose was 10 mmol·L-1, the adequate substrate concentration of UDP was 7 mmol·L-1

  4. Alternative oxidase pathway optimizes photosynthesis during osmotic and temperature stress by regulating cellular ROS, malate valve and antioxidative systems

    Directory of Open Access Journals (Sweden)

    DINAKAR eCHALLABATHULA

    2016-02-01

    Full Text Available The present study reveals the importance of alternative oxidase (AOX pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 µmoles m-2 s-1 at 25 oC under a range of sorbitol concentrations from 0.4 M to 1.0M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 oC to 10 oC to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25 OC, the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation, under both hyper-osmotic (1.0 M sorbitol and sub-optimal temperature stress conditions (10 OC, while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG related to antioxidative system during hyper-osmotic stress. Nevertheless, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD and sub-optimal temperature (NADPH/NADP stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM, the observed changes in NaHCO3 dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(PH/NAD(P and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the

  5. Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocycl opropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

    Institute of Scientific and Technical Information of China (English)

    Jian-Feng LI; Robert QI; Liang-Hu QU; Autar K Mattoo; Ning LI

    2005-01-01

    l-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv.Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the Nterminus of LeACS2. Four C-terminus-deleted (26-50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser460 for this metalloprotease.Furhermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  11. 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.

  12. 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

  13. Reduced plasma adiponectin concentrations may contribute to impaired insulin activation of glycogen synthase in skeletal muscle of patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, K.; Frystyk, J.; Levin, K.;

    2006-01-01

    AIMS/HYPOTHESIS: Circulating levels of adiponectin are negatively associated with multiple indices of insulin resistance, and the concentration is reduced in humans with insulin resistance and type 2 diabetes. However, the mechanisms by which adiponectin improves insulin sensitivity remain unclear...... (ten lean, 21 obese and 20 with type 2 diabetes). RESULTS: Plasma adiponectin was significantly reduced in type 2 diabetic compared with obese and lean subjects. In lean and obese subjects, insulin significantly reduced plasma adiponectin, but this response was blunted in patients with type 2 diabetes...... by improving the capacity to switch from lipid to glucose oxidation and to store glucose as glycogen in response to insulin, and that low adiponectin may contribute to impaired insulin activation of GS in skeletal muscle of patients with type 2 diabetes....

  14. 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

  15. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  16. EPSP合成酶的纯化与制备%Purification and Preparation of EPSP Synthase

    Institute of Scientific and Technical Information of China (English)

    向文胜; 王相晶; 覃兆海; 任天瑞; 张雅莉; 张文吉; 苏少泉

    2000-01-01

    The rapid purification(less than 1.5 h) of EPSP synthase from bean seedling by S ephadex G-50 and Mono-Q chromtography was reported. Specific activity of E PSP synthase obtained by the method was 175.2 nmol.min-1.mg-1.Conc entrated enzyme solution after adjusting to 50% glycerol(V/V) and 1mg.mL -1BSA, was stored at -20℃. EPSP synthase activity was stable at least f or 150 days.The activity of EPSP synthase was inhibited approximately 50% by 6.3 μmol.L-1 glyphosate. It showed that the purified EPSP synth ase as herbicide screening model is possible. This purified method has been used to study enzyme mechanism of the glyphosate resistant bean.

  17. Novel type III polyketide synthases from Aloe arborescens.

    Science.gov (United States)

    Mizuuchi, Yuusuke; Shi, She-Po; Wanibuchi, Kiyofumi; Kojima, Akiko; Morita, Hiroyuki; Noguchi, Hiroshi; Abe, Ikuro

    2009-04-01

    Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85-96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl-CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild-type PKS obtained from A. arborescens. Interestingly, the aloesone-forming activity was maximum at 50 degrees C, and the novel heptaketide pyrone was non-enzymatically converted to aloesone. In PKS3, the active-site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site-directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone. PMID:19348024

  18. 沉默MALAT1基因对蜂毒素诱导HepG2细胞增殖和凋亡的影响%Effects of silencing MALAT1 on proliferation and apoptosis in HepG2 cells induced by Melittin

    Institute of Scientific and Technical Information of China (English)

    赵斌; 吴毓婷; 黄成; 吕雄文; 李俊

    2016-01-01

    Aim To investigate the effects of silencing MALAT1 gene on cell proliferation inhibition and apop-tosis induced by Melittin in human hepatocellular car-cinoma HepG2 cells. Methods The inhibitory rate of cell proliferation treated with Melittin in HepG2 cells was examined by MTT assay. Apoptotic rate was detec-ted by flow cytometry. The MALAT1 expression level in HepG2 cells was measured by qPCR. Specific siR-NAs were utilized to silence MALAT1 expression. The rates of cell proliferation inhibition and apoptosis in HepG2 cells treated with siRNA and Melittin were compared with those of Melittin alone. Results Melit-tin significantly suppressed the growth of HepG2 and induced cell apoptosis in a dose-dependent manner. Compared with normal liver cell lines, MALAT1 was highly expressed in HepG2 cells ( P<0. 05 ) . The ex-pression of MALAT1 in HepG2 cells was inhibited by Melittin, and the inhibitory rate increased with the in-crease of concentration. The rates of cell proliferation inhibition and apoptosis in HepG2 cells treated with siRNA and Melittin were significantly higher than those treated merely with Melittin. Conclusion Melittin can reduce the expression of MALAT1 and silencing MALAT1 can effectively promote proliferation inhibi-tion and apoptosis in HepG2 cells induced by Melittin.%目的:探讨沉默HepG2细胞株中MALAT1基因对蜂毒素诱导的细胞增殖抑制和凋亡的影响。方法采用MTT法检测蜂毒素对HepG2细胞的增殖抑制作用;流式细胞仪检测细胞凋亡率;qPCR法检测HepG2细胞中MALAT1基因的表达;采用特异性siRNA对HepG2细胞的MALAT1基因进行沉默;比较单独用蜂毒素处理和给予蜂毒素同时沉默MALAT1的细胞增殖抑制率和凋亡率变化。结果蜂毒素明显抑制HepG2细胞的增殖并促进细胞凋亡,呈浓度依赖性;和正常肝细胞株L0-2相比,MALAT1 mRNA在HepG2细胞中存在高表达(P<0.05);蜂毒素可下调细胞中MALAT1的表达,并随着浓度的增加

  19. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  20. 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

  1. 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

  2. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    Science.gov (United States)

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

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

    Science.gov (United States)

    2016-01-01

    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 D80DQFD and N218DVRSFAQE. 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 H218O 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 [1-2H2]FDP and (R)-[1-2H]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 were

  4. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  5. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

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

    Directory of Open Access Journals (Sweden)

    Park Jaeok

    2012-12-01

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

  7. 溴氰菊酯对大鼠中枢神经系统一氧化氮合酶活性的影响%Effect of deltamethrin on the activity of nitric oxide synthase in the central nervous system of rat

    Institute of Scientific and Technical Information of China (English)

    牛玉杰; 杨建芳; 张荣

    2003-01-01

    Objective To study the effect of deltamethrin (DM) on the activity of nitric oxide synthase (NOS) from different parts of central nervous system of rat,and the possible mechanism of DM effecting the NOS activity was also explored. Methods The activity of NOS from cerebral cortex, hippocampus and cerebellum was observed by determination of the radioactivity. Furthermore,the possible mechanism of DM effecting the NOS activity was studied by using Nω-nitro-L-arginine(L-NNA), a competitive inhibitor of NOS and nimodipine(NIM), a voltage-dependent Ca2+ channel blocker.Results DM could enhance significantly the activity of NOS from cerebral cortex, hippocampus and cerebellum, respectively compared with the control (P<0.01); L-NNA and NIM could effectively inhibit the enhancement of NOS activity caused by DM. Conclusion DM could enhance the activity of NOS from central nervous system of rat, and further make the injury of neuron.

  8. 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...

  9. 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...

  10. 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 ...

  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. 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 ...

  14. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  15. 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....

  16. 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.

  17. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  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. Alcoholytic cleavage of polyhydroxyalkanoate chains by class IV synthases induced by endogenous and exogenous ethanol.

    Science.gov (United States)

    Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Abe, Hideki; Tsuge, Takeharu

    2014-02-01

    Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature.

  20. QUANTITATIVE ASSAY OF ALMOTRIPTAN MALATE IN PURE DRUG AND PHARMACEUTICAL PREPARATIONS USING SIMPLE AND CONVENIENT VISIBLE SPECTROPHOTOMETRIC METHODS

    Directory of Open Access Journals (Sweden)

    U. VIPLOVA PRASAD

    2012-05-01

    Full Text Available Two direct, simple and sensitive visible spectrophotometric methods (M1&M2 are described for the assay of almotripan malate in pure and solid dosage forms. The method M1 involves oxidative coupling of drug with brucine in presence of sodium meta periodate and purple red colored species is formed and exhibits absorption maxima at 520nm. The method M2 is based on the formation of yellowish brown colored species by the drug with Folin reagent and exhibits absorption maxima at 450nm. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (8.0-24 μg/ml for method M1, (16-48 μg/ml for method M2 respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the reported UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the almotriptan malate in the presence of other ingredients that are usually present in dosageforms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

  1. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

    Directory of Open Access Journals (Sweden)

    Delnia Arshadi

    2007-06-01

    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  2. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

    Science.gov (United States)

    Mott, David M; Stone, Karen; Gessel, Mary C; Bunt, Joy C; Bogardus, Clifton

    2008-02-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

  3. 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...

  4. 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

  5. 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).

  6. 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

  7. 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)

  8. 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)

  9. In vitro evidence that phytanic acid compromises Na(+),K(+)-ATPase activity and the electron flow through the respiratory chain in brain cortex from young rats.

    Science.gov (United States)

    Busanello, Estela Natacha Brandt; Viegas, Carolina Maso; Moura, Alana Pimentel; Tonin, Anelise Miotti; Grings, Mateus; Vargas, Carmen R; Wajner, Moacir

    2010-09-17

    Phytanic acid (Phyt) tissue concentrations are increased in Refsum disease and other peroxisomal disorders characterized by neurologic damage and brain abnormalities. The present work investigated the in vitro effects of Phyt, at concentrations found in these peroxisomal disorders, on important parameters of energy metabolism in brain cortex of young rats. The parameters analyzed were CO(2) production from labeled acetate and glucose, the activities of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, as well as of the respiratory chain complexes I-IV, creatine kinase and Na(+),K(+)-ATPase. Our results show that Phyt did not alter citric acid cycle enzyme activities, or CO(2) production from acetate, reflecting no impairment of the functionality of the citric acid cycle. In contrast, respiratory chain activities were reduced at complexes I, II, I-III, II-III and IV. Membrane synaptical Na(+),K(+)-ATPase activity was also reduced by Phyt, with no alteration of creatine kinase activity. Considering the importance of the electron flow through the respiratory chain for brain energy metabolism (oxidative phosphorylation) and of Na(+),K(+)-ATPase activity for maintaining membrane potential necessary for neurotransmission, the data indicate that Phyt impairs brain bioenergetics at the level of energy formation, as well as neurotransmission. It is presumed that Phyt-induced impairment of these important systems may be involved at least in part in the neurological damage found in patients affected by disorders in which brain Phyt concentrations are increased.

  10. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    Science.gov (United States)

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

  11. 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...

  12. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  13. Inducible nitric oxide synthase and inflammation.

    Science.gov (United States)

    Salvemini, D; Marino, M H

    1998-01-01

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

  14. Nitric Oxide Synthases and Atrial Fibrillation

    OpenAIRE

    CynthiaAnnCarnes; ArunSridhar; SandorGyorke

    2012-01-01

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

  15. Effects of the dicarboxylic acids malate and fumarate on E. coli 0157:H7 and Salmonella enterica Typhimurium populations in pure culture and in mixed ruminal microorganism fermentations

    Science.gov (United States)

    The dicarboxylic organic acids malate and fumarate increase ruminal pH, reduce methane production, increase propionate and total VFA production, and reduce lactic acid accumulation in a manner similar to ionophores. These acids stimulate the ruminal bacterium Selenomonas ruminantium to ferment lact...

  16. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

  17. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

    Science.gov (United States)

    Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

    2014-02-01

    Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. PMID:24112147

  18. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  19. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  20. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  1. Inhibitors of nitric oxide synthase in inflammatory arthritis.

    Science.gov (United States)

    Boughton-Smith, N K; Tinker, A C

    1998-07-01

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

  2. ISOLATION AND IDENTIFICATION OF A THERMOTOLERANT PLANT GROWTH PROMOTING PSEUDOMONAS PUTIDA PRODUCING TREHALOSE SYNTHASE

    OpenAIRE

    Ali Sk.Z.; Sandhya Vardharajula

    2013-01-01

    A thermotolerant plant growth promoting Pseudomonas isolate growing at 40oC producing trehalose synthase (TreS) was isolated from rhizosphere soil under semi arid conditions of India. Trehalose synthase was extracted; purified and enzymatic activity was examined at various temperatures and pH. The optimum temperature and pH was 38oC and pH 7.5 and the activity declined at above or below the optimum pH and temperature. The enzyme was active on maltose and trehalose among saccharides tested. Th...

  3. Tapentadol and nitric oxide synthase systems.

    Science.gov (United States)

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

    2015-04-01

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

  4. A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells.

    Science.gov (United States)

    Huber, S C; Akazawa, T

    1986-08-01

    Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K(m) for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective ;PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible

  5. Molecular evolution of dihydrouridine synthases

    Directory of Open Access Journals (Sweden)

    Kasprzak Joanna M

    2012-06-01

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

  6. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

  7. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.

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

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

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

  9. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  10. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  11. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  12. Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua.

    Science.gov (United States)

    Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg(2+) and Mn(2+) were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn(2+) for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

  13. Functional Inducible Nitric Oxide Synthase Gene Variants Associate With Hypertension

    OpenAIRE

    Nikkari, Seppo T; Määttä, Kirsi M.; Kunnas, Tarja A.

    2015-01-01

    Abstract Increased inducible nitric oxide synthase (iNOS) activity and expression has been associated with hypertension, but less is known whether the 2 known functional polymorphic sites in the iNOS gene (g.–1026 C/A (rs2779249), g.2087 G/A (rs2297518)) affect susceptibility to hypertension. The objective of this study was to investigate the association between the genetic variants of iNOS and diagnosed hypertension in a Finnish cohort. This study included 320 hypertensive cases and 439 heal...

  14. Fatty Acid Synthase Inhibitor C75 Ameliorates Experimental Colitis

    OpenAIRE

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

    2013-01-01

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

  15. Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.

    Science.gov (United States)

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M

    2013-05-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

  16. Ghrelin对体外大鼠血管内皮细胞eNOS活性的影响及机制研究%Effect of Ghrelin on endothelial nitric oxide synthase activity in vascular endothelial cells of cultured rats and its mechanism

    Institute of Scientific and Technical Information of China (English)

    赵荫涛; 邵莉; 杨海波; 李凌; 滕丽莉

    2014-01-01

    Objective To investigate the effect of Ghrelin on endothelial nitric oxide synthase (eNOS) activity and its mechanism.Methods Vascular endothelial cells (ECs) were isolated from aorta in rats and were cultured.ECs were divided into two groups:control group and Ghrelin group.The cells were incubated with Ghrelin in different concentration (10,50,100 and 200 nmol/L) for 3 hour in Ghrelin group.The protein expression levels of nitric oxide synthase (eNOS) and protien kinase C (PKC) were measured by Western blot.Results Ghrelin can upregulate the phosphorylated eNOS expression level,and downregulate signal conduction pathway PKC phosphorylation in a dose dependent manner (P < 0.05).Conclusions Ghrelin can improve the activity of eNOS in ECs,and it may be related to the loss of PKC activity.%目的 探讨Ghrelin对内皮型一氧化氮合成酶(eNOS)活性的影响和作用机制.方法 分离培养大鼠主动脉血管内皮细胞并分为对照组(C组)和Ghrelin组.Ghrelin组加入终浓度为10、50、100和200nmol/L Ghrelin孵育3h收集标本.采用Western blot法测定内皮细胞eNOS和PKC蛋白表达水平.结果 与对照组相比,Ghrelin组eNOS磷酸化水平呈剂量依赖性增加(P<0.05),PKC磷酸化水平呈剂量依赖性降低(P<0.05).结论 Ghrelin可提高内皮细胞eNOS的活性,其机制可能与PKC通路的失活有关.

  17. A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase

    Science.gov (United States)

    Criswell, Jared; Potter, Kevin; Shephard, Freya; Beale, Michael H.; Peters, Reuben J.

    2012-01-01

    Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity. PMID:23167845

  18. Brucine salts of L-alpha-hydroxy acids: brucinium hydrogen (S)-malate pentahydrate and anhydrous brucinium hydrogen (2R,3R)-tartrate at 130 K.

    Science.gov (United States)

    Smith, Graham; Wermuth, Urs D; White, Jonathan M

    2006-06-01

    The structures of two brucinium (2,3-dimethoxy-10-oxostrychnidinium) salts of the alpha-hydroxy acids L-malic acid and L-tartaric acid, namely brucinium hydrogen (S)-malate pentahydrate, C23H27N2O4+.C4H5O5-.5H2O, (I), and anhydrous brucinium hydrogen (2R,3R)-tartrate, C23H27N2O4+.C4H5O6-,(II), have been determined at 130 K. Compound (I) has two brucinium cations, two hydrogen malate anions and ten water molecules of solvation in the asymmetric unit, and forms an extensively hydrogen-bonded three-dimensional framework structure. In compound (II), the brucinium cations form the common undulating brucine sheet substructures, which accommodate parallel chains of head-to-tail hydrogen-bonded tartrate anion species in the interstitial cavities.

  19. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazin