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Sample records for carbamoyl phosphate synthetase

  1. Distribution of glutamine synthetase and carbamoyl-phosphate synthetase I in vertebrate liver.

    OpenAIRE

    Smith, D. D.; Campbell, J. W.

    1988-01-01

    Mitochondrial glutamine synthetase (EC 6.3.1.2) is the primary ammonia-detoxifying enzyme in avian liver and is therefore analogous in function to carbamoyl-phosphate synthetase I (ammonia) (EC 6.3.4.16) in mammalian liver. In mammalian liver, glutamine synthetase is cytosolic and its distribution is restricted to a few hepatocytes around the terminal venules. These cells do not express carbamoyl-phosphate synthetase I. Using immunocytochemistry, we show here that there is little or no zonati...

  2. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-09

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.

  3. Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

    Science.gov (United States)

    Anderson, P M

    1989-07-15

    The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

  4. The T1405N carbamoyl phosphate synthetase polymorphism does not affect plasma arginine concentrations in preterm infants.

    Directory of Open Access Journals (Sweden)

    Rob M J Moonen

    Full Text Available BACKGROUND: A C-to-A nucleotide transversion (T1405N in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1 has been associated with changes in plasma concentrations of L-arginine in term and near term infants but not in adults. In preterm infants homozygosity for the CPS1 Thr1405 variant (CC genotype was associated with an increased risk of having necrotizing enterocolitis (NEC. Plasma L-arginine concentrations are decreased in preterm infants with NEC. AIM: To examine the putative association between the CPS1 T1405N polymorphism and plasma arginine concentrations in preterm infants. METHODS: Prospective multicenter cohort study. Plasma and DNA samples were collected from 128 preterm infants (<30 weeks between 6 and 12 hours after birth. Plasma amino acid and CPS1 T1405N polymorphism analysis were performed. RESULTS: Distribution of genotypes did not differ between the preterm (CC:CA:AA = 55.5%:33.6%:10.9%, n = 128 and term infants (CC:CA:AA = 54.2%:35.4%:10.4%, n = 96. There was no association between the CPS1 genotype and plasma L-arginine or L-citrulline concentration, or the ornithine to citrulline ratio, which varies inversely with CPS1 activity. Also the levels of asymmetric dimethylarginine, and symmetric dimethylarginine were not significantly different among the three genotypes. CONCLUSIONS: The present study in preterm infants did not confirm the earlier reported association between CPS1 genotype and L-arginine levels in term infants.

  5. Detection of circulating tumor cells in hepatocellular carcinoma using antibodies against asialoglycoprotein receptor, carbamoyl phosphate synthetase 1 and pan-cytokeratin.

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    Jun Li

    Full Text Available BACKGROUND: Asialoglycoprotein receptor (ASGPR-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK antibody have been demonstrated to detect circulating tumor cells (CTCs in hepatocellular carcinoma (HCC. The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. METHODS: The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1 antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. RESULTS: ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. CONCLUSIONS: Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.

  6. The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp. citri

    Institute of Scientific and Technical Information of China (English)

    Guo Jing; SonG Xue; Zou Li-fang; Zou Hua-song; CHen Gong-you

    2015-01-01

    Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, carA and carB encode the smal and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper-sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracel ular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri.

  7. Aroclor 1254 treatment and fasting influences on rat liver mitochondrial carbamoyl phosphate synthesis with ADP and ATP.

    Science.gov (United States)

    Ebner, K; Couri, D

    1988-10-01

    Previous studies have shown that the polychlorinated biphenyl mixture, Aroclor 1254 (ARO), -induced wasting in male rats is associated with increased permeability of hepatic mitochondria. This was correlated with hyperuremia and stimulated urea synthesis, hypoglycemia and suppressed glucogenesis after an ammonium acetate injection, and decreased retention of assimilated nitrogen and food intake. For ARO-toxic rats (100 mg/kg, ip, for 1, 2, and 4 days) versus Tween 80-treated, ad libitum-fed controls, mitochondrial carbamoyl phosphate (CP) formation (the initial step in urea synthesis from NH4+) was progressively stimulated for the duration of treatment from NH4+ and ATP but not from NH4+ and ADP. ARO maximal stimulation of CP formation also correlated with significant loss in body weight. Mitochondrial ornithine transcarbamoylase synthesis of citrulline from ornithine and carbamoyl phosphate was also stimulated. In comparison to fasted rats (24 hr), mitochondrial CP synthesis from NH4+ was enhanced with ADP but not with ATP. This ARO uncoupling of mitochondrial NH4+ metabolism and stimulation of CP formation with exogenous ATP and citrulline synthesis may have resulted from increased availability of substrates and cofactors in the matrix space, leakage of enzymes from the matrix, or a combination of these effects. These results are consistent with an increased inner membrane permeability and fragility during isolation and assays. In agreement with our previous studies, the data show that ARO exposure poises hepatic mitochondria toward the synthesis of urea intermediates.

  8. Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1deficiency with N-carbamylglutamate

    Science.gov (United States)

    Ah Mew, Nicholas; McCarter, Robert; Daikhin, Yevgeny; Lichter, Uta; Nissim, Ilana; Yudkoff, Marc; Tuchman, and Mendel

    2014-01-01

    Identical studies employing stable isotopes were performed before and after a 3-day trial of oral N-carbamylglutamate (NCG) in 5 subjects with late onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in one subject. PMID:24880889

  9. Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1deficiency with N-carbamylglutamate

    OpenAIRE

    Ah Mew, Nicholas; McCarter, Robert; Daikhin, Yevgeny; Lichter, Uta; Nissim, Ilana; Yudkoff, Marc; Tuchman, and Mendel

    2014-01-01

    Identical studies employing stable isotopes were performed before and after a 3-day trial of oral N-carbamylglutamate (NCG) in 5 subjects with late onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in one subject.

  10. carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) regulates Notch signaling and vascular development in zebrafish

    NARCIS (Netherlands)

    Coxam, Baptiste; Neyt, Christine; Grassini, Daniela R.; Le Guen, Ludovic; Smith, Kelly A.; Schulte-Merker, Stefan; Hogan, Benjamin M.

    2015-01-01

    Background: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors

  11. carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) regulates Notch signaling and vascular development in zebrafish

    NARCIS (Netherlands)

    Coxam, Baptiste; Neyt, Christine; Grassini, D.R.; Guen, Le Ludovic; Smith, K.A.; Schulte-Merker, Stefan; Hogan, B.M.

    2015-01-01

    Background: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic facto

  12. Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) regulates Notch signaling and vascular development in zebrafish

    NARCIS (Netherlands)

    Coxam, Baptiste; Neyt, Christine; Grassini, D.R.; Guen, Le Ludovic; Smith, K.A.; Schulte-Merker, Stefan; Hogan, B.M.

    2015-01-01

    Background: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic facto

  13. Protein: CAD [Trypanosomes Database

    Lifescience Database Archive (English)

    Full Text Available CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotaseCAD trifunctional prot...eincarbamoylphosphate synthetase 2/aspartate transcarbamylase/dihydroorotasemultifunctional prot

  14. Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

    Science.gov (United States)

    Praschberger, Monika; Hermann, Marcela; Laggner, Christian; Jirovetz, Leopold; Exner, Markus; Kapiotis, Stylianos; Gmeiner, Bernhard M K; Laggner, Hilde

    2013-11-01

    Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

  15. Complete NMR analysis of oxytocin in phosphate buffer.

    Science.gov (United States)

    Ohno, Akiko; Kawasaki, Nana; Fukuhara, Kiyoshi; Okuda, Haruhiro; Yamaguchi, Teruhide

    2010-02-01

    Complete NMR analysis of oxytocin (OXT) in phosphate buffer was elucidated by one-dimensional (1D)- and two-dimensional (2D)-NMR techniques, which involve the assignment of peptide amide NH protons and carbamoyl NH(2) protons. The (1)H-(15)N correlation of seven amide NH protons and three carbamoyl NH(2) protons were also shown by HSQC NMR of OXT without (15)N enrichment.

  16. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  17. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-D-ribosyl α-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  18. Thermal analysis of N-carbamoyl benzotriazole derivatives

    Directory of Open Access Journals (Sweden)

    Kos Ivan

    2015-06-01

    Full Text Available Thermal properties of N-carbamoyl benzotriazole derivatives and N,N’,N’’-tribenzyloxyisocyanuric acid were investigated using thermogravimetric analysis and differential scanning calorimetry. The results revealed a difference between structural analogs of N-carbamoyl benzotriazole derivatives. They seem to be in agreement with the previously proposed formation of N,N’,N’’-tribenzyloxyisocyanuric acid from 1-(N-benzyloxycarbamoyl benzotriazole, via an intermediary N-benzyloxyisocyanate acid, during heating. Substantially different thermal properties were observed for structural analogues, 1-(N-methoxycarbamoyl benzotriazole and 1-(N-ethoxycarbamoyl benzotriazole. In contrast to N-benzyloxyisocyanate, no corresponding reactions were observed for their decomposition products, i.e., methoxyisocyanate and ethoxyisocyanate.

  19. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  20. Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

    DEFF Research Database (Denmark)

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...

  1. [Anti-synthetase syndrome].

    Science.gov (United States)

    Novak, Srdan

    2012-01-01

    Antysynthetase syndrome is considered as a group ofidiopathic inflammatory myositis with charcteristic serologic hallmark--antibodies which recognise the aminoacyl-tRNA synthetasses (ARS). Clinical picture of those patients contains myositis and/or intersticial lung disease (ILD) and/or arthritis and/or fever and/or Raynaud phenomenon and sometimes characteristic look of mechanic's hands. Myositis can be overt, sometimes even absent, while IBP is major cause of morbidity and determines the outcome of the disease. Untill now eight different any-synthetase autoantibodies are recognised, and most frequent are findings of anti-histidyl-tRNa synthetase antibodies. Patients with other ARS autoantibodies usually have severe ILD. Drug of choice are steroids in dosage of 1 mg/kg with immunosupresive agent (azatioprin or methotrexate) while in severe IBP cyclophosphamide is needed. Recently succsesful treatment with rituximab in combination with cyclophosphamide is reported.

  2. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.;

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine......-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5...... phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5...

  3. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  4. Synthesis and antibacterial activity of 4“-O-carbamoyl analogs of clarithromycin

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A series of novel 4"-O-carbamoyl analogs of clarithromycin were synthesized and evaluated for their in vitro antibacterial activity. All of the desired compounds showed excellent activity against erythromycin-susceptible S.pneumoniae.Particularly,4-fluorobenzyl carbamate 7a demonstrated potent activity against erythromycin-resistant S.pneumoniae encoded by the mef gene,and remarkably improved activity against erythromycin-resistant S.pneumoniae encoded by the erm gene,and the erm and mef genes.

  5. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...... enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis...

  6. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.;

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...... of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined...

  7. The discovery of S/GSK1265744: a carbamoyl pyridone HIV-1 integrase inhibitor

    Directory of Open Access Journals (Sweden)

    H Murai

    2012-11-01

    Full Text Available Background: HIV-1 integrase is a virally encoded enzyme essential for lentiviral replication. Assiduous medicinal chemistry efforts culminated in the discovery of raltegravir, the first marketed HIV-1 integrase inhibitor (INI. However, there is significant opportunity for improvement including overall dose burden, dosing interval and potency against resistant viruses. Our molecular design approach used a two-metal binding pharmacophore strategy and succeeded in identification of carbamoyl pyridone HIV-1 INIs. This enriched core scaffold has abundant structural features expanding the opportunity to control drug properties, leading to the discovery of S/GSK1265744. Methods: The carbamoyl pyridone scaffold was derivatized and evaluated for antiviral activity against wild-type virus (±HSA along with key INI-resistant mutants. Animal pharmacokinetic profiles including a key measure of the trough drug concentration over protein-adjusted antiviral potency (C24/PAIC50 along with in vitro DMPK properties, were used along with the virological data for compound selection. Results: The carbamoyl pyridone series inhibitors exhibited potent antiviral profiles with promising DMPK properties. S/GSK1265744 demonstrated good coverage of C24 over PAIC50 predicting low mg unboosted once daily dosing, now validated in phase 2 clinical studies. These preclinical data along with a long human T1/2 of ~30 hours in oral tablet study supports S/GSK1265744 as a long acting parenteral agent for once-monthly or less frequent dosing. Conclusions: A medicinal chemistry approach utilizing key viral mutants in combination with C24/PAIC50 has allowed for discovery of S/GSK1265744. This agent is currently in phase 2 development evaluating a novel, long-acting parenteral route of administration and may enable new approaches to HIV therapy and prevention.

  8. Crystal structure of yeast FAD synthetase (Fad1) in complex with FAD.

    Science.gov (United States)

    Leulliot, Nicolas; Blondeau, Karine; Keller, Jenny; Ulryck, Nathalie; Quevillon-Cheruel, Sophie; van Tilbeurgh, Herman

    2010-05-21

    Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 A resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.

  9. Streptomyces hygroscopicus Has Two Glutamine Synthetase Genes

    NARCIS (Netherlands)

    Kumada, Y.; Takano, E.; Nagaoka, Kozo; Thompson, C.J.

    1990-01-01

    Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli gl

  10. Archaeal RibL: a new FAD synthetase that is air sensitive.

    Science.gov (United States)

    Mashhadi, Zahra; Xu, Huimin; Grochowski, Laura L; White, Robert H

    2010-10-12

    FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

  11. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    Improving crop nitrogen (N) utilization efficiency (NUE) is of major importance in modern agriculture in order to reduce the amount of N fertilizer used for crop production. There is a high demand for development of crops which are able to produce high yields but with a concomitantly lower N...... fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...... and wildtype control. However, when grown to maturity the differences between transgenic lines and wildtype were highly dependent on the growth conditions applied. The transgenic lines had a higher N utilization efficiency (NUtE) than wildtype control, but only when exposed to a mild N stress following...

  12. Genetics Home Reference: phosphoribosylpyrophosphate synthetase superactivity

    Science.gov (United States)

    ... synthetase superactivity ( PRS superactivity ) is characterized by the overproduction and accumulation of uric acid (a waste product ... chemical processes) in the blood and urine. The overproduction of uric acid can lead to gout, which ...

  13. Synthesis, characterization and corrosion inhibition efficiency of N-(4-(Morpholinomethyl Carbamoyl Phenyl Furan-2-Carboxamide

    Directory of Open Access Journals (Sweden)

    N. Zulfareen

    2016-01-01

    Full Text Available A mannich base namely N-(4-(Morpholinomethyl Carbamoyl Phenyl Furan-2-Carboxamide (MFC was synthesized and characterized by FT-IR, 1H NMR, and 13C NMR. The molecular weight of MFC was confirmed by LC-MS. The inhibition effect of MFC on brass in 1 M HCl medium has been investigated by weight loss measurement, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS and cyclic voltametry (CV. Thermodynamic parameters such as free energy, entropy and enthalpy were calculated to describe the mechanism of corrosion inhibitor. The inhibition efficiency of MFC increases with increase in concentration and temperature ranges from 30 °C to 60 °C. Polarization measurements indicated that MFC acts as a mixed type corrosion inhibitor. AC impedance indicates that Rct value increases with increase in the concentration of inhibitor. CV reveals that the oxidation of the copper is controlled by the addition of inhibitor on the brass metal. Surface analysis using scanning electron microscope (SEM shows a significant morphological improvement on the brass surface with the addition of the inhibitor. The adsorption of MFC on brass obeys Langmuir adsorption isotherm. The molecular structure of MFC was distorted to quantum chemical indices using density functional theory (DFT which indicates that the inhibition efficiency of MFC is closely related to quantum parameters.

  14. Structural basis for the binding of succinate to succinyl-CoA synthetase.

    Science.gov (United States)

    Huang, Ji; Fraser, Marie E

    2016-08-01

    Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

  15. Sodium Phosphate

    Science.gov (United States)

    Sodium phosphate is used in adults 18 years of age or older to empty the colon (large intestine, bowel) ... view of the walls of the colon. Sodium phosphate is in a class of medications called saline ...

  16. Phosphate salts

    Science.gov (United States)

    ... levels that are too high, and for preventing kidney stones. They are also taken for treating osteomalacia (often ... But intravenous phosphate salts should not be used. Kidney stones (nephrolithiasis). Taking potassium phosphate by mouth might help ...

  17. Substrate specificity of hybrid modules from peptide synthetases

    NARCIS (Netherlands)

    Elsner, A; Engert, H; Saenger, W; Hamoen, L; Venema, G; Bernhard, F

    1997-01-01

    Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine activating module of surfactin synthetase were constructed by combining the t

  18. The function of the three phosphoribosyl pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

    Science.gov (United States)

    Jiang, Ping; Wei, Wen-Fan; Zhong, Guo-Wei; Zhou, Xiao-Gang; Qiao, Wei-Ran; Fisher, Reinhard; Lu, Ling

    2017-02-01

    Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

  19. The function of the three phosphoribosyl-pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

    Science.gov (United States)

    Jiang, Ping; Wei, Wen-Fan; Zhong, Guo-Wei; Zhou, Xiao-Gang; Qiao, Wei-Ran; Lu, Ling

    2017-01-12

    Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyzes the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total PRPP synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthase-homologous genes (AnprsA, B, and C), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, B and C are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time-point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

  20. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    Science.gov (United States)

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies.

  1. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a,3...... of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands MgATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate....... Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by MgADP indicated that MgADP binding to the allosteric site may occur in competition...

  2. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

    Science.gov (United States)

    Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

    1986-01-01

    The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

  3. Retinal Vasculitis in Anti-Synthetase Syndrome.

    Science.gov (United States)

    Donovan, Christopher P; Pecen, Paula E; Baynes, Kimberly; Ehlers, Justis P; Srivastava, Sunil K

    2016-09-01

    A 31-year-old woman with a history of anti-synthetase syndrome-related myositis and interstitial lung disease presented with acute-onset blurry vision and rash on her hands and feet. Visual acuity was hand motion in her right eye and 20/40 in her left eye. Dilated fundus exam showed extensive retinal vasculitis, diffuse intraretinal hemorrhages, and subretinal fluid. Optical coherence tomography revealed significant macular thickening, and fluorescein angiography revealed vascular leakage with peripheral nonperfusion. Aggressive systemic immunosuppression was initiated, with gradual resolution of her disease during 8 months of follow-up. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:874-879.].

  4. Dissecting and Exploiting Nonribosomal Peptide Synthetases

    Institute of Scientific and Technical Information of China (English)

    Qing-Tao SHEN; Xiu-Lan CHEN; Cai-Yun SUN; Yu-Zhong ZHANG

    2004-01-01

    A large number of therapeutically useful cyclic and linear peptides of bacteria or fungal origin are synthesized via a template-directed, nucleic-acid-independent nonribosomal mechanism. This process is carried out by mega-enzymes called nonribosomal peptide synthetases (NRPSs). NRPSs contain repeated coordinated groups of active sites called modules, and each module is composed of several domains with different catalytic activities. The familiarity to these domains lays base for the future genetic engineering of NRPSs to generate entirely "unnature" Products. The details about NRPSs domain structures and the exploitation of NRPSs are described in this review.

  5. Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

    Directory of Open Access Journals (Sweden)

    Diego A Alonzo

    Full Text Available Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.

  6. The microsomal dicarboxylyl-CoA synthetase.

    Science.gov (United States)

    Vamecq, J; de Hoffmann, E; Van Hoof, F

    1985-09-15

    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo.

  7. Discovery of unique lanthionine synthetases reveals new mechanistic and evolutionary insights.

    Directory of Open Access Journals (Sweden)

    Yuki Goto

    2010-03-01

    Full Text Available Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter's high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds.

  8. Glutamine Synthetase: Role in Neurological Disorders.

    Science.gov (United States)

    Jayakumar, Arumugam R; Norenberg, Michael D

    2016-01-01

    Glutamine synthetase (GS) is an ATP-dependent enzyme found in most species that synthesizes glutamine from glutamate and ammonia. In brain, GS is exclusively located in astrocytes where it serves to maintain the glutamate-glutamine cycle, as well as nitrogen metabolism. Changes in the activity of GS, as well as its gene expression, along with excitotoxicity, have been identified in a number of neurological conditions. The literature describing alterations in the activation and gene expression of GS, as well as its involvement in different neurological disorders, however, is incomplete. This review summarizes changes in GS gene expression/activity and its potential contribution to the pathogenesis of several neurological disorders, including hepatic encephalopathy, ischemia, epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and astroglial neoplasms. This review also explores the possibility of targeting GS in the therapy of these conditions.

  9. Mechanistic issues in asparagine synthetase catalysis.

    Science.gov (United States)

    Richards, N G; Schuster, S M

    1998-01-01

    The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred.

  10. Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations.

    Science.gov (United States)

    Issoglio, Federico M; Campolo, Nicolas; Zeida, Ari; Grune, Tilman; Radi, Rafael; Estrin, Dario A; Bartesaghi, Silvina

    2016-10-13

    Glutamine synthetase is an important enzyme that catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. In mammals, it plays a key role in preventing excitotoxicity in the brain and detoxifying ammonia in the liver. In plants and bacteria, it is fundamental for nitrogen metabolism, being critical for the survival of the organism. In this work, we show how the use of classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics simulations allowed us to examine the structural properties and dynamics of human glutamine synthetase (HsGS), as well as the reaction mechanisms involved in the catalytic process with atomic level detail. Our results suggest that glutamine formation proceeds through a two-step mechanism that includes a first step in which the γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free energy barrier and a -8 kcal/mol reaction free energy, and then a second rate-limiting step involving the ammonia nucleophilic attack, with a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. A detailed analysis of structural features within each step exposed the relevance of the acid-base equilibrium related to protein residues and substrates in the thermodynamics and kinetics of the reactions. These results provide a comprehensive study of HsGS dynamics and establish the groundwork for further analysis regarding changes in HsGS activity, as occur in natural variants and post-translational modifications.

  11. Structural and functional characterization of S-adenosylmethionine (SAM) synthetase from Pichia ciferrii.

    Science.gov (United States)

    Yoon, Sangyoung; Lee, Wonkyu; Kim, Minsoo; Kim, T Doohun; Ryu, Yeonwoo

    2012-01-01

    S-adenosylmethionine synthetase (SAM-s) catalyzes the synthesis of S-adenosylmethionine (SAM), which is essential for methylation, transcription, proliferation, and production of secondary metabolites. Here SAM-s from Pichia ciferrii were selectively cloned using RNA CapFishing and rapid amplification of cDNA ends (RACE). The putative full-length cDNA of SAM-s encoded a 383 amino acid protein (42.6 kDa), which has highly conserved metal binding sites, a phosphate-binding site, and functionally important motifs. The corresponding enzyme was over-expressed in a heterologous host of Pichia pastoris, and then purified to a homogenous form. Enzyme kinetics, immunoblotting, circular dichroism (CD), high performance liquid chromatography (HPLC), and molecular modeling were conducted to characterize the SAM-s from P. ciferrii. Structural and functional studies of SAM-s will provide important insights for industrial applications.

  12. Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy.

    Science.gov (United States)

    Gruenbaum, Shaun E; Wang, Helen; Zaveri, Hitten P; Tang, Amber B; Lee, Tih-Shih W; Eid, Tore; Dhaher, Roni

    2015-10-01

    The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (pglutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.

  13. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    Science.gov (United States)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  14. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  15. Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases.

    Science.gov (United States)

    Sun, Xun; Li, Hao; Alfermann, Jonas; Mootz, Henning D; Yang, Haw

    2014-12-23

    Nonribosomal peptide synthetases (NRPS) incorporate assorted amino acid substrates into complex natural products. The substrate is activated via the formation of a reactive aminoacyl adenylate and is subsequently attached to the protein template via a thioester bond. The reactive nature of such intermediates, however, leads to side reactions that also break down the high-energy anhydride bond. The off-pathway kinetics or their relative weights compared to that of the on-pathway counterpart remains generally elusive. Here, we introduce multiplatform kinetics profiling to quantify the relative weights of on- and off-pathway reactions. Using the well-defined stoichiometry of thioester formation, we integrate a mass spectrometry (MS) kinetics assay, a high-performance liquid chromatography (HPLC) assay, and an ATP-pyrophosphate (PPi) exchange assay to map out a highly efficient on-pathway kinetics profile of the substrate activation and intermediate uploading (>98% relative weight) for wide-type gramicidin S synthetase A (GrsA) and a 87% rate profile for a cysteine-free GrsA mutant. Our kinetics profiling approach complements the existing enzyme-coupled byproduct-release assays, unraveling new mechanistic insights of substrate activation/channeling in NRPS enzymes.

  16. Effects of 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-dihydropyridine-5-carbonitriles on rat liver mitochondrial function.

    Science.gov (United States)

    Fernandes, Maria A S; Santos, Maria S; Moreno, António J M; Chernova, Laura; Krauze, Aivars; Duburs, Gunars; Vicente, Joaquim A F

    2009-10-01

    It is increasingly recognised that mitochondria are potential targets to pharmacological and toxicological actions of membrane-active agents, including some 1,4-dihydropyridines derivatives (DHPs). The 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-dihydropyridine-5-carbonitriles (OSI-1146, OSI-3701, OSI-3761, and OSI-9642) is a new group of DHPs with minor differences on the molecular structure. It has also been shown that OSI-1146 displays cardiovascular, antioxidant, and antiradical activities, whereas OSI-3701 and OSI-3761 display hepatoprotective activity. Due to their protective properties, this group of DHPs may be potentially useful for the treatment of several pathological processes, including those associated with oxidative stress. However, the cellular targets for their pharmacological actions have not been investigated. The presented study, using isolated rat liver mitochondria was designed to investigate if mitochondria are a cellular target for the pharmacological and/or toxicological actions of these new group of DHPs. We studied the direct influence of these DHPs on rat liver mitochondrial function [bioenergetics, membrane permeability transition (MPT), and oxidative stress]. It was shown that OSI-1146, OSI-3761, and OSI-9642, in the concentration range of up to 200 microM, interfered with mitochondrial bioenergetics by affecting complexes I and II of the mitochondrial respiratory chain, the ATPase activity, and mitochondrial inner membrane permeability to protons. However, the effects of OSI-1146 were higher than those of OSI-3761 and OSI-9642. The remaining compound, OSI-3701, had no effect on the bioenergetic parameters tested. All the compounds increased the susceptibility of mitochondria to MPT, but, OSI-3701, not affecting the bioenergetic parameters, was the most potent. Moreover, all the compounds protected mitochondria against lipid peroxidation induced by the oxidant pair ADP/Fe(2+), but OSI-1146 was also the most potent. In conclusion, our

  17. 3-Amino-2-carbamoyl-4,6-diphenyl-4,5- and -4,7-dihydrothieno-(2,3-b)pyridines

    Energy Technology Data Exchange (ETDEWEB)

    Krauze, A.A.; Liepin' sh, E.E.; Dubur, G.Ya.

    1987-10-01

    The treatment of piperidinium salts of 3-cyano-1,4-dihydropyridine-2-thiols with alkyl halides leads to 2-alkylthio-3-cyano-1,4-dihydropyridines. The authors have shown that alkylation of the piperidinium salt of 4,6-diphenyl-3-cyano-1,4-dihydropyridine-2-thiol with iodoacetamide gives carbamoylmethylthio derivative II, which, by the action of an equimolar amount of a base with heating to 50-60/sup 0/C, gives a mixture of 3-amino-2-carbamoyl-4,6-diphenyl-4,7-dihydrothieno(2,3-b)pyridine and 3-amino-2-carbamoyl-4,6-diphenyl-4,5-dihydrothieno(2,3-b)-pyridine in a ratio of 1:1. In the presence of excess base the principal product is IV. It was established by PMR spectroscopy that dihydropyridine II initially undergoes cyclization to 4,7-dihydrothienopyridine III, which then undergoes isomerization to 4,5-dihydrothienopyridine IV. Acidification of a solution of IV in d/sub 6/-DMSO gives rise to reverse isomerization.

  18. The glutamine synthetase gene family in Populus

    Directory of Open Access Journals (Sweden)

    Cánovas Francisco M

    2011-08-01

    Full Text Available Abstract Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1 and 1 which codes for the choroplastic GS isoform (GS2. Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. Conclusions Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

  19. Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

    Science.gov (United States)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

  20. An efficient one-pot procedure for asymmetric bifunctionalization of 5,15-disubstituted porphyrins: a simple preparation of meso acyl-, alkoxycarbonyl-, and carbamoyl-substituted meso-formylporphyrins.

    Science.gov (United States)

    Takanami, Toshikatsu; Wakita, Atsushi; Matsumoto, Jun; Sekine, Sadashige; Suda, Kohji

    2009-01-07

    An efficient one-pot procedure which converts 5,15-disubstituted porphyrins into their corresponding meso acyl-, alkoxycarbonyl-, and carbamoyl-substituted meso-formylporphyrins has been developed, where the procedure involves a sequential S(N)Ar reaction of porphyrins with PyMe(2)SiCH(2)Li, followed by acylation or related reactions and oxidation.

  1. Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

    Directory of Open Access Journals (Sweden)

    Anna P Lucarelli

    Full Text Available The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB and extensively drug-resistant strains (XDR-TB is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+ or Mn(2+ and inhibited by Ca(2+ and Cu(2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+ and Cu(2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

  2. Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

    NARCIS (Netherlands)

    Y. He; T.B.M. Hakvoort; J.L.M. Vermeulen; W.T. Labruyere; D.R. de Waart; W.S. van der Hel; J.M. Ruijter; H.B.M. Uylings; W.H. Lamers

    2010-01-01

    Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/f)l mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in astrocyte

  3. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  4. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

    2014-01-01

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

  5. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling.

    Science.gov (United States)

    Bernard, Stéphanie M; Habash, Dimah Z

    2009-01-01

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  6. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  7. Inhibition of Inositol-1-phosphate Synthetase in Mycobacterium Tuberculosis by Chitosan-antisense Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    LI Yuanyuan; CHEN Zhifei; ZHANG Hongling; LI Xiaobo; SHEN Jie; LU Shi; XU Shunqing

    2009-01-01

    Oligodeoxynucleotides (ODNs) were combined with the biodegradable polymer chitosan to form chitosan-ODN nanoparticles by complex coacervation, in order to improve the sta-bility and intracellular penetration. The diameter of the nanoparticles was light strength size and ranged between 60 and 219 nm with a mean value of 132 nm, while zeta potential was between +12 and + 20 mV at pH 5.5. The chitosan-ODN nanoparticles could partially protect the encapsulated ODN from nuclease degradation. Moreover, chitosan-ODN nanoparticles were much more effective in inhibiting the proliferation of M.tuberculosis than free ODN.

  8. SYNTHESIS OF MONO CARBAMOYL AMIDE OF SQUARIC ACID%氨甲酰类氮氧方酸的合成研究

    Institute of Scientific and Technical Information of China (English)

    李鸿波; 李聚才; 陈益钊

    2000-01-01

    A kind of mono-substituted squaric amide——mono carbamoyl amide of squaric acid 3 (MCASAQ) was prepared by the condensation of squaric acid (SQ) with urea or its derivatives in water. MCASAQs (3) may have some physiological activity for the ureido was brought into them. Meanwhile, the reaction of SQ with arylureain water provided a new route to the preparation of Narylnitrogenoxosquaric acids.%以水为介质,方酸与脲及其衍生物顺利发生脱水缩合,合成了一类方酸单酰胺化合物——氨甲酰类氮氧方酸3.方酸与芳基脲于水中的反应为N-芳基氮氧方酸的制备提供了一条新途径

  9. Phosphate homeostasis and disorders.

    Science.gov (United States)

    Manghat, P; Sodi, R; Swaminathan, R

    2014-11-01

    Recent studies of inherited disorders of phosphate metabolism have shed new light on the understanding of phosphate metabolism. Phosphate has important functions in the body and several mechanisms have evolved to regulate phosphate balance including vitamin D, parathyroid hormone and phosphatonins such as fibroblast growth factor-23 (FGF23). Disorders of phosphate homeostasis leading to hypo- and hyperphosphataemia are common and have clinical and biochemical consequences. Notably, recent studies have linked hyperphosphataemia with an increased risk of cardiovascular disease. This review outlines the recent advances in the understanding of phosphate homeostasis and describes the causes, investigation and management of hypo- and hyperphosphataemia.

  10. Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori

    Science.gov (United States)

    Hu, Jingsheng; Tian, Jianghai; Li, Fanchi; Xue, Bin; Hu, Jiahuan; Cheng, Xiaoyu; Li, Jinxin; Shen, Weide; Li, Bing

    2017-01-01

    Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm. PMID:28134300

  11. Glucose-6-phosphate dehydrogenase

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that helps red ...

  12. Chloroquine Phosphate Oral

    Science.gov (United States)

    Chloroquine phosphate is in a class of drugs called antimalarials and amebicides. It is used to prevent and treat ... Chloroquine phosphate comes as a tablet to take by mouth. For prevention of malaria in adults, one dose is ...

  13. A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.

    Directory of Open Access Journals (Sweden)

    Christine Böttcher

    Full Text Available An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-5'-[2-(1H-indol-3-ylethyl]phosphate (AIEP mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with K(i-values 17-68-fold lower than the respective K(m-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5-20 µM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function.

  14. Aminoacyl-tRNA Synthetase Complexes in Evolution

    Directory of Open Access Journals (Sweden)

    Svitlana Havrylenko

    2015-03-01

    Full Text Available Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS, and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis.

  15. Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency.

    Science.gov (United States)

    Ben Ameur, Salma; Aloulou, Hajer; Nasrallah, Fehmi; Kamoun, Thouraya; Kaabachi, Naziha; Hachicha, Mongia

    2015-02-01

    Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

  16. Glutamine synthetase gene evolution: A good molecular clock

    Energy Technology Data Exchange (ETDEWEB)

    Pesole, G.; Lanvave, C.; Saccone, C. (Consiglio Nazionale delle Richerche, Bari (Italy)); Bozzetti, M.P. (Univ. di Bari (Italy)); Preparata, G. (Univ. di Milano (Italy))

    1991-01-15

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves.

  17. Glutamine synthetase localization in cortisol-induced chick embryo retinas

    OpenAIRE

    1980-01-01

    We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age- dependent changes in the endogenous enzyme activity as well as cortisol- induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.

  18. Glutamine synthetase activity in the ruminal bacterium Succinivibrio dextrinosolvens.

    OpenAIRE

    Patterson, J A; Hespell, R B

    1985-01-01

    Succinivibrio dextrinosolvens C18 was found to possess glutamine synthetase (GS), urease, glutamate dehydrogenase, and several other nitrogen assimilation enzymes. When grown in continuous culture under ammonia limitation, both GS and urease activities were high and glutamate dehydrogenase activity was low, but the opposite activity pattern was observed for growth in the presence of ample ammonia. The addition of high-level (15 mM) ammonium chloride to ammonia-limited cultures resulted in a r...

  19. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

    Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

  20. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Lamers, Wouter H; Chaudhry, Farrukh A; Verlander, Jill W; Weiner, I David

    2016-06-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

  1. The role of glutamine synthetase in energy production and glutamine metabolism during oxidative stress.

    Science.gov (United States)

    Aldarini, Nohaiah; Alhasawi, Azhar A; Thomas, Sean C; Appanna, Vasu D

    2017-01-17

    Oxidative stress is known to severely impede aerobic adenosine triphosphate (ATP) synthesis. However, the metabolically-versatile Pseudomonas fluorescens survives this challenge by invoking alternative ATP-generating networks. When grown in a medium with glutamine as the sole organic nutrient in the presence of H2O2, the microbe utilizes glutamine synthetase (GS) to modulate its energy budget. The activity of this enzyme that mediates the release of energy stored in glutamine was sharply increased in the stressed cells compared to the controls. The enhanced activities of such enzymes as acetate kinase, adenylate kinase and nucleotide diphosphate kinase ensured the efficacy of this ATP producing-machine by transferring the high energy phosphate. The elevated amounts of phosphoenol pyruvate carboxylase and pyruvate orthophosphate dikinase recorded in the H2O2 exposed cells provided another route to ATP independent of the reduction of O2. This is the first demonstration of a metabolic pathway involving GS dedicated to ATP synthesis. The phospho-transfer network that is pivotal to the survival of the microorganism under oxidative stress may reveal therapeutic targets against infectious microbes reliant on glutamine for their proliferation.

  2. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    Science.gov (United States)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Kuranova, I. P.

    2015-09-01

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6322 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  3. Prenatal diagnosis of "dihydrobiopterin synthetase" deficiency, a variant form of phenylketonuria.

    Science.gov (United States)

    Niederwieser, A; Shintaku, H; Hasler, T; Curtius, H C; Lehmann, H; Guardamagna, O; Schmidt, H

    1986-08-01

    Amniocentesis was performed at 19 weeks gestation in a mother who had previously delivered a boy with "dihydrobiopterin synthetase" (DHBS) deficiency. The amniotic fluid contained neopterin in high (136 nmol/l) and biopterin in very low concentrations (1.8 nmol/l). The activity of the phosphate-eliminating enzyme (PEE, also called 6-pyruvoyl tetrahydropterin synthase, substrate: 7,8-dihydroneopterin triphosphate) which is present in liver and erythrocytes and defective in DHBS deficiency, was measured in the erythrocytes of the family members. The fetal sample showed only 2% of the activity of healthy adult controls and was comparable with that of the affected sibling. Obligate heterozygotes had activities around 20% of the controls. Two fetal control samples showed even higher activities than adult erythrocytes, Sepiapterin reductase activities wer normal in all cases. At autopsy, PEE deficiency was confirmed in the liver of the fetus. We concluded that DHBS deficiency (and most probably also GTP cyclohydrolase I deficiency) can be diagnosed by metabolite measurements in amniotic fluid. PEE activity is measurable in erythrocytes, although the assay needs to be improved. Since maternal tetrahydrobiopterin does not cross the placenta, treatment of a tetrahydrobiopterin-deficient fetus with tetrahydrobiopterin in utero is not possible.

  4. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru; Abramchik, Yu. A., E-mail: tostars@mail.ru; Zhukhlistova, N. E., E-mail: ugama@yandex.ru; Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-09-15

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6{sub 3}22 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  5. Why nature chose phosphates.

    Science.gov (United States)

    Westheimer, F H

    1987-03-06

    Phosphate esters and anhydrides dominate the living world but are seldom used as intermediates by organic chemists. Phosphoric acid is specially adapted for its role in nucleic acids because it can link two nucleotides and still ionize; the resulting negative charge serves both to stabilize the diesters against hydrolysis and to retain the molecules within a lipid membrane. A similar explanation for stability and retention also holds for phosphates that are intermediary metabolites and for phosphates that serve as energy sources. Phosphates with multiple negative charges can react by way of the monomeric metaphosphate ion PO3- as an intermediate. No other residue appears to fulfill the multiple roles of phosphate in biochemistry. Stable, negatively charged phosphates react under catalysis by enzymes; organic chemists, who can only rarely use enzymatic catalysis for their reactions, need more highly reactive intermediates than phosphates.

  6. Study of thymidylate synthetase-function by laser Raman spectroscopy.

    Science.gov (United States)

    Sharma, R K; Kisliuk, R L; Verma, S P; Wallach, D F

    1975-05-23

    The Laser-Raman spectra of thymidylate synthetase have been obtained with 488 nm excitation from an argon ion laser. Raman bands observed in the range 600-800 cm-minus-1 have been assigned to functional groups of constituent amino acids. The band positions and intensities in the Amide I (1600-1700 cm-minus-1) and Amide III (1200-1300 cm-minus-1) regions, suggest that the enzyme is a mixture of alpha-helical and unordered conformations. Low levels of beta-structure cannot be excluded. The spectra of the ternary complex formed by reacting thymidylate synthetase with (+)-L-methylenetetrahydrofolate and fluorodeoxyuridylate reveals a new band at 1618 cm-minus-1 assigned to the C=N stretching vibration. This band may be due to formation of dihydrofolate or an iminium ion. The overall secondary structure of thymidylate synthetase does not change on formation of the ternary complex. However, the spectrum of the complex indicates local changes in groups such as ionized carboxyl (1400 cm-minus-1), tryptophan (1003 cm-minus-1) and CH-3, CH-2 deformation modes (1440-1470 cm-minus-1).

  7. Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.

    Science.gov (United States)

    Van Dommelen, Anne; Spaepen, Stijn; Vanderleyden, Jozef

    2009-04-01

    Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

  8. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    Science.gov (United States)

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-04-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.

  9. Mitochondrial aminoacyl-tRNA synthetases in human disease.

    Science.gov (United States)

    Konovalova, Svetlana; Tyynismaa, Henna

    2013-04-01

    Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential in the process of transferring genetic information from mitochondrial DNA to the complexes of the oxidative phosphorylation system. These synthetases perform an integral step in the initiation of mitochondrial protein synthesis by charging tRNAs with their cognate amino acids. All mtARSs are encoded by nuclear genes, nine of which have recently been described as disease genes for mitochondrial disorders. Unexpectedly, the clinical presentations of these diseases are highly specific to the affected synthetase. Encephalopathy is the most common manifestation but again with gene-specific outcomes. Other clinical presentations include myopathy with anemia, cardiomyopathy, tubulopathy and hearing loss with female ovarian dysgenesis. Here we review the described mutation types and the associated patient phenotypes. The identified mutation spectrum suggests that only mutation types that allow some residual tRNA-charging activity can result in the described mtARS diseases but the molecular mechanisms behind the selective tissue involvement are not currently understood.

  10. Expression of glutamine synthetase in balloon cells: a basis of their antiepileptic role?

    Science.gov (United States)

    Buccoliero, Anna Maria; Barba, Carmen; Giordano, Flavio; Baroni, Gianna; Genitori, Lorenzo; Guerrini, Renzo; Taddei, Gian Luigi

    2015-01-01

    Glutamine synthetase is an enzyme involved in the clearance of glutamate, the most potent excitatory neurotransmitter. We studied the immunohistochemical expression of glutamine synthetase in neocortical samples from 5 children who underwent surgery for pharmacoresistant epilepsy and a histological diagnosis of focal cortical dysplasia IIb. In all cases, balloon cells, but not dysmorphic neurons, were immunopositive for glutamine synthetase. This finding suggests that balloon cells can be involved in the neutralization of glutamate and play a protective anti-seizure role.

  11. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

    OpenAIRE

    Merritt, Ethan A.; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T.; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J.; Kim, Jessica; Li ZHANG; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S.; Van Voorhis, Wesley C.; Hol, Wim G. J.

    2010-01-01

    Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in b...

  12. PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon

    OpenAIRE

    Lulu Li; Ping Zhang; Zhengxi Bao; Tongxin Wang; Shuang Liu; Feiruo Huang

    2016-01-01

    Excess ammonia is produced during fasting when amino acids are used for glucogenesis. Together with ureagenesis, glucogenesis occurs in periportal hepatocytes mediated mainly through the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In vivo experiments showed that fasting strongly stimulated mice glucagon secretion, hepatic PGC-1α, sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) expression and ureagenesis enzymatic activity such as carbamoyl phosphate synthetase 1 (CP...

  13. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    Science.gov (United States)

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  14. Unusual domain architecture of aminoacyl tRNA synthetases and their paralogs from Leishmania major

    Directory of Open Access Journals (Sweden)

    Gowri V S

    2012-11-01

    Full Text Available Abstract Background Leishmania major, a protozoan parasite, is the causative agent of cutaneous leishmaniasis. Due to the development of resistance against the currently available anti-leishmanial drugs, there is a growing need for specific inhibitors and novel drug targets. In this regards, aminoacyl tRNA synthetases, the linchpins of protein synthesis, have received recent attention among the kinetoplastid research community. This is the first comprehensive survey of the aminoacyl tRNA synthetases, their paralogs and other associated proteins from L. major. Results A total of 26 aminoacyl tRNA synthetases were identified using various computational and bioinformatics tools. Phylogenetic analysis and domain architectures of the L. major aminoacyl tRNA synthetases suggest a probable archaeal/eukaryotic origin. Presence of additional domains or N- or C-terminal extensions in 11 aminoacyl tRNA synthetases from L. major suggests possibilities such as additional tRNA binding or oligomerization or editing activity. Five freestanding editing domains were identified in L. major. Domain assignment revealed a novel asparagine tRNA synthetase paralog, asparagine synthetase A which has been so far reported from prokaryotes and archaea. Conclusions A comprehensive bioinformatic analysis revealed 26 aminoacyl tRNA synthetases and five freestanding editing domains in L. major. Identification of two EMAP (endothelial monocyte-activating polypeptide II-like proteins similar to human EMAP II-like proteins suggests their participation in multisynthetase complex formation. While the phylogeny of tRNA synthetases suggests a probable archaeal/eukaryotic origin, phylogeny of asparagine synthetase A strongly suggests a bacterial origin. The unique features identified in this work provide rationale for designing inhibitors against parasite aminoacyl tRNA synthetases and their paralogs.

  15. Discussion about magnesium phosphating

    Directory of Open Access Journals (Sweden)

    P. Pokorny

    2016-07-01

    Full Text Available The paper describes results from recently published research focused on production of non-conventional magnesium phosphate Mg3(PO42・4H2O – bobierrite, or MgHPO4・3H2O – newberyite coating for both magnesium alloys and/or mild steel. This new kind of coating is categorized in the context of current state of phosphating technology and its potential advantages and crystal structure is discussed. At the same time, the suitable comparison techniques for magnesium phosphate coating and conventional zinc phosphate coating are discussed.

  16. Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

    Science.gov (United States)

    Soriano-Maldonado, Pablo; Las Heras-Vazquez, Francisco Javier; Clemente-Jimenez, Josefa María; Rodriguez-Vico, Felipe; Martínez-Rodríguez, Sergio

    2015-01-01

    Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

  17. DFT electronic structure calculations, spectroscopic studies, and normal coordinate analysis of 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl acetate.

    Science.gov (United States)

    Muthu, S; Elamuruguporchelvi, E; Varghese, Anitha

    2015-03-05

    The solid phase FTIR and FT-Raman spectra of 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl acetate (25N2LCPA) have been recorded 450-4000cm(-1) and 100-4000cm(-1) respectively. The normal coordinate analysis was carried out to confirm the precision of the assignments. DFT calculations have been performed giving energies, optimized structures, harmonic vibrational frequencies and IR intensities. The structure of the molecule was optimized and the structural characteristics were determined by density functional theory (DFT) using B3LYP method with 6-31+G(d,p) basis set. The detailed interpretation of the vibrational spectra has been carried out with aid of normal coordinate analysis (NCA) following the scaled quantum mechanical force field methodology. The Vibrational frequencies are calculated in the above method and are compared with experimental frequencies which yield good agreement between observed and calculated frequencies. Stability of the molecule arising from hyper conjugative interactions, charge delocalization has been analyzed using natural bond orbital (NBO) analysis. In addition, Frontiers molecular orbital and molecular electrostatic potential were computed by using Density Functional Theory (DFT) B3LYP/6-31+G(d,p) basis set. The calculated HOMO and LUMO energies show that charge transfer occurs in the molecule.

  18. Oxidation of 2'-deoxycytidine to four inter-converting diastereomers of N-1-carbamoyl-4,5-dihydroxy-2-oxo-imidazolidine nucleosides

    Energy Technology Data Exchange (ETDEWEB)

    Tremblay, S.; Gantchev, T.; Cadet, J.; Wagner, J.R. [Univ Sherbrooke, Fac Med and Hlth Sci, Dept Med Nucl and Radiobiol, Sherbrooke, PQ J1H 5N4, (Canada); Tremblay, L.; Lavigne, P. [Univ Sherbrooke, Fac Med and Hlth Sci, Inst Pharmacol, Sherbrooke, PQ J1H 5N4, (Canada); Cadet, J. [CEA Grenoble, Dept Rech Fondamentale Mat Condensee, SCIB/LAN, UMR-E 3, CEA-UJF, F-38054 Grenoble, (France)

    2007-07-01

    Modification of 2'-deoxycytidine (dCyd) by hydroxyl radicals and direct ionization leads to the formation of various oxidation products, including dCyd 5,6-glycols, 5-hydroxy-2'-deoxycytidine, and ring fragmentation products. The mechanism of oxidation is complex and poorly understood. In the present work, we have prepared four cis- and trans-diastereomers of N-1-(2-deoxy-{beta}-D-erythro-pentofuranosyl)-1-carbamoyl-2-oxo-4,5-dihydro oxy-imidazolidine by bromination of dCyd followed by peroxidation of the resulting dCyd bromo-hydrins. The structure and stereochemistry of each product were determined by {sup 1}H NMR, {sup 13}C NMR, and 2D NOE analyses. The formation of imidazolidine products involves rearrangement of initial 5(6)-hydroxy-6(5)-hydro-peroxides to C6-C2 endo-peroxides, which subsequently decompose by a concerted pathway to imidazolidine products. A remarkable feature of the four diastereomers was their ability to inter-convert via single and successive cycles of ring-chain tautomerism at N1-C5 and N3-C4, leading to epimerization of C5 and C4, respectively. The rate of isomerization was greater for cis-diastereomers compared to trans-diastereomers, and the rate sharply increased with pH (pH 9.0 {>=} 7.0 {>=} 5.5). (authors)

  19. Therapeutic Use of 3β-[N-(N',N'-Dimethylaminoethane) Carbamoyl] Cholesterol-Modified PLGA Nanospheres as Gene Delivery Vehicles for Spinal Cord Injury.

    Science.gov (United States)

    Gwak, So-Jung; Yun, Yeomin; Yoon, Do Heum; Kim, Keung Nyun; Ha, Yoon

    2016-01-01

    Gene delivery holds therapeutic promise for the treatment of neurological diseases and spinal cord injury. Although several studies have investigated the use of non-viral vectors, such as polyethylenimine (PEI), their clinical value is limited by their cytotoxicity. Recently, biodegradable poly (lactide-co-glycolide) (PLGA) nanospheres have been explored as non-viral vectors. Here, we show that modification of PLGA nanospheres with 3β-[N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) enhances gene transfection efficiency. PLGA/DC-Chol nanospheres encapsulating DNA were prepared using a double emulsion-solvent evaporation method. PLGA/DC-Chol nanospheres were less cytotoxic than PEI both in vitro and in vivo. DC-Chol modification improved the uptake of nanospheres, thereby increasing their transfection efficiency in mouse neural stem cells in vitro and rat spinal cord in vivo. Also, transgene expression induced by PLGA nanospheres was higher and longer-lasting than that induced by PEI. In a rat model of spinal cord injury, PLGA/DC-Chol nanospheres loaded with vascular endothelial growth factor gene increased angiogenesis at the injury site, improved tissue regeneration, and resulted in better recovery of locomotor function. These results suggest that DC-Chol-modified PLGA nanospheres could serve as therapeutic gene delivery vehicles for spinal cord injury.

  20. Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti

    Directory of Open Access Journals (Sweden)

    Kelly Lynn Hagberg

    2016-11-01

    Full Text Available Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the Nitrogen Stress Response (NSR and Phosphate Stress Response (PSR have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow very slowly and pseudoreversion of the slow growth phenotype is common. To understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.

  1. Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti

    Science.gov (United States)

    Hagberg, Kelly L.; Yurgel, Svetlana N.; Mulder, Monika; Kahn, Michael L.

    2016-01-01

    Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the nitrogen stress response (NSR) and phosphate stress response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow very slowly and pseudoreversion of the slow growth phenotype is common. To understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation. PMID:27965651

  2. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin.

    Science.gov (United States)

    Pascual, María Belén; Jing, Zhong Ping; Kirby, Edward G; Cánovas, Francisco M; Gallardo, Fernando

    2008-01-01

    Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth

  3. Glutamine versus ammonia utilization in the NAD synthetase family.

    Directory of Open Access Journals (Sweden)

    Jessica De Ingeniis

    Full Text Available NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS. Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS

  4. Roles of Long-chain Acyl Coenzyme A Synthetase in Absorption and Transport of Fatty Acid

    Institute of Scientific and Technical Information of China (English)

    Fan Gao; Xue-feng Yang; Nian Fu; Yang Hu; Yan Ouyang; Kai Qing

    2016-01-01

    Abstract Long-chain acyl coenzyme A synthetase (ACSL) is a member of the synthetase family encoded by a multigene family; it plays an important role in the absorption and transport of fatty acid. Here we review the roles of ACSL in the regulating absorption and transport of fatty acid, as well as the connection between ACSL and some metabolic diseases.

  5. Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

    OpenAIRE

    Wax, Richard; Synder, Linda; Kaplan, Louis

    1982-01-01

    In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.

  6. [The anti-synthetase syndrome: muscle disease and multisystem disorder at the same time

    NARCIS (Netherlands)

    Hengstman, G.J.D.; Venrooij, W.J.W. van; Hoogen, F.H.J. van den; Engelen, B.G.M. van

    2003-01-01

    In three women, aged 60, 45 and 38 years, who presented with exertional dyspnoea (due to lung fibrosis) and Raynaud's phenomenon, dermatomyopathy and Raynaud's phenomenon, and symmetrical arthralgia and myalgia, respectively, the anti-synthetase syndrome was diagnosed. The anti-synthetase syndrome c

  7. Primer Dependent and Independent Forms of Soluble Starch Synthetase from Developing Barley Endosperms

    DEFF Research Database (Denmark)

    Kreis, M.

    1980-01-01

    The activity of soluble starch synthetase (ADP-glucose: agr-1,4-glucan agr-4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purif...

  8. Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya.

    Science.gov (United States)

    Wax, R; Synder, L; Kaplan, L

    1982-10-01

    In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.

  9. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...... groups of rats. These changes may partly explain the demonstrated training-induced increase in glucose tolerance. None of the findings could be ascribed to differences in foold intake or body weight....

  10. The Mitochondrial Aminoacyl tRNA Synthetases: Genes and Syndromes.

    Science.gov (United States)

    Diodato, Daria; Ghezzi, Daniele; Tiranti, Valeria

    2014-01-01

    Mitochondrial respiratory chain (RC) disorders are a group of genetically and clinically heterogeneous diseases. This is because protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis and maintenance of mitochondria, including mitochondrial DNA (mtDNA) replication, transcription, and translation, require nuclear-encoded genes. In the past decade, a growing number of syndromes associated with dysfunction of mtDNA translation have been reported. This paper reviews the current knowledge of mutations affecting mitochondrial aminoacyl tRNAs synthetases and their role in the pathogenic mechanisms underlying the different clinical presentations.

  11. The Mitochondrial Aminoacyl tRNA Synthetases: Genes and Syndromes

    Directory of Open Access Journals (Sweden)

    Daria Diodato

    2014-01-01

    Full Text Available Mitochondrial respiratory chain (RC disorders are a group of genetically and clinically heterogeneous diseases. This is because protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis and maintenance of mitochondria, including mitochondrial DNA (mtDNA replication, transcription, and translation, require nuclear-encoded genes. In the past decade, a growing number of syndromes associated with dysfunction of mtDNA translation have been reported. This paper reviews the current knowledge of mutations affecting mitochondrial aminoacyl tRNAs synthetases and their role in the pathogenic mechanisms underlying the different clinical presentations.

  12. Circumstantial evidence for a role of glutamine-synthetase in suicide.

    Science.gov (United States)

    Kalkman, Hans O

    2011-06-01

    Suicide occurs during depression, schizophrenia, diabetes and epilepsy. A common denominator of these disorders is the presence of inflammation. Inflammatory cytokines affect function and expression of the glial enzyme glutamine synthetase and post mortem studies indicate that brain glutamine synthetase function is suppressed in mood disorders and epilepsy. In a study of schizophrenia brains, the expression of glutamine synthetase was reduced in those cases where the cause of death was suicide. The glycogen synthase kinase 3 (GSK3) inhibitor, lithium, which has a proven efficacy against suicide, increased in an animal experiment the expression of glutamine synthetase. Based on these data one could reason that suicide may be prevented by centrally acting GSK3 inhibitors. However, since inhibition of glutamine synthetase may lead to a deficit in glutamine and as consequence a GABA and glutamate deficit, even simple food supplementation with glutamine might help to reduce suicide.

  13. Radioactivity of phosphate mineral products

    OpenAIRE

    Mitrović Branislava; Vitorović Gordana; Stojanović Mirjana; Vitorović Duško

    2011-01-01

    The phosphate industry is one of the biggest polluters of the environment with uranium. Different products are derived after processing phosphoric ore, such as mineral and phosphate fertilizers and phosphate mineral supplements (dicalcium-and monocalcium phosphate) for animal feeding. Phosphate mineral additives used in animal food may contain a high activity of uranium. Research in this study should provide an answer to the extent in which phosphate minera...

  14. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  15. Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.

    Science.gov (United States)

    Wu, C

    1977-04-01

    Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and alpha-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the gamma-glutamyl transfer reaction of the enzyme. Inhibition of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.

  16. Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Noinaj, Nicholas; Wattanasak, Rut; Lee, Duck-Yeon; Wally, Jeremy L.; Piszczek, Grzegorz; Chock, P. Boon; Stadtman, Thressa C.; Buchanan, Susan K. (NIH)

    2012-03-26

    Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg{sup 2+} and K{sup +} and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS{sup C17S}) in apo form (without ATP bound). EcSPS{sup C17S} crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

  17. Enzymatic syntheses of carbamyl phosphate, L-citrulline, and N-carbamyl L-aspartate labeled with either 13N or 11C.

    Science.gov (United States)

    Gelbard, A S; Kaseman, D S; Rosenspire, K C; Meister, A

    1985-01-01

    [13N]- and [11C]carbamyl phosphate, L-[omega-13N]citrulline, L-[ureido-11C]citrulline, [carbamyl-13N]- and [carbamyl-11C]carbamyl-L-aspartate were synthesized using carbamyl phosphate synthetase co-immobilized with either aspartate transcarbamylase or ornithine transcarbamylase. Carbamyl L-[13N]aspartate was enzymatically prepared from carbamyl phosphate and L-[13N]aspartate. The tissue distribution of radioactivity in mice after injection of radiolabeled ammonia, carbamyl phosphate or citrulline was studied. The tissue distribution of isotope derived from [13N]carbamyl phosphate and [13N]ammonia were similar, with the exception of liver, brain and pancreas, in which 13NH3 uptake was higher after retroorbital injection. The distribution of label derived from L-[omega-13N]- and L-[ureido-11C]citrulline was similar. Substantial tumor (Sarcoma-180) uptake of label from L-citrulline was observed.

  18. Synthesis of phosphinoferrocene amides and thioamides from carbamoyl chlorides and the structural chemistry of Group 11 metal complexes with these mixed-donor ligands.

    Science.gov (United States)

    Fernandes, Tiago A; Solařová, Hana; Císařová, Ivana; Uhlík, Filip; Štícha, Martin; Štěpnička, Petr

    2015-02-21

    The reaction of in situ generated 1'-(diphenylphosphino)-1-lithioferrocene with carbamoyl chlorides, ClC(E)NMe2, affords the corresponding (thio)amides, Ph2PfcC(E)NMe2 (E = O (), S (); fc = ferrocene-1,1'-diyl). These compounds as well as their analogues, Ph2PfcC(O)NHMe () and Ph2PfcC(O)NH2 (), prepared from 1'-(diphenylphosphino)ferrocene-1-carboxylic acid (Hdpf) were studied as ligands for the Group 11 metal ions. In the reactions with [Cu(MeCN)4][BF4], the amides give rise to bis-chelate complexes of the type [Cu(L-κ(2)O,P)2][BF4]. Similar products, [Ag(L-κ(2)O,P)2]ClO4, are obtained from silver(i) perchlorate and , or . In contrast, the reaction of AgClO4 with produces a unique molecular dimer [Ag()(ClO4-κO)]2, where the metal centres are bridged by the sulfur atoms of the P,S-chelating thioamides. The reactions of with [AuCl(tht)] (tht = tetrahydrothiophene) afford the expected gold(i)-phosphine complexes, [AuCl(L-κP)], containing uncoordinated (thio)amide moieties. Hemilabile coordination of the phosphinoamide ligands in complexes with the soft Group 11 metal ions is established by the crystal structure of a solvento complex, [Cu(-κ(2)O,P)(-κP)(CHCl3-κCl)][BF4], which was isolated serendipitously during an attempted crystallisation of [Cu(-κ(2)O,P)2][BF4]. All of the compounds are characterised by spectroscopic methods, and the structures of several representatives of both the free phosphinoamides and their complexes are determined by X-ray diffraction analysis and further studied by DFT calculations and cyclic voltammetry.

  19. Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

    Science.gov (United States)

    Savilov, P N; Yakovlev, V N

    2016-01-01

    Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

  20. Encapsulation of glutamine synthetase in mouse erythrocytes: a new procedure for ammonia detoxification.

    Science.gov (United States)

    Kosenko, Elena A; Venediktova, Natalia I; Kudryavtsev, Andrey A; Ataullakhanov, Fazoil I; Kaminsky, Yury G; Felipo, Vicente; Montoliu, Carmina

    2008-12-01

    There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 +/- 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations.

  1. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    Energy Technology Data Exchange (ETDEWEB)

    Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

    2016-03-15

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  2. Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.

    Science.gov (United States)

    Hanley, K; Chappell, J

    1992-01-01

    Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

  3. Metal-phosphate binders

    Science.gov (United States)

    Howe, Beth Ann [Lewistown, IL; Chaps-Cabrera, Jesus Guadalupe [Coahuila, MX

    2009-05-12

    A metal-phosphate binder is provided. The binder may include an aqueous phosphoric acid solution, a metal-cation donor including a metal other than aluminum, an aluminum-cation donor, and a non-carbohydrate electron donor.

  4. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  5. Phosphate control in dialysis.

    Science.gov (United States)

    Cupisti, Adamasco; Gallieni, Maurizio; Rizzo, Maria Antonietta; Caria, Stefania; Meola, Mario; Bolasco, Piergiorgio

    2013-10-04

    Prevention and correction of hyperphosphatemia is a major goal of chronic kidney disease-mineral and bone disorder (CKD-MBD) management, achievable through avoidance of a positive phosphate balance. To this aim, optimal dialysis removal, careful use of phosphate binders, and dietary phosphate control are needed to optimize the control of phosphate balance in well-nourished patients on a standard three-times-a-week hemodialysis schedule. Using a mixed diffusive-convective hemodialysis tecniques, and increasing the number and/or the duration of dialysis tecniques are all measures able to enhance phosphorus (P) mass removal through dialysis. However, dialytic removal does not equal the high P intake linked to the high dietary protein requirement of dialysis patients; hence, the use of intestinal P binders is mandatory to reduce P net intestinal absorption. Unfortunately, even a large dose of P binders is able to bind approximately 200-300 mg of P on a daily basis, so it is evident that their efficacy is limited in the case of an uncontrolled dietary P load. Hence, limitation of dietary P intake is needed to reach the goal of neutral phosphate balance in dialysis, coupled to an adequate protein intake. To this aim, patients should be informed and educated to avoid foods that are naturally rich in phosphate and also processed food with P-containing preservatives. In addition, patients should preferentially choose food with a low P-to-protein ratio. For example, patients could choose egg white or protein from a vegetable source. Finally, boiling should be the preferred cooking procedure, because it induces food demineralization, including phosphate loss. The integrated approach outlined in this article should be actively adapted as a therapeutic alliance by clinicians, dieticians, and patients for an effective control of phosphate balance in dialysis patients.

  6. Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

    Science.gov (United States)

    Jeitner, Thomas M; Battaile, Kevin; Cooper, Arthur J L

    2015-12-01

    The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

  7. Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation.

    Science.gov (United States)

    Bott, Alex J; Peng, I-Chen; Fan, Yongjun; Faubert, Brandon; Zhao, Lu; Li, Jinyu; Neidler, Sarah; Sun, Yu; Jaber, Nadia; Krokowski, Dawid; Lu, Wenyun; Pan, Ji-An; Powers, Scott; Rabinowitz, Joshua; Hatzoglou, Maria; Murphy, Daniel J; Jones, Russell; Wu, Song; Girnun, Geoffrey; Zong, Wei-Xing

    2015-12-01

    c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

  8. Astrocyte glutamine synthetase: pivotal in health and disease.

    Science.gov (United States)

    Rose, Christopher F; Verkhratsky, Alexei; Parpura, Vladimir

    2013-12-01

    The multifunctional properties of astrocytes signify their importance in brain physiology and neurological function. In addition to defining the brain architecture, astrocytes are primary elements of brain ion, pH and neurotransmitter homoeostasis. GS (glutamine synthetase), which catalyses the ATP-dependent condensation of ammonia and glutamate to form glutamine, is an enzyme particularly found in astrocytes. GS plays a pivotal role in glutamate and glutamine homoeostasis, orchestrating astrocyte glutamate uptake/release and the glutamate-glutamine cycle. Furthermore, astrocytes bear the brunt of clearing ammonia in the brain, preventing neurotoxicity. The present review depicts the central function of astrocytes, concentrating on the importance of GS in glutamate/glutamine metabolism and ammonia detoxification in health and disease.

  9. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    Science.gov (United States)

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

  10. In Silico Discovery of Aminoacyl-tRNA Synthetase Inhibitors

    Directory of Open Access Journals (Sweden)

    Yaxue Zhao

    2014-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNA. They play a pivotal role in protein synthesis and are essential for cell growth and survival. The aaRSs are one of the leading targets for development of antibiotic agents. In this review, we mainly focused on aaRS inhibitor discovery and development using in silico methods including virtual screening and structure-based drug design. These computational methods are relatively fast and cheap, and are proving to be of great benefit for the rational development of more potent aaRS inhibitors and other pharmaceutical agents that may usher in a much needed generation of new antibiotics.

  11. Common peptides study of aminoacyl-tRNA synthetases.

    Directory of Open Access Journals (Sweden)

    Assaf Gottlieb

    Full Text Available BACKGROUND: Aminoacyl tRNA synthetases (aaRSs constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains. RESULTS: We utilized the Common Peptides (CPs framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS-class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA enzyme overlapping binding sites in both families. CONCLUSIONS: The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.

  12. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward M.

    1980-01-01

    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy/sup +/ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy/sup +/ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy/sup +/ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  13. High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency : Implications for treatment?

    NARCIS (Netherlands)

    van Spronsen, F. J.; Reijngoud, D. J.; Verhoeven, N. M.; Soorani-Lunsing, R. J.; Jakobs, C.; Sijens, P. E.

    2006-01-01

    Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was increa

  14. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    Energy Technology Data Exchange (ETDEWEB)

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara; (Maryland)

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  15. Phosphate control in dialysis

    Directory of Open Access Journals (Sweden)

    Cupisti A

    2013-10-01

    Full Text Available Adamasco Cupisti,1 Maurizio Gallieni,2 Maria Antonietta Rizzo,2 Stefania Caria,3 Mario Meola,4 Piergiorgio Bolasco31Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; 2Nephrology and Dialysis Unit, San Carlo Borromeo Hospital, Milan, Italy; 3Territorial Department of Nephrology and Dialysis, ASL Cagliari, Italy; 4Sant'Anna School of Advanced Studies, University of Pisa, Pisa, ItalyAbstract: Prevention and correction of hyperphosphatemia is a major goal of chronic kidney disease–mineral and bone disorder (CKD–MBD management, achievable through avoidance of a positive phosphate balance. To this aim, optimal dialysis removal, careful use of phosphate binders, and dietary phosphate control are needed to optimize the control of phosphate balance in well-nourished patients on a standard three-times-a-week hemodialysis schedule. Using a mixed diffusive–convective hemodialysis tecniques, and increasing the number and/or the duration of dialysis tecniques are all measures able to enhance phosphorus (P mass removal through dialysis. However, dialytic removal does not equal the high P intake linked to the high dietary protein requirement of dialysis patients; hence, the use of intestinal P binders is mandatory to reduce P net intestinal absorption. Unfortunately, even a large dose of P binders is able to bind approximately 200–300 mg of P on a daily basis, so it is evident that their efficacy is limited in the case of an uncontrolled dietary P load. Hence, limitation of dietary P intake is needed to reach the goal of neutral phosphate balance in dialysis, coupled to an adequate protein intake. To this aim, patients should be informed and educated to avoid foods that are naturally rich in phosphate and also processed food with P-containing preservatives. In addition, patients should preferentially choose food with a low P-to-protein ratio. For example, patients could choose egg white or protein from a vegetable source

  16. Diffuse glutamine synthetase overexpression restricted to areas of peliosis in a β-catenin-activated hepatocellular adenoma: a potential pitfall in glutamine synthetase interpretation.

    Science.gov (United States)

    Berry, Ryan S; Gullapalli, Rama R; Wu, Jin; Morris, Katherine; Hanson, Joshua A

    2014-08-01

    Hepatocellular adenomas have recently been classified into four subtypes based on molecular findings: hepatocyte nuclear factor 1α (HNF1α) inactivated, inflammatory/telangiectatic, β-catenin activated, and unclassifiable. β-catenin-activated adenomas have the potential for malignant transformation and are thus important to recognize. Diffuse glutamine synthetase immunohistochemical positivity has been shown to be a reliable surrogate marker for β-catenin activation, though variations in staining patterns may be difficult to interpret. We report a case of a peliotic adenoma that was morphologically consistent with a β-catenin wild-type hepatocellular adenoma but harbored a β-catenin mutation by molecular analysis. The tumor lacked nuclear β-catenin positivity and demonstrated a hitherto undescribed pattern of glutamine synthetase overexpression restricted to areas of peliosis with mostly negative staining in non-peliotic areas. This pattern was initially interpreted as physiologic and may represent a potential pitfall in glutamine synthetase interpretation.

  17. Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus

    OpenAIRE

    Anna eShiyan; Melanie eThompson; Saskia eKöcher; Michaela eTausendschön; Helena eSantos; Inga eHänelt; Volker eMüller

    2014-01-01

    Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride....

  18. The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence.

    Science.gov (United States)

    Avila-Ospina, Liliana; Marmagne, Anne; Talbotec, Joël; Krupinska, Karin; Masclaux-Daubresse, Céline

    2015-04-01

    Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions.

  19. N-Acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L-cysteine (iso-GAMA) a further product of human metabolism of acrylamide: comparison with the simultaneously excreted other mercaptuic acids.

    Science.gov (United States)

    Hartmann, Eva C; Boettcher, Melanie I; Bolt, Hermann M; Drexler, Hans; Angerer, Jürgen

    2009-07-01

    The N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L: -cysteine (iso-GAMA) could be identified as a further human metabolite of acrylamide. In this study, we report the excretion of d(3)-iso-GAMA in human urine after single oral administration of deuterium labelled acrylamide (d(3)-AA). One healthy male volunteer ingested a dose of about 1 mg d(3)-AA which is equivalent to a dose of 13 microg/kg bodyweight. Over a period of 46 h the urine was collected and the d(3)-iso-GAMA levels analysed by LC-ESI-MS/MS. The excretion of iso-GAMA begins five hours after application. It rises to a maximum concentration (c (max)) of 43 microg/l which was quantified in the urine excreted after 22 h (t (max)). The excretion pattern is parallel to that of the major oxidative metabolite N-acetyl-S-(2-carbamoyl-2-hydroxy-ethyl)-L-cysteine (GAMA). Total recovery of iso-GAMA was about 1% of the applied dose. Together with N-acetyl-S-(2-carbamoylethyl)-L: -cysteine (AAMA) and GAMA, 57% of the applied dose is eliminated as mercapturic acids. The elimination kinetics of the three mercapturic acids of AA are compared. We show that dietary doses of acrylamide (AA) cause an overload of detoxification via AAMA and lead to the formation of carcinogenic glycidamide (GA) in the human body.

  20. Phosphate Mines, Jordan

    Science.gov (United States)

    2008-01-01

    Jordan's leading industry and export commodities are phosphate and potash, ranked in the top three in the world. These are used to make fertilizer. The Jordan Phosphate Mines Company is the sole producer, having started operations in 1935. In addition to mining activities, the company produces phosphoric acid (for fertilizers, detergents, pharmaceuticals), diammonium phosphate (for fertilizer), sulphuric acid (many uses), and aluminum fluoride (a catalyst to make aluminum and magnesium). The image covers an area of 27.5 x 49.4 km, was acquired on September 17, 2005, and is located near 30.8 degrees north latitude, 36.1 degrees east longitude. The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

  1. Recurrent seizures and brain pathology after inhibition of glutamine synthetase in the hippocampus in rats.

    Science.gov (United States)

    Eid, Tore; Ghosh, Arko; Wang, Yue; Beckström, Henning; Zaveri, Hitten P; Lee, Tih-Shih W; Lai, James C K; Malthankar-Phatak, Gauri H; de Lanerolle, Nihal C

    2008-08-01

    An excess of extracellular glutamate in the hippocampus has been linked to the generation of recurrent seizures and brain pathology in patients with medically intractable mesial temporal lobe epilepsy (MTLE). However, the mechanism which results in glutamate excess in MTLE remains unknown. We recently reported that the glutamate-metabolizing enzyme glutamine synthetase is deficient in the hippocampus in patients with MTLE, and we postulated that this deficiency is critically involved in the pathophysiology of the disease. To further explore the role of glutamine synthetase in MTLE we created a novel animal model of hippocampal glutamine synthetase deficiency by continuous (approximately 28 days) microinfusion of methionine sulfoximine (MSO: 0.625 to 2.5 microg/h) unilaterally into the hippocampus in rats. This treatment led to a deficiency in hippocampal glutamine synthetase activity by 82-97% versus saline. The majority (>95%) of the MSO-treated animals exhibited recurrent seizures that continued for several weeks. Some of the MSO-treated animals exhibited neuropathological features that were similar to mesial temporal sclerosis, such as hippocampal atrophy and patterned loss of hippocampal neurons. However, many MSO-treated animals displayed only minimal injury to the hippocampus, with no clear evidence of mesial temporal sclerosis. These findings support the hypothesis that a deficiency in hippocampal glutamine synthetase causes recurrent seizures, even in the absence of classical mesial temporal sclerosis, and that restoration of glutamine synthetase may represent a novel approach to therapeutic intervention in this disease.

  2. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  3. Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution.

    Science.gov (United States)

    Dock-Bregeon, Anne-Catherine; Rees, Bernard; Torres-Larios, Alfredo; Bey, Gilbert; Caillet, Joel; Moras, Dino

    2004-11-05

    The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.

  4. Aminoacyl-tRNA Synthetases in the Bacterial World.

    Science.gov (United States)

    Giegé, Richard; Springer, Mathias

    2016-05-01

    Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation. Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g., in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show huge structural plasticity related to function and limited idiosyncrasies that are kingdom or even species specific (e.g., the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS). Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably between distant groups such as Gram-positive and Gram-negative Bacteria. The review focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation, and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulated in last two decades is reviewed, showing how the field moved from essentially reductionist biology towards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRS paralogs (e.g., during cell wall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointed throughout the review and

  5. Actinobacterial acyl coenzyme A synthetases involved in steroid side-chain catabolism.

    Science.gov (United States)

    Casabon, Israël; Swain, Kendra; Crowe, Adam M; Eltis, Lindsay D; Mohn, William W

    2014-02-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2'-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 10(5) ± 0.1 × 10(5) M(-1) s(-1) and 3.2 × 10(5) ± 0.3 × 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid

  6. Glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... medlineplus.gov/ency/article/000528.htm Glucose-6-phosphate dehydrogenase deficiency To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a condition in which ...

  7. Research on Uncrystallized Phosphating Film

    Institute of Scientific and Technical Information of China (English)

    TANG En-jun; XING Ze-kuan

    2004-01-01

    This article excogitated a kind of uncrystallized phosphating film bears wearing capacity goodly by adding Ca2 + in normal phosphating solution. This technology is very useful to protect steel parts working in oil from abrasion.

  8. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.

    Science.gov (United States)

    Verlander, Jill W; Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E; Weiner, I David

    2013-09-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.

  9. Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase.

    OpenAIRE

    Rothstein, D M; Pahel, G; Tyler, B.; Magasanik, B

    1980-01-01

    One of the suspected regulators of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is glutamine synthetase itself. We isolated Escherichia coli strains carrying fusions of the beta-galactosidase structural gene to the promoter of the glutamine synthetase gene, with the aid of the Casadaban Mud1 (ApR, lac, cts62) phage. Some aspects of regulation were retained in haploid fusion strains despite the absence of glutamine synthetase, whereas other as...

  10. Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

    Institute of Scientific and Technical Information of China (English)

    Sun Hui; Huang Qiman; Su Jin

    2005-01-01

    Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.

  11. The plastidial folylpolyglutamate synthetase and root apical meristem maintenance

    Science.gov (United States)

    Srivastava, Avinash C; Tang, Yuhong; Díaz de la Garza, Rocío I

    2011-01-01

    Folylpolyglutamate synthetase (FPGS) catalyzes the attachment of glutamate residues to the folate molecule in plants. Three isoforms of FPGS have been identified in Arabidopsis and these are localized in the plastid (AtDFB), mitochondria (AtDFC) and cytosol (AtDFD). We recently determined that mutants in the AtDFB (At5G05980) gene disrupt primary root development in Arabidopsis thaliana seedlings. Transient expression of AtDFB-green fluorescent protein (GFP) fusion under the control of the native AtDFB promoter in Nicotiana tabacum leaf epidermal cells verified the plastid localization of AtDFB. Furthermore, low concentrations of methotrexate (MTX), a compound commonly used as a folate antagonist in plant and mammalian cells induced primary root defects in wild type seedlings that were similar to atdfb. In addition, atdfb seedlings were more sensitive to MTX when compared to wild type. Quantitative (q) RT-PCR showed lower transcript levels of the mitochondrial and cytosolic FPGS in roots of 7-day-old atdfb seedling suggesting feedback regulation of AtDFB on the expression of other FPGS isoforms during early seedling development. The primary root defects of atdfb, which can be traced in part to altered quiescent center (QC) identity, pave the way for future studies that could link cell type specific folate and FPGS isoform requirements to whole organ development. PMID:21502816

  12. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    Science.gov (United States)

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  13. Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

    LENUS (Irish Health Repository)

    Kell, M R

    2012-02-03

    BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

  14. Prostaglandin synthetase inhibitors in the treatment of nephrogenic diabetes insipidus.

    Science.gov (United States)

    Monn, E

    1981-01-01

    Two boys with classical NDI have been treated with prostaglandin synthetase inhibitors. A boy, 7 years old, was treated with low solute-load diet and diuretics from his first year of life. His main complaint was nocturnal enuresis. He responded within one day to indomethacin 25 mg twice daily, and the urine volume was reduced from 4 1/2--6 litre/day to 2 1/2--3 litre/day. There is almost no enuresis. A boy, 7 months old, had a basal daily urine volume of 1.6--1.8 litre. A low solute-load diet and diuretics reduced urine volume to 1 litre, but he still needed gastric tube feeding. With the addition of acetylsalicylic acid, 75 mg three times daily, the urine volume was reduced to 600 ml, and he needed no more tube feeding. Both boys are doing well on the above-mentioned regimens, and no side effects have been observed after 1 year of treatment.

  15. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  16. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Energy Technology Data Exchange (ETDEWEB)

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  17. Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis.

    Science.gov (United States)

    Paress, P S; Streicher, S L

    1985-08-01

    Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.

  18. Synthesis and preliminary evaluation of (R,S)-1-[2-((carbamoyl-4-hydroxy)phenoxy)-ethylamino]-3-[4-(1-[11C]-met hyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol ([11C]CGP 20712A) as a selective beta 1-adrenoceptor ligand for PET

    NARCIS (Netherlands)

    Elsinga, P H; Van Waarde, A; Visser, Gerben; Vaalburg, W

    1994-01-01

    The most selective beta 1-adrenoceptor ligand known at this moment is (S)-1-[2-((carbamoyl-4-hydroxy) phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol (CGP 26505), the S-isomer of CGP 20712A. We prepared the racemic 11C analogue by methylation with [11C]CH3I of

  19. Practical application of phosphate treatment

    Energy Technology Data Exchange (ETDEWEB)

    Caravaggio, Mike [Integrated Chemistry Solutions Pte. Ltd., Singapore (Singapore)

    2011-05-15

    Phosphate treatment has been applied to subcritical fossil power boilers for well over half a century, as well as being used frequently in heat recovery steam generators. The use of this treatment has evolved over the decades, with the operating sodium to phosphate ratio being the defining factor for the evolution of the treatment. The evolving prescribed sodium to phosphate ratios have been based on the scientific research results and operating experience available at the time, and in the latest EPRI Guidelines issued in 2004 are set at a minimum sodium to phosphate ratio of 3:1, with provision to add up to 1 mg . L{sup -1} of additional free caustic. The ratio limitation has always been set in an effort to minimize the potential for corrosion caused by the potential misapplication of the treatment. Typically, the operating ranges for phosphate treatments are depicted on an x-y plot with the x-axis the phosphate concentration and the y-axis the corrected pH value based on the maximum sodium to phosphate ratio allowed for by the treatment. These operating range plots define the theoretical operating range of a phosphate treatment. This paper briefly discusses the origin of the current phosphate control limits in the EPRI Guidelines, discusses phosphate chemistry, outlines the limitations involved when applying a phosphate treatment and provides additional practical guidance for overcoming these limitations and minimizing the potential for corrosion induced by the incorrect application of a phosphate treatment. (orig.)

  20. Optimality in the zonation of ammonia detoxification in rodent liver.

    Science.gov (United States)

    Bartl, Martin; Pfaff, Michael; Ghallab, Ahmed; Driesch, Dominik; Henkel, Sebastian G; Hengstler, Jan G; Schuster, Stefan; Kaleta, Christoph; Gebhardt, Rolf; Zellmer, Sebastian; Li, Pu

    2015-11-01

    The rodent liver eliminates toxic ammonia. In mammals, three enzymes (or enzyme systems) are involved in this process: glutaminase, glutamine synthetase and the urea cycle enzymes, represented by carbamoyl phosphate synthetase. The distribution of these enzymes for optimal ammonia detoxification was determined by numerical optimization. This in silico approach predicted that the enzymes have to be zonated in order to achieve maximal removal of toxic ammonia and minimal changes in glutamine concentration. Using 13 compartments, representing hepatocytes, the following predictions were generated: glutamine synthetase is active only within a narrow pericentral zone. Glutaminase and carbamoyl phosphate synthetase are located in the periportal zone in a non-homogeneous distribution. This correlates well with the paradoxical observation that in a first step glutamine-bound ammonia is released (by glutaminase) although one of the functions of the liver is detoxification by ammonia fixation. The in silico approach correctly predicted the in vivo enzyme distributions also for non-physiological conditions (e.g. starvation) and during regeneration after tetrachloromethane (CCl4) intoxication. Metabolite concentrations of glutamine, ammonia and urea in each compartment, representing individual hepatocytes, were predicted. Finally, a sensitivity analysis showed a striking robustness of the results. These bioinformatics predictions were validated experimentally by immunohistochemistry and are supported by the literature. In summary, optimization approaches like the one applied can provide valuable explanations and high-quality predictions for in vivo enzyme and metabolite distributions in tissues and can reveal unknown metabolic functions.

  1. Calcium Phosphate Biomaterials: An Update

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Current calcium phosphate (CaP) biomaterials for bone repair, substitution, augmentation and regeneration include hydroxyapatite ( HA ) from synthetic or biologic origin, beta-tricalcium phosphate ( β-TCP ) , biphasic calcium phosphate (BCP), and are available as granules, porous blocks, components of composites (CaP/polymer) cements, and as coatings on orthopedic and dental implants. Experimental calcium phosphate biomaterials include CO3- and F-substituted apatites, Mg-and Zn-substituted β-TCP, calcium phosphate glasses. This paper is a brief review of the different types of CaP biomaterials and their properties such as bioactivity, osteoconductivity, osteoinductivity.

  2. The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases.

    Science.gov (United States)

    Chatton, B; Walter, P; Ebel, J P; Lacroute, F; Fasiolo, F

    1988-01-05

    S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

  3. The effect of glial glutamine synthetase inhibition on recognition and temporal memories in the rat.

    Science.gov (United States)

    Kant, Deepika; Tripathi, Shweta; Qureshi, Munazah F; Tripathi, Shweta; Pandey, Swati; Singh, Gunjan; Kumar, Tankesh; Mir, Fayaz A; Jha, Sushil K

    2014-02-07

    The glutamate neurotransmitter is intrinsically involved in learning and memory. Glial glutamine synthetase enzyme synthesizes glutamine, which helps maintain the optimal neuronal glutamate level. However, the role of glutamine synthetase in learning and memory remains unclear. Using associative trace learning task, we investigated the effects of methionine sulfoximine (MSO) (glutamine synthetase inhibitor) on recognition and temporal memories. MSO and vehicle were injected (i.p.) three hours before training in separate groups of male Wistar rats (n=11). Animals were trained to obtain fruit juice after following a set of sequential events. Initially, house-light was presented for 15s followed by 5s trace interval. Thereafter, juice was given for 20s followed by 20s inter-presentation interval. A total of 75 presentations were made over five sessions during the training and testing periods. The average number of head entries to obtain juice per session and during individual phases at different time intervals was accounted as an outcome measure of recognition and temporal memories. The total head entries in MSO and vehicle treated animals were comparable on training and testing days. However, it was 174.90% (p=0.08), 270.61% (pGlutamine synthetase inhibition did not induce recognition memory deficit, while temporal memory was altered, suggesting that glutamine synthetase modulates some aspects of mnemonic processes.

  4. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

    Science.gov (United States)

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

  5. Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene

    Indian Academy of Sciences (India)

    Jyoti K Jaiswal; Vidyanand Nanjundiah

    2003-12-01

    In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution of GlnRS and AsnRS in eukaryotes.

  6. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  7. Biomediated continuous release phosphate fertilizer

    Science.gov (United States)

    Goldstein, Alan H.; Rogers, Robert D.

    1999-01-01

    A composition is disclosed for providing phosphate fertilizer to the root zone of plants. The composition comprises a microorganism capable of producing and secreting a solubilization agent, a carbon source for providing raw material for the microorganism to convert into the solubilization agent, and rock phosphate ore for providing a source of insoluble phosphate that is solubilized by the solubilization agent and released as soluble phosphate. The composition is provided in a physical form, such as a granule, that retains the microorganism, carbon source, and rock phosphate ore, but permits water and soluble phosphate to diffuse into the soil. A method of using the composition for providing phosphate fertilizer to plants is also disclosed.

  8. Functional interactions between a glutamine synthetase promoter and MYB proteins.

    Science.gov (United States)

    Gómez-Maldonado, Josefa; Avila, Concepción; Torre, Fernando; Cañas, Rafael; Cánovas, Francisco M; Campbell, Malcolm M

    2004-08-01

    In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

  9. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

    Science.gov (United States)

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M R K; Freund, Yvonne R; DeRisi, Joseph; Cusack, Stephen; Rosenthal, Philip J

    2016-08-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

  10. Functional analysis of Leishmania cyclopropane fatty acid synthetase.

    Directory of Open Access Journals (Sweden)

    Samuel O Oyola

    Full Text Available The single gene encoding cyclopropane fatty acid synthetase (CFAS is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular and host (intracellular stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.

  11. Blockade of Glutamine Synthetase Enhances Inflammatory Response in Microglial Cells

    Science.gov (United States)

    Palmieri, Erika M.; Menga, Alessio; Lebrun, Aurore; Hooper, Douglas C.; Butterfield, D. Allan

    2017-01-01

    Abstract Aims: Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia. Results: GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced. Innovation: Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target. Conclusions: Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351–363. PMID:27758118

  12. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  13. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2015-10-20

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  14. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    Science.gov (United States)

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  15. 19-Year Follow-up of A Patient With Severe Glutathione Synthetase Deficiency

    Science.gov (United States)

    Atwal, Paldeep S.; Medina, Casey R.; Burrage, Lindsay C.; Sutton, V. Reid

    2016-01-01

    Glutathione synthetase deficiency is a rare autosomal recessive disorder resulting in low levels of glutathione and an increased susceptibility to oxidative stress. Patients with glutathione synthetase deficiency typically present in the neonatal period with hemolytic anemia, metabolic acidosis and neurological impairment. Lifelong treatment with antioxidants has been recommended in an attempt to prevent morbidity and mortality associated with the disorder. Here we present a 19-year-old female who was diagnosed with glutathione synthetase deficiency shortly after birth and who has been closely followed in our metabolic clinic. Despite an initial severe presentation, she has had normal intellectual development and few complications of her disorder with a treatment regimen that includes polycitra (citric acid, potassium citrate and sodium citrate), vitamin C, vitamin E and selenium. PMID:26984560

  16. Identification of autoantibodies to tyrosil-tRNA synthetase in heart disfunctions

    Directory of Open Access Journals (Sweden)

    Ryabenko D. V.

    2010-09-01

    Full Text Available Aim. To investigate the levels of specific autoantibodies against tyrosyl-tRNA synthetase and its individual modules in the blood serum of people with heart failure caused by dilated cardiomyopathy, myocarditis and ischemic heart disease compared with healthy donors. Methods. Recombinant proteins were obtained using bacterial strains transformed with appropriate plasmid vectors and were purified by chromatography on Ni-NTA-agarose. The levels of specific autoantibodies were investigated by ELISA. Results. The increased levels of autoantibodies specific to tyrosyl-tRNA synthetase, its N-terminal catalytic module and non-catalytic C-module, were found in the blood serum of patients, compared with healthy donors. Conclusions. The results obtained demonstrate the possible role of tyrosyl-tRNA synthetase in adaptive changes of the myocardium in response to stress factors.

  17. Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE.

    Science.gov (United States)

    Jonsson, Anders; Nordlund, Stefan; Teixeira, Pedro Filipe

    2009-10-01

    In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

  18. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced......, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  19. Sequence determination and modeling of structural motifs for the smallest monomeric aminoacyl-tRNA synthetase.

    OpenAIRE

    Hou, Y M; Shiba, K; Mottes, C; Schimmel, P.

    1991-01-01

    Polypeptide chains of 19 previously studied Escherichia coli aminoacyl-tRNA synthetases are as large as 951 amino acids and, depending on the enzyme, have quaternary structures of alpha, alpha 2, alpha 2 beta 2, and alpha 4. These enzymes have been organized into two classes which are defined by sequence motifs that are associated with specific three-dimensional structures. We isolated, cloned, and sequenced the previously uncharacterized gene for E. coli cysteine-tRNA synthetase (EC 6.1.1.16...

  20. Brain and Liver Glutamine Synthetase of Rana catesbeiana and Rana cancrivora.

    Science.gov (United States)

    1983-07-01

    ammonia into urea in marine Chondrichthyes liver 7 Table 1--Liver and brain glutamine synthetase of urea-retaining and non-urea-retaining amphibians 8...for assimilation of ammonia into urea in marine Chondrichthyes liver (from Webb (15)) C1 klA IWE LO, AT? VH3 tLt TKATICAkMUIYL PUOSPULArL I CflALLLINE... elasmobranchii . Biol. Rev. Cambridge Philos. Soc. 11, 49-82. 15. Webb, J.T. 1979. "An assimilatory role of glutamine synthetase in the liver of urea

  1. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D;

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...... in the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that formation...

  2. Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides.

    Science.gov (United States)

    Iamurri, Samantha M; Daugherty, Ashley B; Edmondson, Dale E; Lutz, Stefan

    2013-12-01

    The preparation of flavin mononucleotide (FMN) and FMN analogs from their corresponding riboflavin precursors is traditionally performed in a two-step procedure. After initial enzymatic conversion of riboflavin to flavin adenine dinucleotide (FAD) by a bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. To simplify the protocol, we have engineered the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain. The newly created biocatalyst is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogs to their corresponding FMN cofactors at preparative-scale.

  3. Pyridoxal phosphate-dependent neonatal epileptic encephalopathy.

    Science.gov (United States)

    Bagci, S; Zschocke, J; Hoffmann, G F; Bast, T; Klepper, J; Müller, A; Heep, A; Bartmann, P; Franz, A R

    2008-03-01

    Pyridox(am)ine-5'-phosphate oxidase converts pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate, a cofactor in many metabolic reactions, including neurotransmitter synthesis. A family with a mutation in the pyridox(am)ine-5'-phosphate oxidase gene presenting with neonatal seizures unresponsive to pyridoxine and anticonvulsant treatment but responsive to pyridoxal phosphate is described. Pyridoxal phosphate should be considered in neonatal epileptic encephalopathy unresponsive to pyridoxine.

  4. Pyridoxal phosphate-dependent neonatal epileptic encephalopathy

    OpenAIRE

    2009-01-01

    Pyridox(am)ine-5′-phosphate oxidase converts pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate, a cofactor in many metabolic reactions, including neurotransmitter synthesis. A family with a mutation in the pyridox(am)ine-5′-phosphate oxidase gene presenting with neonatal seizures unresponsive to pyridoxine and anticonvulsant treatment but responsive to pyridoxal phosphate is described. Pyridoxal phosphate should be considered in neonatal epileptic encephalopathy unrespons...

  5. Correlation of exon 3 β-catenin mutations with glutamine synthetase staining patterns in hepatocellular adenoma and hepatocellular carcinoma.

    Science.gov (United States)

    Hale, Gillian; Liu, Xinxin; Hu, Junjie; Xu, Zhong; Che, Li; Solomon, David; Tsokos, Christos; Shafizadeh, Nafis; Chen, Xin; Gill, Ryan; Kakar, Sanjay

    2016-11-01

    The current clinical practice is based on the assumption of strong correlation between diffuse glutamine synthetase expression and β-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data and may represent an oversimplification as glutamine synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse glutamine synthetase patterns, and the association between each pattern and β-catenin mutations is not clearly established. This study examines the correlation between glutamine synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified into one of the three patterns: (a) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate-to-strong staining in 50-90% of lesional cells, without a map-like pattern, and (c) patchy: moderate-to-strong staining in glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 β-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 β-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates >50% in both hepatocellular adenoma and hepatocellular carcinoma. The interpretation of β-catenin activation based on glutamine synthetase staining should be performed with caution, and the undetermined significance of various glutamine synthetase patterns should be highlighted in pathology reports.

  6. Triphenyl phosphate allergy from spectacle frames

    DEFF Research Database (Denmark)

    Carlsen, L; Andersen, K E; Egsgaard, Helge

    1986-01-01

    A case of triphenyl phosphate allergy from spectacle frames is reported. Patch tests with analytical grade triphenyl phosphate, tri-m-cresyl phosphate, and tri-p-cresyl phosphate in the concentrations 5%, 0.5% and 0.05% pet. showed positive reactions to 0.05% triphenyl phosphate and 0.5% tri-m-cr...

  7. Draft Genome Sequences of Five Novel Polyketide Synthetase-Containing Mouse Escherichia coli Strains

    Science.gov (United States)

    Mannion, Anthony; Shen, Zeli; Feng, Yan; Garcia, Alexis

    2016-01-01

    We report herein the draft genomes of five novel Escherichia coli strains isolated from surveillance and experimental mice housed at MIT and the Whitehead Institute and describe their genomic characteristics in context with the polyketide synthetase (PKS)-containing pathogenic E. coli strains NC101, IHE3034, and A192PP.

  8. Glutamine synthetase sequence evolution in the mycobacteria and their use as molecular markers for Actinobacteria speciation

    Directory of Open Access Journals (Sweden)

    Wiid Ian JF

    2009-02-01

    Full Text Available Abstract Background Although the gene encoding for glutamine synthetase (glnA is essential in several organisms, multiple glnA copies have been identified in bacterial genomes such as those of the phylum Actinobacteria, notably the mycobacterial species. Intriguingly, previous reports have shown that only one copy (glnA1 is essential for growth in M. tuberculosis, while the other copies (glnA2, glnA3 and glnA4 are not. Results In this report it is shown that the glnA1 and glnA2 encoded glutamine synthetase sequences were inherited from an Actinobacteria ancestor, while the glnA4 and glnA3 encoded GS sequences were sequentially acquired during Actinobacteria speciation. The glutamine synthetase sequences encoded by glnA4 and glnA3 are undergoing reductive evolution in the mycobacteria, whilst those encoded by glnA1 and glnA2 are more conserved. Conclusion Different selective pressures by the ecological niche that the organisms occupy may influence the sequence evolution of glnA1 and glnA2 and thereby affecting phylogenies based on the protein sequences they encode. The findings in this report may impact the use of similar sequences as molecular markers, as well as shed some light on the evolution of glutamine synthetase in the mycobacteria.

  9. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  10. Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production in Streptomyces coelicolor A3(2)

    OpenAIRE

    D'Alia, Davide; Nieselt, Kay; Steigele, Stephan; Mueller, Jonas; Verburg, Ilse; Takano, Eriko; Alia, Davide D’; Müller, Jonas

    2010-01-01

    Overexpression of antisense chromosomal cis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production in Streptomyces coelicolor. In addition, we predicted 3,597 cis-encoded ncRNAs and validated 13 of them experimentally, including several ncRNAs that are differentially expressed in bacterial hormone-defective mutants.

  11. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA>) in Salmonella typhimurium

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

    1988-01-01

    The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to...

  12. A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

    Directory of Open Access Journals (Sweden)

    Byung Sun Park

    2015-01-01

    Full Text Available Aminoacyl-tRNA synthetases (AminoARSs are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, AminoARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, AminoARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using AminoARSs-specific primers, we screened mRNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 AminoARSs, we found that phenylalanyl-tRNA synthetase beta chain (FARSB, isoleucyl-tRNA synthetase (IARS and methionyl-tRNA synthetase (MARS mRNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.

  13. Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

  14. Effects of univalent cations on the activity of particulate starch synthetase.

    Science.gov (United States)

    Nitsos, R E; Evans, H J

    1969-09-01

    An investigation was made to determine the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCl. Rubidium, cesium, and ammonium were 80% as effective as potassium while sodium and lithium were respectively 21% and 8% as effective as potassium. The K(A) for potassium was determined to be 6 mM. In the case of the particulate starch synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, considerable stimulation of enzyme activity was obtained by the addition of potassium to the reaction mixture. In these studies, low enzyme activity was observed in the absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been negative.

  15. ISOLATION AND CHARACTERIZATION OF THE RAT GENE FOR CARBAMOYLPHOSPHATE SYNTHETASE-I

    NARCIS (Netherlands)

    VANDENHOFF, MJB; VANDEZANDE, LPWGM; DINGEMANSE, MA; DAS, AT; LABRUYERE, W; MOORMAN, AFM; CHARLES, R; LAMERS, WH; Jacobus Mgn Van De Zande, Louis

    1995-01-01

    Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene

  16. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera

    DEFF Research Database (Denmark)

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just;

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have...

  17. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    Science.gov (United States)

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.

  18. EFFECT OF UP-REGULATION OF S-ADOMET SYNTHETASE ON TAXOL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    Chen Lirong; Zheng Shu; Fan Weimin; Zhang Suzhan

    1998-01-01

    Objective:To investigate the gene regulation of taxolinduced apoptosis. Methods: Northern blot hybridization,enzyme activity assay of S-AdoMet synthetase and flow cytometry were performed in the investigation of expression in the Mrna level and biological action of SAdoMet synthetase in taxol-induced apoptosis in human breast cancer cell line (Bcap 37). Results: Up-regulation of S-AdoMet synthetase expression was resulted by taxol treatment and the expression peaked at 48hours. Moreover,the up-regulation of S-AdoMet synthetase was associated with cytotoxicity of antimicrotubule agents including taxol and colchicine.Inhibition rate of S-AdoMet synthetase activity by 1%DMSO was 34% in taxol-treated cells and 14% in taxoluntreated cells compared to control groups, respectively.Posttreatment with 1% DMSO following pretreatment with individual antitumor agent for 3 hrs promoted apoptotic cell death of taxol-,colchicine-,and adriamycintreated Bcap37 cells. Conclusion : The induction of apoptosis enhanced by post-treatment with DMSO in taxol-treated cells is probably linked to its inhibition on enzyme activity of S-AdoMet synthetase ,suggesting that the increased expression of S-AdoMet synthetase possibly plays an important role in protecting cells from DNA fragmentation in taxol-induced apoptosis.

  19. Light weight phosphate cements

    Science.gov (United States)

    Wagh, Arun S.; Natarajan, Ramkumar,; Kahn, David

    2010-03-09

    A sealant having a specific gravity in the range of from about 0.7 to about 1.6 for heavy oil and/or coal bed methane fields is disclosed. The sealant has a binder including an oxide or hydroxide of Al or of Fe and a phosphoric acid solution. The binder may have MgO or an oxide of Fe and/or an acid phosphate. The binder is present from about 20 to about 50% by weight of the sealant with a lightweight additive present in the range of from about 1 to about 10% by weight of said sealant, a filler, and water sufficient to provide chemically bound water present in the range of from about 9 to about 36% by weight of the sealant when set. A porous ceramic is also disclosed.

  20. Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum.

    Science.gov (United States)

    Qiu, Chunhui; Hong, Yang; Cao, Yan; Wang, Fei; Fu, Zhiqiang; Shi, Yaojun; Wei, Meimei; Liu, Shengfa; Lin, Jiaojiao

    2012-12-01

    Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic β-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.μg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

  1. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara (SSRL); (Maryland)

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  2. Crystallo-chemical analyses of calcium phosphates

    Energy Technology Data Exchange (ETDEWEB)

    Sakae, Toshiro; Hayakawa, Tohru; Maruyama, Fumiaki; Nemoto, Kimiya; Kozawa, Yukishige [Nihon Univ., Matsudo, Chiba (Japan). School of Dentistry

    1997-12-01

    Several analytical techniques, methodology and their practical data processing were briefly described to investigate the crystallographic properties of calcium phosphates which are encountered in the field of dental sciences. The applied analytical techniques were X-ray fluorescence spectrometry (XFS), energy dispersive spectrometry (EDS), Fourier transform infrared spectrometry (FT-IR) and X-ray diffraction (XRD). The used materials were tetracalcium phosphate, hydroxyapatite, fluorapatite, {alpha}-tricalcium phosphate, {beta}-tricalcium phosphate, octacalcium phosphate, monetite, brushite and monocalcium phosphate monohydrate. (author)

  3. Triphenyl phosphate allergy from spectacle frames

    DEFF Research Database (Denmark)

    Carlsen, Lars; Andersen, Klaus E.; Egsgaard, Helge

    1986-01-01

    A case of triphenyl phosphate allergy from spectacle frames is reported. Patch tests with analytical grade triphenyl phosphate, tri-m-cresyl phosphate, and tri-p-cresyl phosphate in the concentrations 5%, 0.5% and 0.05% pet. showed positive reactions to 0.05% triphenyl phosphate and 0.5% tri......-m-cresyl phosphate, but no reaction to tri-p-cresyl phosphate. Gas chromatography of the tricresyl phosphate 5% pet. patch test material supplied from Trolab showed that it contained a mixture of a wide range of triaryl phosphates, including 0.08% triphenyl phosphate which is above the threshold for detecting...... triphenyl phosphate allergy in our patient....

  4. Preparation of the multienzyme system gramicidin S-synthetase 2 with an aqueous three-phase system.

    Science.gov (United States)

    Kirchner, A; Simonis, M; von Döhren, H

    1987-06-19

    The distribution of gramicidin S-synthetase activity from disrupted cells suspended in aqueous two- and three-phase systems was investigated. An optimized three-phase system containing 5% dextran, 8% Ficoll, 11% PEG and 6.7% disrupted cells was found to be effective in extracting gramicidin S-synthetase activity. The activity yield achieved was higher in comparison to other preparation methods, and the subsequent purification steps were greatly facilitated. The time needed for the preparation of the labile gramicidin S-synthetase was considerably reduced. The combination of the aqueous phase extraction with chromatographic methods yielded 19 mg gramicidin S-synthetase 2 in essentially pure form from 30 g (wet weight) of cells.

  5. ars1, an Arabidopsis mutant exhibiting increased tolerance to arsenate and increased phosphate uptake.

    Science.gov (United States)

    Lee, David A; Chen, Alice; Schroeder, Julian I

    2003-09-01

    Arsenic is one of the most toxic pollutants at contaminated sites, yet little is known about the mechanisms by which certain plants survive exposure to high arsenic levels. To gain insight into the mechanisms of arsenic tolerance in plants, we developed a genetic screen to isolate Arabidopsis thaliana mutants with altered tolerance to arsenic. We report here on the isolation of a mutant arsenic resisant 1 (ars1) with increased tolerance to arsenate. ars1 germinates and develops under conditions that completely inhibit growth of wild-type plants and shows a semi-dominant arsenic resistance phenotype. ars1 accumulates levels of arsenic similar to that accumulated by wild-type plants, suggesting that ars1 plants have an increased ability to detoxify arsenate. However, ars1 plants produce phytochelatin levels similar to levels produced by the wild type, and the enhanced resistance of ars1 is not abolished by the gamma-glutamylcysteine synthetase inhibitor l-buthionine sulfoxime (BSO). Furthermore, ars1 plants do not show resistance to arsenite or other toxic metals such as cadmium and chromium. However, ars1 plants do show a higher rate of phosphate uptake than that shown by wild-type plants, and wild-type plants grown with an excess of phosphate show increased tolerance to arsenate. Traditional models of arsenate tolerance in plants are based on the suppression of phosphate uptake pathways and consequently on the reduced uptake of arsenate. Our data suggest that arsenate tolerance in ars1 could be due to a new mechanism mediated by increased phosphate uptake in ars1. Models discussing how increased phosphate uptake could contribute to arsenate tolerance are discussed.

  6. Extrapontine myelinolysis resulting in transient cortical blindness.

    Science.gov (United States)

    Langer, Jennifer E; Wilson, William G; Raghavan, Prashant; Rust, Robert S; Goodkin, Howard P

    2010-02-01

    Central pontine myelinolysis and extrapontine myelinolysis are characterized by symmetric demyelination subsequent to rapid shifts in serum osmolality. Described here is a novel case of transient cortical blindness in association with imaging features of extrapontine myelinolysis, which occurred in a child with carbamoyl phosphate synthetase deficiency after rapid correction of hyperammonemia. Serum sodium levels were within normal limits at presentation and throughout the period of ammonia correction. A potential pathogenic mechanism of osmotic demyelination in the setting of acute treatment for hyperammonemia in a patient with a urea cycle abnormality includes disruption of the blood-brain barrier and re-equilibration of organic osmolytes, particularly glutamine.

  7. Recent advances in phosphate biosensors.

    Science.gov (United States)

    Upadhyay, Lata Sheo Bachan; Verma, Nishant

    2015-07-01

    A number of biosensors have been developed for phosphate analysis particularly, concerning its negative impact within the environmental and biological systems. Enzymatic biosensors comprising either a single or multiple enzymatic system have been extensively used for the direct and indirect analysis of phosphate ions. Furthermore, some non-enzymatic biosensors, such as affinity-based biosensors, provide an alternative analytical approach with a higher selectivity. This article reviews the recent advances in the field of biosensor developed for phosphate estimation in clinical and environmental samples, concerning the techniques involved, and the sensitivity toward phosphate ions. The biosensors have been classified and discussed on the basis of the number of enzymes used to develop the analytical system, and a comparative analysis has been performed.

  8. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

    Science.gov (United States)

    Wray, Lewis V; Fisher, Susan H

    2008-04-01

    The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

  9. 21 CFR 573.320 - Diammonium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diammonium phosphate. 573.320 Section 573.320 Food... Additive Listing § 573.320 Diammonium phosphate. The food additive diammonium phosphate may be safely used... crude protein from diammonium phosphate, adequate directions for use and a prominent statement,...

  10. 21 CFR 184.1434 - Magnesium phosphate.

    Science.gov (United States)

    2010-04-01

    ... solution of magnesite with phosphoric acid. (b) Magnesium phosphate, dibasic, meets the specifications of... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Magnesium phosphate. 184.1434 Section 184.1434 Food... Specific Substances Affirmed as GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes...

  11. Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

    Science.gov (United States)

    Huang, Y; Matsunaga, H; Toriba, A; Santa, T; Fukushima, T; Imai, K

    1999-06-01

    A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.

  12. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    Science.gov (United States)

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  13. Acacia senegal and Prosopis chilensis-nodulating rhizobia Sinorhizobium arboris HAMBI 2361 and S. kostiense HAMBI 2362 produce tetra- and pentameric LCOs that are N-methylated, O-6-carbamoylated and partially sulfated.

    Science.gov (United States)

    Nowak, Petri; Soupas, Laura; Thomas-Oates, Jane; Lindström, Kristina

    2004-04-28

    Sinorhizobium arboris and S. kostiense are rhizobia that nodulate the tropical leguminous trees Acacia senegal and Prosopis chilensis. The lipochito-oligosaccharidic signalling molecules (LCOs) of S. arboris HAMBI 2361 and S. kostiense HAMBI 2362 were analyzed by mass spectrometry. The major LCOs produced by the strains were shown to be pentameric, acylated with common fatty acids, N-methylated, O-6-carbamoylated and partially sulfated, as are the LCOs characterized to date for other Acacia-nodulating rhizobia. Besides the major LCOs the two strains produced (i) tetrameric LCOs, (ii) LCOs acylated with fatty acids other than those commonly found, (iii) LCOs with only an acyl substituent and (iv) noncarbamoylated LCOs. Production of LCOs (i) to (iii) are novel among Acacia-nodulating rhizobia. The roles of the different structural characteristics of LCOs in the rhizobium-A. senegal symbiosis are discussed. Specific structural features of the LCOs are proposed to be important in the selection of effective nitrogen-fixing rhizobia by A. senegal.

  14. The Novel [4,5-e][1,3]Diazepine-4,8-dione and Acyclic Carbamoyl Imino-Ureido Derivatives of Imidazole: Synthesis, Anti-Viral and Anti-Tumor Activity Evaluations

    Directory of Open Access Journals (Sweden)

    Karlo Wittine

    2013-10-01

    Full Text Available In the present paper, we report on the synthesis, and in vitro antiviral and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4,8-dione (compounds 9–11 and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13 derivatives. These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM with no concomitant cytotoxic effects on human normal fibroblasts (BJ. Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3 showed a selective cytostatic effect toward cervical carcinoma (HeLa cells (IC50 = 9.5 µM with no apparent cytotoxicity on human normal fibroblasts (BJ. This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.

  15. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

    Science.gov (United States)

    Mirando, Adam C; Fang, Pengfei; Williams, Tamara F; Baldor, Linda C; Howe, Alan K; Ebert, Alicia M; Wilkinson, Barrie; Lounsbury, Karen M; Guo, Min; Francklyn, Christopher S

    2015-08-14

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

  16. Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

    Science.gov (United States)

    Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

    1987-01-01

    The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

  17. Structure of Human Phosphopantothenoylcysteine Synthetase at 2.3 Å Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Manoj, N.; Strauss, E.; Begley, T.P.; Ealick, S.E.

    2010-12-01

    The structure of human phosphopantothenoylcysteine (PPC) synthetase was determined at 2.3 {angstrom} resolution. PPC synthetase is a dimer with identical monomers. Some features of the monomer fold resemble a group of NAD-dependent enzymes, while other features resemble the ribokinase fold. The ATP, phosphopantothenate, and cysteine binding sites were deduced from modeling studies. Highly conserved ATP binding residues include Gly43, Ser61, Gly63, Gly66, Phe230, and Asn258. Highly conserved phosphopantothenate binding residues include Asn59, Ala179, Ala180, and Asp183 from one monomer and Arg55 from the adjacent monomer. The structure predicts a ping pong mechanism with initial formation of an acyladenylate intermediate, followed by release of pyrophosphate and attack by cysteine to form the final products PPC and AMP.

  18. let-65 is cytoplasmic methionyl tRNA synthetase in C. elegans

    Directory of Open Access Journals (Sweden)

    Maha Z. Alriyami

    2014-12-01

    Full Text Available Cytoplasmic methionyl tRNA synthetase (MetRS is one of more than 20 cytoplasmic aminoacyl tRNA synthetase enzymes (ARS. This family of enzymes catalyzes a process fundamental for protein translation. Using a combination of genetic mapping, oligonucleotide array comparative genomic hybridization, and phenotypic correlation, we show that mutations in the essential gene, let-65, reside within the predicted Caenorhabditis elegans homologue of MetRS, which we have named mars-1. We demonstrate that the lethality associated with alleles of let-65 is fully rescued by a transgenic array that spans the mars-1 genomic region. Furthermore, sequence analysis reveals that six let-65 alleles lead to the alteration of highly conserved amino acids.

  19. Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

    Indian Academy of Sciences (India)

    A. Wiest; D. Barchers; M. Eaton; R. Henderson; R. Schnittker; K. Mccluskey

    2013-12-01

    Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.

  20. Natural aminoacyl tRNA synthetase fragment enhances cardiac function after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Margaret E McCormick

    Full Text Available A naturally-occurring fragment of tyrosyl-tRNA synthetase (TyrRS has been shown in higher eukaryotes to 'moonlight' as a pro-angiogenic cytokine in addition to its primary role in protein translation. Pro-angiogenic cytokines have previously been proposed to be promising therapeutic mechanisms for the treatment of myocardial infarction. Here, we show that systemic delivery of the natural fragment of TyRS, mini-TyrRS, improves heart function in mice after myocardial infarction. This improvement is associated with reduced formation of scar tissue, increased angiogenesis of cardiac capillaries, recruitment of c-kitpos cells and proliferation of myocardial fibroblasts. This work demonstrates that mini-TyrRS has beneficial effects on cardiac repair and regeneration and offers support for the notion that elucidation of the ever expanding repertoire of noncanonical functions of aminoacyl tRNA synthetases offers unique opportunities for development of novel therapeutics.

  1. Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

    Science.gov (United States)

    D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  2. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

    Science.gov (United States)

    Pérez-Delgado, Carmen M; García-Calderón, Margarita; Márquez, Antonio J; Betti, Marco

    2015-01-01

    It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.

  3. Glutamine synthetase immunor present in oligodendroglia of regions of the central nervous system

    Science.gov (United States)

    D'Amelio, Fernando; Eng, Lawrence F.; Gibbs, Michael A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  4. Expression of glutamine synthetase and cell proliferation in human idiopathic epiretinal membrane

    OpenAIRE

    Kase, S; Saito, W; Yokoi, M; K. Yoshida; Furudate, N; Muramatsu, M; Saito, A.; Kase, M; Ohno, S

    2006-01-01

    Background/aim: The mechanisms of the cellular origin and cell proliferation in the idiopathic epiretinal membrane (ERM) are unsolved. The aim of this study was to examine the expression of cell cycle related molecules and glutamine synthetase (GS), which is expressed in Müller cells and their processes, in ERM tissues. Methods: The ERMs were surgically removed using pars plana vitrectomy. Formalin fixed, paraffin embedded ERM tissues were analysed by immunohistochemistry with anti-cycli...

  5. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

    OpenAIRE

    Bowles, Tawnya L.; Kim, Randie; Galante, Joseph; Parsons, Colin M.; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J.

    2008-01-01

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is under-expressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers ha...

  6. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

    Directory of Open Access Journals (Sweden)

    Carmen M Pérez-Delgado

    Full Text Available It is well established that the plastidic isoform of glutamine synthetase (GS2 is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1, glutamate dehydrogenase (GDH and asparagine synthetase (ASN was observed in the mutant in correspondence with the diminishment of NH4(+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H-dependent GDH activity.

  7. [Isolation of tyrosyl-tRNA-synthetase from Thermus thermophilus HB-27].

    Science.gov (United States)

    Iaremchuk, A D; Tukalo, M A; Egorova, S P; Konovalenko, A V; Matsuka, G Kh

    1990-01-01

    A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

  8. Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

    OpenAIRE

    Caroline Hoff-Risseti; Felipe Augusto Dörr; Patricia Dayane Carvalho Schaker; Ernani Pinto; Vera Regina Werner; Marli Fatima Fiore

    2013-01-01

    The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene seq...

  9. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    OpenAIRE

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a ...

  10. Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase

    OpenAIRE

    Hamblin, Karleigh; Standley, Daron M; Rogers, Matthew B; Stechmann, Alexandra; Andrew J. Roger; Maytum, Robin; Giezen, Mark van der

    2008-01-01

    The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Althoug...

  11. Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

    Science.gov (United States)

    Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

    2012-06-01

    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

  12. Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase

    OpenAIRE

    Fang, Pengfei; Yu, Xue; Jeong, Seung Jae; Mirando, Adam; Chen, Kaige; Chen, Xin; Kim, Sunghoon; Francklyn, Christopher S.; Guo, Min

    2015-01-01

    The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsit...

  13. Effect of post-silking drought on nitrogen partitioning and gene expression patterns of glutamine synthetase and asparagine synthetase in two maize (Zea mays L.) varieties.

    Science.gov (United States)

    Li, Yajun; Wang, Meiling; Zhang, Fengxia; Xu, Yadong; Chen, Xiaohong; Qin, Xiaoliang; Wen, Xiaoxia

    2016-05-01

    Glutamine synthetase (GS) and asparagine synthetase (AS) are proposed to have important function in plant nitrogen (N) remobilization, but their roles under drought stress are not well defined. In this study, the expression dynamics of GS and AS genes were analyzed in two maize varieties (ZD958 and NH101) in relation to post-silking drought stress induced nitrogen partitioning. ZD958 was a 'stay-green' variety with 5% nitrogen harvest index (NHI) lower than NH101. From silking to maturity, the amount of nitrogen remobilized from ear-leaves in ZD958 was evidently lower than NH101, and post-silking drought stress increased the nitrogen remobilization for both varieties. In ear-leaves, the expression of ZmGln1-3 was enhanced under drought stress. Three AS genes (ZmAS1, ZmAS2 and ZmAS3) were differentially regulated by post-silking drought treatment, of which the expression of ZmAS3 was stimulated at late stage of leaf senescence. In NH101, the expression level of ZmAS3 was markedly higher than that in ZD958. In developing grains, there were no significant differences in expression patterns of GS and AS genes between well water and drought treated plants. Drought stress altered maize N partitioning at the whole-plant level, and the up-regulation of GS and AS genes may contribute to the higher leaf nitrogen remobilization when exposed to drought treatments.

  14. Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue.

    Science.gov (United States)

    Levine, R L

    1983-10-10

    Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

  15. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

    DEFF Research Database (Denmark)

    Elo, Jenni M; Yadavalli, Srujana S; Euro, Liliya

    2012-01-01

    Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochon......Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding...... the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial...... was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial...

  16. Food safety: Structure and expression of the asparagine synthetase gene family of wheat.

    Science.gov (United States)

    Gao, Runhong; Curtis, Tanya Y; Powers, Stephen J; Xu, Hongwei; Huang, Jianhua; Halford, Nigel G

    2016-03-01

    Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat.

  17. Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

    Science.gov (United States)

    Ranade, Ranae M.; Zhang, Zhongsheng; Dranow, David M.; Myers, Janette B.; Choi, Ryan; Nakazawa Hewitt, Steve; Edwards, Thomas E.; Davies, Douglas R.; Lorimer, Donald; Boyle, Stephen M.; Barrett, Lynn K.; Buckner, Frederick S.; Fan, Erkang; Van Voorhis, Wesley C.

    2016-01-01

    We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment. PMID:27500735

  18. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    Science.gov (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development.

  19. (p)ppGpp synthetases regulate the pathogenesis of zoonotic Streptococcus suis.

    Science.gov (United States)

    Zhu, Jiawen; Zhang, Tengfei; Su, Zhipeng; Li, Lu; Wang, Dong; Xiao, Ran; Teng, Muye; Tan, Meifang; Zhou, Rui

    2016-10-01

    (p)ppGpp-mediated stringent response is one of the main adaption mechanism in bacteria, and the ability to adapt to environment is linked to the pathogenesis of bacterial pathogens. In the zoonotic pathogen Streptococcus suis, there are two (p)ppGpp synthetases, RelA and RelQ. To investigate the regulatory functions of (p)ppGpp/(p)ppGpp synthetases on the pathogenesis of S. suis, the phenotypes of the [(p)ppGpp(0)] mutant ΔrelAΔrelQ and its parental strain were compared. Light and electron microscopy observation showed that the mutant strain had a longer chain-length than its parental strain. Disruption of relA and relQ led to decreased adhesive and invasive ability to HEp-2 cells, and increased sensitivity to the blood killing and phagocytosis. Mouse infection experiments showed that the mutant strain was attenuated and easier to be cleaned up in vivo. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expressions of virulence related genes involving in morphology and virulence were down-regulated in the mutant strain. Our study demonstrated that the (p)ppGpp synthetases or (p)ppGpp can regulate the pathogenesis of this important zoonotic pathogen.

  20. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes

    OpenAIRE

    Wang, Yanxin; Watford, Malcolm

    2006-01-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. C...

  1. Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.

    OpenAIRE

    Walsh, J P; Caldwell, K K; Majerus, P W

    1991-01-01

    We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was a...

  2. [Phosphate metabolism and iron deficiency].

    Science.gov (United States)

    Yokoyama, Keitaro

    2016-02-01

    Autosomal dominant hypophosphatemic rickets(ADHR)is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage. Fibroblast growth factor 23(FGF23)is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. Low iron status plays a role in the pathophysiology of ADHR. Iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. It was reported that FGF23 elevation in patients with CKD, who are often iron deficient. In patients with nondialysis-dependent CKD, treatment with ferric citrate hydrate resulted in significant reductions in serum phosphate and FGF23.

  3. Phosphorus release from phosphate rock and iron phosphate by low-molecular-weight organic acids

    Institute of Scientific and Technical Information of China (English)

    XU Ren-kou; ZHU Yong-guan; David Chittleborough

    2004-01-01

    Low-molecular-weight(LMW) organic acids widely exist in soils, particularly in the rhizosphere. A series of batch experiments were carried out to investigate the phosphorus release from rock phosphate and iron phosphate by Iow-molecular-weight organic acids.Results showed that citric acid had the highest capacity to solubilize P from both rock and iron phosphate. P solubilization from rock phosphate and iron phosphate resulted in net proton consumption. P release from rock phosphate was positively correlated with the pKa values. P release from iron phosphate was positively correlated with Fe-organic acid stability constants except for aromatic acids, but was not correlated with PKa. Increase in the concentrations of organic acids enhanced P solubilization from both rock and iron phosphate almost linearrly. Addition of phenolic compounds further increased the P release from iron phosphate. Initial solution pH had much more substantial effect on P release from rock phosphate than from iron phosphate.

  4. Photorelease of phosphates: Mild methods for protecting phosphate derivatives

    Directory of Open Access Journals (Sweden)

    Sanjeewa N. Senadheera

    2014-08-01

    Full Text Available We have developed a new photoremovable protecting group for caging phosphates in the near UV. Diethyl 2-(4-hydroxy-1-naphthyl-2-oxoethyl phosphate (14a quantitatively releases diethyl phosphate upon irradiation in aq MeOH or aq MeCN at 350 nm, with quantum efficiencies ranging from 0.021 to 0.067 depending on the solvent composition. The deprotection reactions originate from the triplet excited state, are robust under ambient conditions and can be carried on to 100% conversion. Similar results were found with diethyl 2-(4-methoxy-1-naphthyl-2-oxoethyl phosphate (14b, although it was significantly less efficient compared with 14a. A key step in the deprotection reaction in aq MeOH is considered to be a Favorskii rearrangement of the naphthyl ketone motif of 14a,b to naphthylacetate esters 25 and 26. Disruption of the ketone-naphthyl ring conjugation significantly shifts the photoproduct absorption away from the effective incident wavelength for decaging of 14, driving the reaction to completion. The Favorskii rearrangement does not occur in aqueous acetonitrile although diethyl phosphate is released. Other substitution patterns on the naphthyl or quinolin-5-yl core, such as the 2,6-naphthyl 10 or 8-benzyloxyquinolin-5-yl 24 platforms, also do not rearrange by aryl migration upon photolysis and, therefore, do not proceed to completion. The 2,6-naphthyl ketone platform instead remains intact whereas the quinolin-5-yl ketone fragments to a much more complex, highly absorbing reaction mixture that competes for the incident light.

  5. Independent transcription of glutamine synthetase (glnA2) and glutamine synthetase adenylyltransferase (glnE) in Mycobacterium bovis and Mycobacterium tuberculosis.

    Science.gov (United States)

    Hotter, Grant S; Mouat, Pania; Collins, Desmond M

    2008-09-01

    Mycobacterium bovis and Mycobacterium tuberculosis possess four glutamine synthetase homologues, two of which, glnA1 and glnA2, are required for virulence and are located on the bacterial chromosome on either side of glutamine synthetase adenylyltransferase (glnE). While glnA1 is encoded on the complementary strand, glnA2 is located 48bp upstream from glnE, raising the possibility that glnA2 and glnE may be co-transcribed. However, previous studies in M. bovis and M. tuberculosis have painted a contradictory picture of the (co)transcriptional status of glnA2 and glnE. Given the importance of the genes at the glnA1-glnE-glnA2 locus, we sought to clarify the transcriptional status of glnA2 and glnE in both M. bovis and M. tuberculosis. Reverse transcription-PCR demonstrated that glnA2 and glnE were independently transcribed in all six M. bovis and M. tuberculosis strains examined. Northern analysis of the glnA2 transcript in M. bovis AF2122/97 and M. tuberculosis H37Rv showed that it was monocistronic. These results predicted the presence of a glnE transcriptional start site in the glnA2-glnE intergenic region. An identical start site was confirmed in M. bovis AF2122/97 and M. tuberculosis H37Rv using 5' rapid amplification of cDNA ends. Typical mycobacterial -10 and -35 sequences are associated with this start site.

  6. Phosphate Recognition in Structural Biology

    NARCIS (Netherlands)

    Hirsch, Anna K.H.; Fischer, Felix R.; Diederich, François

    2007-01-01

    Drug-discovery research in the past decade has seen an increased selection of targets with phosphate recognition sites, such as protein kinases and phosphatases, in the past decade. This review attempts, with the help of database-mining tools, to give an overview of the most important principles in

  7. Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus).

    Science.gov (United States)

    Dalessandro, G; Northcote, D H

    1981-01-01

    Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-(14)C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-(14)C]xylose released all the radioactivity as xylose. β-1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4-β xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[(14)C]xylose and tri-O-methyl-[(14)C]xylose, suggesting a 1→4-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent Km value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg(2+) and Mn(2+) ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.

  8. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

    Directory of Open Access Journals (Sweden)

    Caroline Hoff-Risseti

    Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

  9. The McbB component of microcin B17 synthetase is a zinc metalloprotein.

    Science.gov (United States)

    Zamble, D B; McClure, C P; Penner-Hahn, J E; Walsh, C T

    2000-12-26

    The microcin B17 synthetase converts glycine, serine, and cysteine residues in a polypeptide precursor into oxazoles and thiazoles during the maturation of the Escherichia coli antibiotic Microcin B17. This multimeric enzyme is composed of three subunits (McbB, McbC, and McbD), and it employs both ATP and FMN as cofactors. The McbB subunit was purified as a fusion with the maltose-binding protein (MBP), and metal analysis revealed that this protein binds 0.91+/-0.17 zinc atoms. Upon incubation of MBP-McbB with excess zinc, the stoichiometry increased to two atoms of zinc bound, but metal binding to the second site resulted in a decrease in the heterocyclization activity when MBP-McbB was reconstituted with the other components of the synthetase. Apo-protein was prepared by using p-hydroxymercuriphenylsulfonic acid (PMPS), and loss of the metal caused a severe reduction in enzymatic activity. However, if dithiothreitol was added to the PMPS reactions within a few minutes, enzymatic activity was retained and MBP-McbB could be reconstituted with zinc. Spectroscopic analysis of the cobalt-containing protein and extended X-ray absorption fine structure analysis of the zinc-containing protein both provide evidence for a tetrathiolate coordination sphere. Site-directed mutants of MBP-McbB as well as the synthetase tagged with the calmodulin-binding peptide were constructed. Activity assays and metal analysis were used to determine which of the six cysteines in McbB are metal ligands. These results suggest that the zinc cofactor in McbB plays a structural role.

  10. Primary structure of the succinyl-CoA synthetase of Escherichia coli.

    Science.gov (United States)

    Buck, D; Spencer, M E; Guest, J R

    1985-10-22

    The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes. The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex. The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read-through transcript that extends from the suc promoter to a potential rho-independent terminator at the distal end of sucD. The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes. The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase. The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase. The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme. This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit.

  11. Synthesis of Glu-tRNA(Gln) by engineered and natural aminoacyl-tRNA synthetases.

    Science.gov (United States)

    Rodríguez-Hernández, Annia; Bhaskaran, Hari; Hadd, Andrew; Perona, John J

    2010-08-10

    A protein engineering approach to delineating which distinct elements of homologous tRNA synthetase architectures are responsible for divergent RNA-amino acid pairing specificities is described. Previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) were replaced with the corresponding residues of human glutamyl-tRNA synthetase (GluRS). The engineered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNA(Gln) more than 10(4)-fold more efficiently than GlnRS. Detailed comparison of kinetic parameters between GlnRS S1/L1/L2 and the naturally occurring Methanothermobacter thermautotrophicus GluRS(ND), which is also capable of Glu-tRNA(Gln) synthesis, now shows that both k(cat) and K(m) for glutamate are recapitulated in the engineered enzyme, but that K(m) for tRNA is 200-fold higher. Thus, the simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. We infer that the GlnRS architecture has differentiated to match only cognate amino acid-RNA pairs, and that the substrate selection functions do not operate independently of each other. Design and characterization of four additional hybrids identify further residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites. The robust catalytic function demonstrated in this engineered system offers a novel platform for exploring the stereochemical origins of coding as a property of the ancient Rossmann fold.

  12. Phosphate based oil well cements

    Science.gov (United States)

    Natarajan, Ramkumar

    The main application of the cement in an oil well is to stabilize the steel casing in the borehole and protect it from corrosion. The cement is pumped through the borehole and is pushed upwards through the annulus between the casing and the formation. The cement will be exposed to temperature and pressure gradients of the borehole. Modified Portland cement that is being used presently has several shortcomings for borehole sealant. The setting of the Portland cement in permafrost regions is poor because the water in it will freeze even before the cement sets and because of high porosity and calcium oxide, a major ingredient it gets easily affected by the down hole gases such as carbon dioxide. The concept of phosphate bonded cements was born out of considerable work at Argonne National Laboratory (ANL) on their use in stabilization of radioactive and hazardous wastes. Novel cements were synthesized by an acid base reaction between a metal oxide and acid phosphate solution. The major objective of this research is to develop phosphate based oil well cements. We have used thermodynamics along with solution chemistry principles to select calcined magnesium oxide as candidate metal oxide for temperatures up to 200°F (93.3°C) and alumina for temperatures greater than 200°F (93.3°C). Solution chemistry helped us in selecting mono potassium phosphate as the acid component for temperatures less than 200°F (93.3°C) and phosphoric acid solution greater than 200°F (93.3°C). These phosphate cements have performance superior to common Portland well cements in providing suitable thickening time, better mechanical and physical properties.

  13. Sintering of calcium phosphate bioceramics.

    Science.gov (United States)

    Champion, E

    2013-04-01

    Calcium phosphate ceramics have become of prime importance for biological applications in the field of bone tissue engineering. This paper reviews the sintering behaviour of these bioceramics. Conventional pressureless sintering of hydroxyapatite, Ca10(PO4)6(OH)2, a reference compound, has been extensively studied. Its physico-chemistry is detailed. It can be seen as a competition between two thermally activated phenomena that proceed by solid-state diffusion of matter: densification and grain growth. Usually, the objective is to promote the first and prevent the second. Literature data are analysed from sintering maps (i.e. grain growth vs. densification). Sintering trajectories of hydroxyapatite produced by conventional pressureless sintering and non-conventional techniques, including two-step sintering, liquid phase sintering, hot pressing, hot isostatic pressing, ultrahigh pressure, microwave and spark plasma sintering, are presented. Whatever the sintering technique may be, grain growth occurs mainly during the last step of sintering, when the relative bulk density reaches 95% of the maximum value. Though often considered very advantageous, most assisted sintering techniques do not appear very superior to conventional pressureless sintering. Sintering of tricalcium phosphate or biphasic calcium phosphates is also discussed. The chemical composition of calcium phosphate influences the behaviour. Similarly, ionic substitutions in hydroxyapatite or in tricalcium phosphate create lattice defects that modify the sintering rate. Depending on their nature, they can either accelerate or slow down the sintering rate. The thermal stability of compounds at the sintering temperature must also be taken into account. Controlled atmospheres may be required to prevent thermal decomposition, and flash sintering techniques, which allow consolidation at low temperature, can be helpful.

  14. 21 CFR 184.1301 - Ferric phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ferric phosphate. 184.1301 Section 184.1301 Food... Specific Substances Affirmed as GRAS § 184.1301 Ferric phosphate. (a) Ferric phosphate (ferric orthophosphate, iron (III) phosphate, FePO4·xH2O, CAS Reg. No. 10045-86-0) is an odorless, yellowish-white...

  15. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    Science.gov (United States)

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  16. delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    van der Lende, T.R.; de Kamp, M.; den Berg, M.van; Sjollema, K.; Bovenberg, R.A.L.; Veenhuis, M; Konings, W.N; Driessen, A.J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteme, and L-

  17. δ-(L-α-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    Lende, Ted R. van der; Kamp, Mart van de; Berg, Marco van den; Sjollema, Klaas; Bovenberg, Roel A.L.; Veenhuis, Marten; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-α-aminoadipate, L-cysteine, and L-valine into

  18. Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger.

    Science.gov (United States)

    Sealy-Lewis, H M; Fairhurst, V

    1998-07-01

    Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

  19. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  20. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.

    Science.gov (United States)

    Jeitner, Thomas M; Cooper, Arthur J L

    2014-12-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.

  1. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    Energy Technology Data Exchange (ETDEWEB)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Jeyakanthan, Jeyaraman [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Baba, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Kuramitsu, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Shiro, Yoshitsugu [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Sekar, Kanagaraj, E-mail: sekar@serc.iisc.ernet.in [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012 (India); Yokoyama, Shigeyuki, E-mail: sekar@serc.iisc.ernet.in [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India)

    2007-02-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

  2. Total glutamine synthetase levels in cerebrospinal fluid of Alzheimer's disease patients are unchanged.

    Science.gov (United States)

    Timmer, Nienke M; Herbert, Megan K; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2015-03-01

    Decreased cerebral protein and activity levels of glutamine synthetase (GS) have been reported for Alzheimer's disease (AD) patients. Using a recently established method, we quantified total GS levels in cerebrospinal fluid (CSF) from AD patients and control subjects. Furthermore, we investigated if total GS levels in CSF could differentiate AD from frontotemperal dementia and dementia with Lewy bodies patients. As we found no significantly altered total GS levels in any of the patient groups compared with control subjects, we conclude that levels of total GS in CSF have no diagnostic value for AD, dementia with Lewy bodies, or frontotemperal dementia.

  3. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    Science.gov (United States)

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  4. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

    OpenAIRE

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-01-01

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

  5. Evolution of the 2'-5'-Oligoadenylate Synthetase family in eukaryotes and bacteria

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Poulsen, Jesper Buchhave; Reitamm, Tonu

    2009-01-01

    The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon...... may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may...

  6. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    Science.gov (United States)

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  7. Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

    1989-01-01

    Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine...... represented a primary stimulus in all subjects. First, basal 2',5'A activity increased severalfold in response to yellow fever vaccination. In IDDM subjects, this increase was significantly lower (P = .025). Second, the 2',5'A activity increased proportionately to the higher basal 2',5'A activity in IDDM...

  8. 21 CFR 582.5217 - Calcium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phosphate. 582.5217 Section 582.5217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  9. 21 CFR 182.8217 - Calcium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium phosphate. 182.8217 Section 182.8217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic). (b) Conditions of use. This...

  10. 40 CFR 721.5995 - Polyalkyl phosphate.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Polyalkyl phosphate. 721.5995 Section... Substances § 721.5995 Polyalkyl phosphate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a polyalkyl phosphate (PMN P-95-1772)...

  11. 21 CFR 582.6285 - Dipotassium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Dipotassium phosphate. 582.6285 Section 582.6285 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Dipotassium phosphate. (a) Product. Dipotassium phosphate. (b) Conditions of use. This substance is...

  12. 21 CFR 582.1141 - Ammonium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ammonium phosphate. 582.1141 Section 582.1141 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1141 Ammonium phosphate. (a) Product. Ammonium phosphate (mono- and dibasic). (b)...

  13. 21 CFR 582.1217 - Calcium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  14. 21 CFR 582.6290 - Disodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Disodium phosphate. 582.6290 Section 582.6290 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Disodium phosphate. (a) Product. Disodium phosphate. (b) Conditions of use. This substance is...

  15. 21 CFR 182.1217 - Calcium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  16. 21 CFR 582.5434 - Magnesium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Magnesium phosphate. 582.5434 Section 582.5434 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5434 Magnesium phosphate. (a) Product. Magnesium phosphate (di- and tribasic)....

  17. 21 CFR 182.6285 - Dipotassium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dipotassium phosphate. 182.6285 Section 182.6285 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Dipotassium phosphate. (a) Product. Dipotassium phosphate. (b) Conditions of use. This substance is...

  18. 21 CFR 182.6290 - Disodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Disodium phosphate. 182.6290 Section 182.6290 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... phosphate. (a) Product. Disodium phosphate. (b) Conditions of use. This substance is generally recognized...

  19. 21 CFR 182.1778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium phosphate. 182.1778 Section 182.1778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1778 Sodium phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic)....

  20. 21 CFR 582.5301 - Ferric phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ferric phosphate. 582.5301 Section 582.5301 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5301 Ferric phosphate. (a) Product. Ferric phosphate. (b) Conditions of use....

  1. 21 CFR 182.8778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium phosphate. 182.8778 Section 182.8778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic). (b) Conditions of use. This...

  2. 21 CFR 582.6778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium phosphate. 582.6778 Section 582.6778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic). (b) Conditions of use....

  3. 21 CFR 582.1778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium phosphate. 582.1778 Section 582.1778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1778 Sodium phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic)....

  4. 21 CFR 582.5778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium phosphate. 582.5778 Section 582.5778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5778 Sodium phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic)....

  5. 21 CFR 182.6778 - Sodium phosphate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium phosphate. 182.6778 Section 182.6778 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... phosphate. (a) Product. Sodium phosphate (mono-, di-, and tribasic). (b) Conditions of use. This...

  6. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  7. Calcium phosphate cements: study of the beta-tricalcium phosphate--monocalcium phosphate system.

    Science.gov (United States)

    Mirtchi, A A; Lemaitre, J; Terao, N

    1989-09-01

    The possibility of making cements based on beta-tricalcium phosphate (beta-TCP), a promising bone graft material, was investigated. Upon admixture with water, beta-TCP/monocalcium phosphate monohydrate (MCPM) mixtures were found to set and harden like conventional hydraulic cements. Beta-TCP powders with larger particle size, obtained by sintering at higher temperatures, increased the ultimate strength of the cement. Results show that setting occurs after dissolution of MCPM, as a result of the precipitation of dicalcium phosphate dihydrate (DCPD) in the paste. The ultimate tensile strength of the hardened cement is proportional to the amount of DCPD formed. Upon ageing above 40 degrees C, DCPD transforms progressively into anhydrous dicalcium phosphate (DCP), thereby decreasing the strength. Ageing of the pastes in 100% r.h. results in a decay of the mechanical properties. This can be ascribed to an intergranular dissolution of the beta-TCP aggregates as a result of the pH lowering brought about by the MCPM to DCPD conversion.

  8. N-acetylglutamate synthetase deficiency: diagnosis, management and follow-up of a rare disorder of ammonia detoxication.

    Science.gov (United States)

    Schubiger, G; Bachmann, C; Barben, P; Colombo, J P; Tönz, O; Schüpbach, D

    1991-03-01

    We report the 9-year follow-up of a patient suffering from N-acetylglutamate synthetase deficiency, an urea cycle disorder leading to severe neonatal hyperammonaemia. Hitherto two patients from two families with this inborn error of metabolism had been observed. Our management consisted mainly of a protein-restricted diet and oral treatment with N-carbamylglutamate, an activator of carbamylphosphate synthetase, together with arginine or citrulline. The somatic development was normal whereas a moderate psychomotor retardation was diagnosed. The patient died after an episode of coma and prolonged generalized convulsions at the age of 9.5 years.

  9. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    Science.gov (United States)

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  10. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Aoki, Takashi [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  11. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  12. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Marta Spodenkiewicz

    2016-10-01

    Full Text Available Glutamine synthetase (GS is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  13. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.

    Science.gov (United States)

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-10-19

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  14. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

    Directory of Open Access Journals (Sweden)

    Ana Rita Seabra

    2015-07-01

    Full Text Available Glutamine Synthetase (GS catalyses the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of glutamine synthetase isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  15. Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus.

    Science.gov (United States)

    Shiyan, Anna; Thompson, Melanie; Köcher, Saskia; Tausendschön, Michaela; Santos, Helena; Hänelt, Inga; Müller, Volker

    2014-01-01

    Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

  16. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles.

    Science.gov (United States)

    Piel, Jörn

    2002-10-29

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges.

  17. Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli.

    Science.gov (United States)

    Jayalakshmi, R; Sumathy, K; Balaram, Hemalatha

    2002-06-01

    Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.

  18. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion.

    Science.gov (United States)

    Szabó, C; Dawson, V L

    1998-07-01

    Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.

  19. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.

    Science.gov (United States)

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; van Voorhis, Wesley C; Hol, Wim G J

    2010-03-26

    Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

  20. Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: properties of acetohydroxy acid synthetase.

    Science.gov (United States)

    McDonald, R A; Satyanarayana, T; Kaplan, J G

    1973-04-01

    The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.

  1. Comparison of histidine recognition in human and trypanosomatid histidyl-tRNA synthetases.

    Science.gov (United States)

    Koh, Cho Yeow; Wetzel, Allan B; de van der Schueren, Will J; Hol, Wim G J

    2014-11-01

    As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS.

  2. H2S synthetase AtD-CDes involves in ethylene and drought regulated stomatal movement

    Institute of Scientific and Technical Information of China (English)

    Lixia Hou; Dan Zhu; Qian Ma; Dandan Zhang; Xin Liu

    2016-01-01

    The endogenous hydrogen sulfide (H2S),as a new gasotransmitter,participates in many plant physiological processes.In this study,we found that both ethylene and drought strongly induced H2S synthetase AtD-CDes gene expression and enzymatic activity.H2S synthesis inhibitors restrained the ethylene and drought induced stomatal closure in Arabidopsis.The H2S synthetase mutant Atd-cdes was insensitive to ethylene and drought,and overexpression of AtD-CDes conferred the transgenic plants more sensitive to ethylene and drought.Sequence analysis of AtD-CDes promoter showed that it contained ethylene response cis element ERE,and abiotic stress responsive cis elements MBS,LTR,and ABRE.The AtDCDes promoter fused with GUS was transformed into Arabidopsis thaliana to get AtD-CDes promoter::GUS transgenic plants.When treated with ethylene and drought stress,the enzymatic activity of β-glucuronidase was higher in the leaves and stomata of transgenic Arabidopsis.Analyses of GUS activity from the transgenic plants harboring different fragments of the promoter shows that the key section of AtD-CDes promoter response to ethylene was from the-697 to-408 bp,and the key section response to drought stress was from-90 to-1 bp.These results suggested that the H2S produced from AtD-CDes may mediate ethylene and drought-induced stomatal movement.

  3. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

    Science.gov (United States)

    Steinchen, Wieland; Schuhmacher, Jan S; Altegoer, Florian; Fage, Christopher D; Srinivasan, Vasundara; Linne, Uwe; Marahiel, Mohamed A; Bange, Gert

    2015-10-27

    Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

  4. Calcium phosphate polymer hybrid materials

    OpenAIRE

    2011-01-01

    Calcium phosphate (CaP) is of strong interest to the medical field because of its potential for bone repair, gene transfection, etc.1-3 Nowadays, the majority of the commercially available materials are fabricated via “classical” materials science approaches, i.e. via high temperature or high pressure approaches, from rather poorly defined slurries, or from organic solvents.3,4 Precipitation of inorganics with (polymeric) additives from aqueous solution on the other hand enables the synthesis...

  5. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix.

    Science.gov (United States)

    Yoshimura, Yukihiro; Araki, Aya; Maruta, Hitomi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-12-21

    Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

  6. Nitrogen Control in Pseudomonas aeruginosa : Mutants Affected in the Synthesis of Glutamine Synthetase, Urease, and NADP-Dependent Glutamate Dehydrogenase

    NARCIS (Netherlands)

    Janssen, Dick B.; Habets, Winand J.A.; Marugg, Joey T.; Drift, Chris van der

    1982-01-01

    Mutants were isolated from Pseudomonas aeruginosa that were impaired in the utilization of a number of nitrogen sources. In contrast to the wild-type strain, these mutants appeared to be unable to derepress the formation of glutamine synthetase and urease under nitrogen-limited growth conditions, wh

  7. Purification and Properties of a Prokaryote Type Glutamine Synthetase from the Bialaphos Producer Streptomyces hygroscopicus SF1293

    NARCIS (Netherlands)

    Kumada, Yoichi; Takano, Eriko; Nagaoka, Kozo

    1990-01-01

    A prokaryote type glutamine synthetase (GS) was purified from a bialaphos (BA)-producing organism, Streptomyces hygroscopicus SF1293 (SF1293). The GS (GS I) consisted of a 55,000 dalton subunit, and its N-terminal amino acid sequence was similar to that of S. coelicolor GS. GS I was highly sensitive

  8. Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production in Streptomyces coelicolor A3(2)▿ ‡

    OpenAIRE

    D'Alia, Davide; Nieselt, Kay; Steigele, Stephan; Müller, Jonas; Verburg, Ilse; Takano, Eriko

    2009-01-01

    Overexpression of antisense chromosomal cis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production in Streptomyces coelicolor. In addition, we predicted 3,597 cis-encoded ncRNAs and validated 13 of them experimentally, including several ncRNAs that are differentially expressed in bacterial hormone-defective mutants.

  9. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  10. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

    1988-01-01

    in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional...... significance....

  11. The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis.

    Science.gov (United States)

    Oves-Costales, Daniel; Kadi, Nadia; Challis, Gregory L

    2009-11-21

    Siderophores are high-affinity ferric iron chelators biosynthesised and excreted by most microorganisms that play an important role in iron acquisition. Siderophore-mediated scavenging of ferric iron from hosts contributes significantly to the virulence of pathogenic microbes. As a consequence siderophore biosynthesis is an attractive target for chemotherapeutic intervention. Two main pathways for siderophore biosynthesis exist in microbes. One pathway involves nonribosomal peptide synthetase (NRPS) multienzymes while the other is NRPS-independent. The enzymology of NRPS-mediated siderophore biosynthesis has been extensively studied for more than a decade. In contrast, the enzymology of NRPS-independent siderophore (NIS) biosynthesis was overlooked for almost thirty years since the first genetic characterisation of the NIS biosynthetic pathway to aerobactin. However, the past three years have witnessed an explosion of interest in the enzymology of NIS synthetases, the key enzymes in the assembly of siderophores via the NIS pathway. The biochemical characterisation of ten purified recombinant synthetases has been reported since 2007, along with the first structural characterisation of a synthetase by X-ray crystallography in 2009. In this feature article we summarise the recent progress that has been made in understanding the long-overlooked enzymology of NRPS-independent siderophore biosynthesis, highlight important remaining questions, and suggest likely directions for future research.

  12. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.;

    2006-01-01

    an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored...

  13. Novel expression pattern of cytosolic gln synthetase in nitrogen-fixing root nodules of the actinorhizal host, Datisca glomerata

    NARCIS (Netherlands)

    Berry, A.M.; Murphy, T.M.; Okubara, P.A.; Jacobsen, K.R.; Swensen, S.M.; Pawlowski, K.

    2004-01-01

    Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform, DgGS1

  14. Cloning, molecular characterization, and phylogeny of two evolutionary distinct glutamine synthetase isoforms in the green microalga Haematococcus pluvialis (Chlorophyceae)

    NARCIS (Netherlands)

    Reinecke, Diana L.; Zarka, Aliza; Leu, Stefan; Boussiba, Sammy

    2016-01-01

    Haematococcus pluvialis (Chlorophyta) is a widely used microalga of great economic potential, yet its molecular genetics and evolution are largely unknown. We present new detailed molecular and phylogenetic analysis of two glutamine synthetase (GS) enzymes and genes (gln) under the Astaxanthin-induc

  15. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat ( Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Bernard, Stéphanie M; Møller, Anders Laurell Blom; Dionisio, Giuseppe

    2008-01-01

    We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...

  16. Phosphate: are we squandering a scarce commodity?

    Science.gov (United States)

    Ferro, Charles J; Ritz, Eberhard; Townend, Jonathan N

    2015-02-01

    Phosphorus is an essential element for life but is a rare element in the universe. On Earth, it occurs mostly in the form of phosphates that are widespread but predominantly at very low concentration. This relative rarity has resulted in a survival advantage, in evolutionary terms, to organisms that conserve phosphate. When phosphate is made available in excess it becomes a cause for disease, perhaps best recognized as a potential cardiovascular and renal risk factor. As a reaction to the emerging public health issue caused by phosphate additives to food items, there have been calls for a public education programme and regulation to bring about a reduction of phosphate additives to food. During the Paleoproterzoic era, an increase in the bioavailability of phosphate is thought to have contributed significantly to the oxygenation of our atmosphere and a dramatic increase in the evolution of new species. Currently, phosphate is used poorly and often wasted with phosphate fertilizers washing this scarce commodity into water bodies causing eutrophication and algal blooms. Ironically, this is leading to the extinction of hundreds of species. The unchecked exploitation of phosphate rock, which is an increasingly rare natural resource, and our dependence on it for agriculture may lead to a strange situation in which phosphate might become a commodity to be fought over whilst at the same time, health and environmental experts are likely to recommend reductions in its use.

  17. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    Science.gov (United States)

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  18. Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.

    2013-07-01

    Full Text Available In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA rather than usual 4 or 5 nucleotides (type 1 tRNA. In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong

  19. Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2 defects predicts differential effects on aminoacylation

    Directory of Open Access Journals (Sweden)

    Liliya eEuro

    2015-02-01

    Full Text Available The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19 is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations.The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change p.R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

  20. Protein: MPA5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA5 Pyrimidine biosynthesis Tc00.1047053511647.4 Glutamine-dependent carbamoyl-pho...sphate synthetase, putative 353153 Trypanosoma cruzi (strain CL Brener) 3540844 Q4D6X8 2209850 ...

  1. Can Phosphate Salts Recovered from Manure Replace Conventional Phosphate Fertilizer?

    Directory of Open Access Journals (Sweden)

    Andrea Ehmann

    2017-01-01

    Full Text Available Pig farming produces more manure than can reasonably be spread onto surrounding fields, particularly in regions with high livestock densities and limited land availability. Nutrient recycling offers an attractive solution for dealing with manure excesses and is one main objective of the European commission-funded project “BioEcoSIM”. Phosphate salts (“P-Salt” were recovered from the separated liquid manure fraction. The solid fraction was dried and carbonized to biochar. This study compared the fertilizing performance of P-Salt and conventional phosphate fertilizer and determined whether additional biochar application further increased biomass yields. The fertilizers and biochar were tested in pot experiments with spring barley and faba beans using two nutrient-poor soils. The crops were fertilized with P-Salt at three levels and biochar in two concentrations. Biomass yield was determined after six weeks. Plant and soil samples were analysed for nitrogen, phosphorus and potassium contents. The P-Salt had similar or even better effects than mineral fertilizer on growth in both crops and soils. Slow release of nutrients can prevent leaching, rendering P-Salt a particularly suitable fertilizer for light sandy soils. Biochar can enhance its fertilizing effect, but the underlying mechanisms need further investigation. These novel products are concluded to be promising candidates for efficient fertilization strategies.

  2. Insight into biological phosphate recovery from sewage.

    Science.gov (United States)

    Ye, Yuanyao; Ngo, Huu Hao; Guo, Wenshan; Liu, Yiwen; Zhang, Xinbo; Guo, Jianbo; Ni, Bing-Jie; Chang, Soon Woong; Nguyen, Dinh Duc

    2016-10-01

    The world's increasing population means that more food production is required. A more sustainable supply of fertilizers mainly consisting of phosphate is needed. Due to the rising consumption of scarce resources and limited natural supply of phosphate, the recovery of phosphate and their re-use has potentially high market value. Sewage has high potential to recover a large amount of phosphate in a circular economy approach. This paper focuses on utilization of biological process integrated with various subsequent processes to concentrate and recycle phosphate which are derived from liquid and sludge phases. The phosphate accumulation and recovery are discussed in terms of mechanism and governing parameters, recovery efficiency, application at plant-scale and economy.

  3. Tetracalcium phosphate: Synthesis, properties and biomedical applications.

    Science.gov (United States)

    Moseke, C; Gbureck, U

    2010-10-01

    Monoclinic tetracalcium phosphate (TTCP, Ca(4)(PO(4))(2)O), also known by the mineral name hilgenstockite, is formed in the (CaO-P(2)O(5)) system at temperatures>1300 degrees C. TTCP is the only calcium phosphate with a Ca/P ratio greater than hydroxyapatite (HA). It appears as a by-product in plasma-sprayed HA coatings and shows moderate reactivity and concurrent solubility when combined with acidic calcium phosphates such as dicalcium phosphate anhydrous (DCPA, monetite) or dicalcium phosphate dihydrate (DCPD, brushite). Therefore it is widely used in self-setting calcium phosphate bone cements, which form HA under physiological conditions. This paper aims to review the synthesis and properties of TTCP in biomaterials applications such as cements, sintered ceramics and coatings on implant metals.

  4. Application of Calcium Phosphate Materials in Dentistry

    Directory of Open Access Journals (Sweden)

    Jabr S. Al-Sanabani

    2013-01-01

    Full Text Available Calcium phosphate materials are similar to bone in composition and in having bioactive and osteoconductive properties. Calcium phosphate materials in different forms, as cements, composites, and coatings, are used in many medical and dental applications. This paper reviews the applications of these materials in dentistry. It presents a brief history, dental applications, and methods for improving their mechanical properties. Notable research is highlighted regarding (1 application of calcium phosphate into various fields in dentistry; (2 improving mechanical properties of calcium phosphate; (3 biomimetic process and functionally graded materials. This paper deals with most common types of the calcium phosphate materials such as hydroxyapatite and tricalcium phosphate which are currently used in dental and medical fields.

  5. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.;

    1994-01-01

    Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically...... in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine...

  6. Covalent aspartylation of aspartyl-tRNA synthetase from Bakers' yeast by its cognat aspartyl adenylate: identification of the labeled residues

    Energy Technology Data Exchange (ETDEWEB)

    Mejdoub, H.; Kern, D.; Giege, R.; Ebel, J.P.; Boulanger, Y.; Reinbolt, J.

    1987-04-07

    Aspartyl-tRNA synthetase from bakers' yeast gives an unstable complex with the cognate adenylate, which reacts after dissociation with amino acid side chains of the protein. This leads to a covalent incorporation of (/sup 14/C)-aspartic acid into aspartyl-tRNA synthetase via amide or ester bonds formed between the ..cap alpha..-carboxyl group of activated aspartic acid and accessible lysines, serines, and threonines. This property is used to label the peptides at the surface of the enzyme. The main labeled residues have been identified, and their location in the primary structure is discussed in relation to structural properties of aspartyl-tRNA synthetase.

  7. Matrix Extracellular Phosphoglycoprotein Inhibits Phosphate Transport

    OpenAIRE

    Marks, J; Churchill, L J; Debnam, E. S.; Unwin, R J

    2008-01-01

    The role of putative humoral factors, known as phosphatonins, in phosphate homeostasis and the relationship between phosphate handling by the kidney and gastrointestinal tract are incompletely understood. Matrix extracellular phosphoglycoprotein (MEPE), one of several candidate phosphatonins, promotes phosphaturia, but whether it also affects intestinal phosphate absorption is unknown. Here, using the in situ intestinal loop technique, we demonstrated that short-term infusion of MEPE inhibits...

  8. Cytosolic glutamine synthetase: a target for improvement of crop nitrogen use efficiency?

    Science.gov (United States)

    Thomsen, Hanne C; Eriksson, Dennis; Møller, Inge S; Schjoerring, Jan K

    2014-10-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude that GS1 activity may be downregulated via a chain of processes elicited by metabolic imbalances and environmental constraints. We suggest that a pivotal role of GS1 may be related to the maintenance of essential nitrogen (N) flows and internal N sensing during critical stages of plant development. A number of more refined overexpression strategies exploiting gene stacking combined with tissue and cell specific targeting to overcome metabolic bottlenecks are considered along with their potential in relation to new N management strategies.

  9. Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels.

    Science.gov (United States)

    Palmieri, Erika Mariana; Spera, Iolanda; Menga, Alessio; Infantino, Vittoria; Iacobazzi, Vito; Castegna, Alessandra

    2014-12-20

    The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.

  10. p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates.

    Science.gov (United States)

    Nguyen, Thang Van; Li, Jing; Lu, Chin-Chun Jean; Mamrosh, Jennifer L; Lu, Gang; Cathers, Brian E; Deshaies, Raymond J

    2017-03-20

    Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4(CRBN) and p97 pathways.

  11. Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Sherry L. Mowbray

    2014-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  12. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

    DEFF Research Database (Denmark)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne

    2014-01-01

    of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine......Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains...... but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia....

  13. Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases.

    Science.gov (United States)

    Bloudoff, Kristjan; Alonzo, Diego A; Schmeing, T Martin

    2016-03-17

    Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

  14. Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

    Directory of Open Access Journals (Sweden)

    Marco Betti

    2012-06-01

    Full Text Available Glutamine synthetase (GS is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1 or plastidic (GS2 isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

  15. Prokaryotic Expression and Preparation of Polyantibody of Human Histydyl-tRNA Synthetase Related Gene

    Institute of Scientific and Technical Information of China (English)

    孟宪芳; 施静; 刘晓春; 陈金中

    2004-01-01

    The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryoticexpression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. Afterpurified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.

  16. Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions.

    Science.gov (United States)

    Harden, Bradley J; Frueh, Dominique P

    2017-01-24

    Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.

  17. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly;

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles......, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA(Glu) was able to protect GluRS1 against oxidative...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

  18. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    Science.gov (United States)

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  19. Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase

    Science.gov (United States)

    Fang, Pengfei; Yu, Xue; Jeong, Seung Jae; Mirando, Adam; Chen, Kaige; Chen, Xin; Kim, Sunghoon; Francklyn, Christopher S.; Guo, Min

    2015-01-01

    The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth ‘orthogonal’ subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes. PMID:25824639

  20. Plant growth is influenced by glutamine synthetase-catalyzed nitrogen metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Langston-Unkefer, P.J.

    1991-06-11

    Ammonia assimilation has been implicated as participating in regulation of nitrogen fixation in free-living bacteria. In fact, these simple organisms utilize an integrated regulation of carbon and nitrogen metabolism; we except to observe an integration of nitrogen and carbon fixation in plants; how could these complex systems grow efficiently and compete in the ecosystem without coordinating these two crucial activities We have been investigating the role of ammonia assimilation regulating the complex symbiotic nitrogen fixation of legumes. Just as is observed in the simple bacterial systems, perturbation of ammonia assimilation in legumes results in increased overall nitrogen fixation. The perturbed plants have increased growth and total nitrogen fixation capability. Because we have targeted the first enyzme in ammonia assimilation, glutamine synthetase, this provides a marker that could be used to assist selection or screening for increased biomass yield. 45 refs., 4 tabs.

  1. PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carter, Andrew T.; Beiche, Flora; Hove-Jensen, Bjarne;

    1997-01-01

    In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS......) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has...... a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed...

  2. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J;

    2012-01-01

    that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS...... with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate......)-triphosphatase 205, thiamine monophosphate kinase 179, pyruvate formate lyase family activating protein 298, 3-hydroxy-3-methylglutaryl-CoA reductase (mevanolate), N(2), N(2)-dimethylguanosine tRNA methyltransferase 145, N2, N2-dimethylguanosine tRNA methyltransferase 170, putative 5-methylcytosine restriction...

  3. Regulation of glutamine synthetase activity by adenylylation in the Gram-positive bacterium Streptomyces cattleya.

    Science.gov (United States)

    Streicher, S L; Tyler, B

    1981-01-01

    The enzymatic activity of glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from the Gram-positive bacterium Streptomyces cattleya is regulated by covalent modification. In whole cells containing high levels of GS the addition of ammonium chloride leads to a rapid decline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels of GS activity as measured by biosynthetic and gamma-glutamyltransferase assays. Incubation of the crude extracts with snake venom phosphodiesterase restored GS activity. In cell extracts, GS was also inactivated by an ATP- and glutamine-dependent reaction. Radioactive labeling studies demonstrated the incorporation of an AmP moiety into GS protein upon modification. Our results suggest a covalent modification of GS in a Gram-positive bacterium. This modification appears to be adenylylation of the GS subunit similar to that found in the Gram-negative bacteria.

  4. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ute Hentschel

    2012-05-01

    Full Text Available Genomic mining revealed one major nonribosomal peptide synthetase (NRPS phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.

  5. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

    Science.gov (United States)

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just; Kelve, Merike; Uriz, Maria Jesus

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

  6. Mineral induced formation of sugar phosphates

    Science.gov (United States)

    Pitsch, S.; Eschenmoser, A.; Gedulin, B.; Hui, S.; Arrhenius, G.

    1995-01-01

    Glycolaldehyde phosphate, sorbed from highly dilute, weakly alkaline solution into the interlayer of common expanding sheet structure metal hydroxide minerals, condenses extensively to racemic aldotetrose-2, 4-diphophates, and aldohexose-2, 4, 6-triphosphates. The reaction proceeds mainly through racemic erythrose-2, 4-phosphate, and terminates with a large fraction of racemic altrose-2, 4, 6-phosphate. In the absence of an inductive mineral phase, no detectable homogeneous reaction takes place in the concentration- and pH range used. The reactant glycolaldehyde phosphate is practically completely sorbed within an hour from solutions with concentrations as low as 50 micron; the half-time for conversion to hexose phosphates is of the order of two days at room temperature and pH 9.5. Total production of sugar phosphates in the mineral interlayer is largely independent of the glycolaldehyde phosphate concentration in the external solution, but is determined by the total amount of GAP offered for sorption up to the capacity of the mineral. In the presence of equimolar amounts of rac-glyceraldehyde-2-phosphate, but under otherwise similar conditions, aldopentose-2, 4, -diphosphates also form, but only as a small fraction of the hexose-2, 4, 6-phosphates.

  7. Preparation of porous lanthanum phosphate with templates

    Energy Technology Data Exchange (ETDEWEB)

    Onoda, Hiroaki, E-mail: onoda@kpu.ac.jp [Department of Informatics and Environmental Sciences, Faculty of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Shimogamo Nakaragi-cyo, Sakyo-ku, Kyoto 606-8522 (Japan); Ishima, Yuya [Department of Applied Chemistry, Faculty of Life Sciences, Ritsumeikan University, 1-1-1, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Takenaka, Atsushi [Department of Materials Science, Yonago National College of Technology, 4448, Hikona-cho, Yonago, Tottori 683-8502 (Japan); Tanaka, Isao [Department of Materials Science and Engineering, Faculty of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501 (Japan)

    2009-08-05

    Malonic acid, propionic acid, glycine, n-butylamine, and urea were added to the preparation of lanthanum phosphate from lanthanum nitrate and phosphoric acid solutions. All additives were taken into lanthanum phosphate particles. The additives that have a basic site were easy to contain in precipitates. The addition of templates improved the specific surface area of lanthanum phosphate. The amount of pore, with radius smaller than 4 nm, increased with the addition of templates. The remained additives had influence on the acidic properties of lanthanum phosphate.

  8. Immunotherapeutic potential of N-formylated peptides of ESAT-6 and glutamine synthetase in experimental tuberculosis.

    Science.gov (United States)

    Mir, Shabir Ahmad; Sharma, Sadhna

    2014-02-01

    Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host anti mycobacterial innate and/or adaptive immune response have been supposed to be useful in the treatment of tuberculosis. Since N-formyl peptides are products of bacterial metabolism, and their binding to a specific phagocyte receptor (FPR) induces chemotaxis and activation of phagocytes that are critical effectors in our innate immune system, it is reasonable to assume that the interaction between these two counterparts (i.e. formylated peptides and FPR) is also important in host defence against M. tuberculosis. In the present study the direct immunotherapeutic potential of N-formylated peptides of two non-classically secreted proteins (early secreted antigenic target-6 and glutamine synthetase) of M. tuberculosis H37Rv was evaluated. Treatment of M. tuberculosis H37Rv infected mice with N-formylated peptides of early secreted antigenic target-6 (ESAT-6) and glutamine synthetase (GS) markedly reduced the bacilli load in their lungs (p < 0.001) and spleen (p < 0.01) as compared to the untreated mice. In addition, the histopathological changes were observed to be in correlation with the CFU data with minor areas of consolidation in the lung sections of N-formylated peptide treated infected mice as compared to those of the untreated mice. Further, these N-formylated peptides were able to confer an additional therapeutic effect when given in combination with the anti tuberculosis drugs and hence can be used as an adjunct to the conventional chemotherapy against tuberculosis.

  9. Proximal Tubule Glutamine Synthetase Expression is Necessary for the Normal Response to Dietary Protein Restriction.

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Verlander, Jill W; Weiner, I David

    2017-03-22

    Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and ¬decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of NH4+ and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The current study's purpose was to determine proximal tubule GS's role in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule glutamine synthetase expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

  10. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.

    2011-12-01

    Full Text Available Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa. Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates

  11. Succinyl-CoA Synthetase: New Antigen Candidate of Bartonella bacilliformis

    Science.gov (United States)

    Gomes, Cláudia; Palma, Noemí; Pons, Maria J.; Magallón-Tejada, Ariel; Sandoval, Isabel; Tinco-Valdez, Carmen; Gutarra, Carlos; del Valle-Mendoza, Juana; Ruiz, Joaquim; Matsuoka, Mayumi

    2016-01-01

    Background Bartonella bacilliformis is the causative agent of Carrion’s disease, a neglected illness with mortality rates of 40–85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas. Methodology/Principal Findings Blood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion’s disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit β (SCS-β). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-β interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31). Conclusions/Significance RT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion’s disease and identify asymptomatic carriers. PMID:27627803

  12. D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea.

    Science.gov (United States)

    Riederer, B; Han, M; Keller, U

    1996-11-01

    The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

  13. The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

    Science.gov (United States)

    Berthet-Colominas, C; Seignovert, L; Härtlein, M; Grotli, M; Cusack, S; Leberman, R

    1998-01-01

    The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor. PMID:9582288

  14. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...

  15. Drug-pyridoxal phosphate interactions.

    Science.gov (United States)

    Ebadi, M; Gessert, C F; Al-Sayegh, A

    1982-01-01

    phosphate. Some interesting relationships are pointed out between vitamin B6, picolinic acid, and zinc. It is postulated that the intestinal absorption of zinc is facilitated by picolinic acid, a metabolite of tryptophan. The derivation of picolinic acid from tryptophan depends on the action of the enzyme kynureninase, which is dependent on pyridoxal phosphate; therefore, the adequate absorption of zinc is indirectly dependent on an adequate supply of vitamin B6. The formation of pyridoxal phosphate, on the other hand, appears to be indirectly dependent on Zn2++ which activates pyridoxal kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

  16. Electrochemical phosphate recovery from nanofiltration concentrates

    NARCIS (Netherlands)

    Kappel, C.; Yasadi, K.; Temmink, B.G.; Metz, S.J.; Kemperman, A.J.B.; Nijmeijer, K.; Zwijnenburg, A.; Witkamp, G.J.; Rijnaarts, H.

    2013-01-01

    The high total phosphorus content of raw domestic wastewater with its significant eutrophication potential offers an excellent possibility for phosphate recovery. Continuous recirculation of NF concentrate to an MBR and simultaneous phosphate recovery from the NF concentrate can be applied to produc

  17. 21 CFR 520.823 - Erythromycin phosphate.

    Science.gov (United States)

    2010-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other means... chickens—(i) Amount. 0.500 gram per gallon. (ii) Indications for use. As an aid in the control of...

  18. Stable Development of Phosphate Fertilizer Sector

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ The rapid growth of China's economy in recent years gave rise to a sound external environment for the development of the phosphate fertilizer industry. With quite a few state agricultural incentives, the initiative of farmers in grain production is much higher, and consumption of phosphate fertilizers has increased constantly.

  19. Phosphate recycling in the phosphorus industry

    NARCIS (Netherlands)

    Schipper, W.J.; Klapwijk, A.; Potjer, B.; Rulkens, W.H.; Temmink, B.G.; Kiestra, F.D.G.; Lijmbach, A.C.M.

    2004-01-01

    The article describes the potential and limitations for recovery of phosphate from secondary materials in the production process for white phosphorus. This thermal process involves the feeding of phosphate rock, cokes and pebbles to a furnace. The reducing conditions in the furnace promote the forma

  20. Phosphate recycling in the phosphorus industry

    NARCIS (Netherlands)

    Schipper, W.J.; Klapwijk, A.; Potjer, A.; Rulkens, W.H.; Temmink, B.G.; Kiestra, F.D.G.; Lijmbach, A.C.M.

    2001-01-01

    The feasibility of phosphate recycling in the white phosphorus production process is discussed. Several types of materials may be recycled, provided they are dry inorganic materials, low in iron, copper and zinc. Sewage sludge ash may be used if no iron is used for phosphate precipitation in the tre

  1. Adsorption of Phosphate on Variable Charge Soils

    Institute of Scientific and Technical Information of China (English)

    HUGUO-SONG; ZHUZU-XIANG; 等

    1992-01-01

    The study about the adsorption of phosphate on four variable charge soils and some minerals revealed that two stage adsorption appeared in the adsorption isothems of phosphate on 4 soils and there was a maximum adsorption on Al-oxide-typed surfaces between pH 3.5 to pH 5.5 as suspension pH changed from 2 to 9,but the adsorption amount of phosphate decreased continually as pH rose on Fe-oxide typed surfaces.The adsorption amount of phosphate and the maximum phosphate adsorption pH decreased in the order of yellow-red soil> lateritic red soil> red soil> paddy soil,which was coincided with the content order of amorphous Al oxide.The removement of organic matter and Fe oxide made the maximum phosphate adsorption pH rise from 4.0 to 5.0 and 4.5,respectively.The desorption curves with pH of four soils showed that phosphate desorbed least at pH 5.Generally the desorption was contrary to the adsorption with pH changing.There was a good accordance between adsorption or desorption and the concentration of Al in the suspension.The possible mechanisms of phosphate adsorption are discussed.

  2. Genetics Home Reference: pyridoxal 5'-phosphate-dependent epilepsy

    Science.gov (United States)

    ... 5'-phosphate-dependent epilepsy pyridoxal 5'-phosphate-dependent epilepsy Enable Javascript to view the expand/collapse boxes. ... All Close All Description Pyridoxal 5'-phosphate-dependent epilepsy is a condition that involves seizures beginning soon ...

  3. Interferon titer and the 2',5'-oligoadenylate-synthetase activity in rat thymus lymphocytes in conditions of Omeprazol-caused hypergastrinemia

    Directory of Open Access Journals (Sweden)

    Kompanets I. V.

    2013-01-01

    Full Text Available The aim of this work was the determination of rat thymocytes response to hypergastrinemia evoked by hypoacidity and multiprobiotic «Symbiter® acidophilic concentrated» (symbiter treatment via the estimation of the interferon (IFN titer and 2', 5'-oligoadenylate (OA-synthetase activity in lymphocytes. 2', 5'-OA-synthetase is the IFN-induced enzyme. Methods. The micromethod of IFN titer determination by antiviral activity, spectrophotometrical method of 2', 5'-OA-synthetase activity determination. Results. It was shown that the IFN production by cultivated thymocytes is amplified while the 2', 5'-OA-synthetase activity decreases in these cells in conditions of hypoacidity caused by the 28-days omeprazol treatment. The treatment of animals by symbiter against a background of hypoacidity causes the augmentation of IFN production by thymocytes, but does not stimulate the 2', 5'-OA-synthetase activity. The IFN production by thymocytes in response to IFN inducers (PHA and cycloferone in vitro is intensified comparatively to the control at hypoacidity and symbiter treatment. Conclusions. The multiprobiotic symbiter exhibits interferonogenic properties. The IFN synthesis in response to induction in vitro is intensified in comparison with healthy animals at both hypoacidity and symbiter treatment while the 2', 5'-OA-synthetase acivity in thymocytes decreases.

  4. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections

    Energy Technology Data Exchange (ETDEWEB)

    De Benedetti, A.; Pytel, B.A.; Baglioni, C.

    1987-02-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested.

  5. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections.

    Science.gov (United States)

    De Benedetti, A; Pytel, B A; Baglioni, C

    1987-01-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested. Images PMID:2433688

  6. Hydrolysis of dicalcium phosphate dihydrate to hydroxyapatite.

    Science.gov (United States)

    Fulmer, M T; Brown, P W

    1998-04-01

    Dicalcium phosphate dihydrate (DCPD) was hydrolysed in water and in 1 M Na2HPO4 solution at temperatures from 25-60 degrees C. Hydrolysis was incomplete in water. At 25 degrees C, DCPD partially hydrolysed to hydroxyapatite (HAp). Formation of HAp is indicative of incongruent DCPD dissolution. At the higher temperatures, hydrolysis to HAp was more extensive and was accompanied by the formation of anhydrous dicalcium phosphate (DCP). Both of these processes are endothermic. When hydrolysis was carried out in 1 M Na2HPO4 solution, heat absorption was greater at any given temperature than for hydrolysis in water. Complete hydrolysis to HAp occurred in this solution. The hydrolysis of DCPD to HAp in sodium phosphate solution was also endothermic. The complete conversion of DCPD to HAp in sodium phosphate solution would not be expected if the only effect of this solution was to cause DCPD dissolution to become congruent. Because of the buffering capacity of a dibasic sodium phosphate solution, DCPD hydrolysed completely to HAp. Complete conversion to HAp was accompanied by the conversion of dibasic sodium phosphate to monobasic sodium phosphate. The formation of DCP was not observed indicating that the sodium phosphate solution precluded the DCPD-to-DCP dehydration reaction. In addition to affecting the extent of hydrolysis, reaction in the sodium phosphate solution also caused a morphological change in the HAp which formed. HAp formed by hydrolysis in water was needle-like to globular while that formed in the sodium phosphate solution exhibited a florette-like morphology.

  7. Dominant oceanic bacteria secure phosphate using a large extracellular buffer

    OpenAIRE

    M. V. Zubkov; Martin, A. P.; Hartmann, M.; Grob, C.; Scanlan, D.J.

    2015-01-01

    The ubiquitous SAR11 and Prochlorococcus bacteria manage to maintain a sufficient supply of phosphate in phosphate-poor surface waters of the North Atlantic subtropical gyre. Furthermore, it seems that their phosphate uptake may counter-intuitively be lower in more productive tropical waters, as if their cellular demand for phosphate decreases there. By flow sorting 33P-phosphate-pulsed 32P-phosphate-chased cells, we demonstrate that both Prochlorococcus and SAR11 cells exploit an extracellul...

  8. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2016-01-15

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  9. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Science.gov (United States)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-01

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  10. Altered alpha subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichis coli.

    Science.gov (United States)

    Hennecke, H; Böck, A

    1975-07-01

    Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenyalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The alpha and beta subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant alpha subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation.

  11. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product...... of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased...... sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P

  12. Nanomolar inhibitors of Mycobacterium tuberculosis glutamine synthetase 1: synthesis, biological evaluation and X-ray crystallographic studies.

    Science.gov (United States)

    Couturier, Cédric; Silve, Sandra; Morales, Renaud; Pessegue, Bernard; Llopart, Sylvie; Nair, Anil; Bauer, Armin; Scheiper, Bodo; Pöverlein, Christoph; Ganzhorn, Axel; Lagrange, Sophie; Bacqué, Eric

    2015-04-01

    A series of imidazo[1,2-a]indeno[1,2-e]pyrazin-4-ones that potently inhibit M. tuberculosis glutamine synthetase (GlnA1) has been identified by high throughput screening. Exploration of this series was performed owing to a short chemistry program. Despite possibly nanomolar inhibitions, none of these compounds was active on whole cell Mtb, suggesting that GlnA1 may not be a suitable target to find new anti-tubercular drugs.

  13. DNA Polymerases and Aminoacyl-tRNA Synthetases: Shared Mechanisms for Ensuring the Fidelity of Gene Expression

    OpenAIRE

    Francklyn, Christopher S.

    2008-01-01

    DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critical roles in the transformation of genetic information from DNA to RNA to protein. DNA polymerases carry out replication and collaborate in the repair of the genome, while ARSs provide aminoacylated tRNA precursors for protein synthesis. Enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high...

  14. [Effect of reproduction of the LPP-3 cyanophage on glutamate dehydrogenase and glutamine synthetase activity in the cyanobacterium Plectonema boryanum].

    Science.gov (United States)

    Mendzhul, M I; Koltukova, N V; Lysenko, T G; Shainskaia, O A; Perepelitsa, S I

    1995-01-01

    The effect of cyanophage LPP-3 reproduction on glutamate dehydrogenase and glutamine synthetase (GS) in P boryanum cells have been studied. It was determined that the both reactions are intensified by 135% and 220%, accordingly. Isoenzymes of GS were purified from native and infected cell of cyanobacteria. Their physical-and-chemical properties are different. The cyanophage development probably causes specific modification of the cell enzymes.

  15. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2...

  16. Inhibition of Mycobacterium tuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo

    OpenAIRE

    2003-01-01

    Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibi...

  17. Structural and functional characterization of the 5' upstream region of a glutamine synthetase gene from Scots pine

    OpenAIRE

    Avila, Concepción; Cantón, Francisco; Barnestein, Pilar; Suárez, María-Fernanda; Marraccini, Pierre; Rey, Manuel; Humara, Jaime; Ordás, Ricardo; Cánovas, Francisco

    2002-01-01

    International audience; We report here the isolation and characterization of a genomic clone encoding Scots pine (P. sylvestris) cytosolic glutamine synthetase GS1a. The clone contains the 5' half of the gene including part of the coding region organized in seven exons, interrupted by 6 introns and 980 bp upstream of the translation initiation codon. Earlier experiments carried out in our lab have shown that the GS1a gene is expressed in a light dependent fashion during the initial stages of ...

  18. Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential uppS Gene

    OpenAIRE

    Apfel, Christian M.; Takács, Béla; Fountoulakis, Michael; Stieger, Martin; Keck, Wolfgang

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E,E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide ge...

  19. Biochemical analyses of ppGpp effect on adenylosuccinate synthetases, key enzymes in purine biosynthesis in rice.

    Science.gov (United States)

    Nomura, Yuhta; Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    The ppGpp-signaling system functions in plant chloroplasts. In bacteria, a negative effect of ppGpp on adenylosuccinate synthetase (AdSS) has been suggested. Our biochemical analysis also revealed rice AdSS homologs are apparently sensitive to ppGpp. However, further investigation clarified that this phenomenon is cancelled by the high substrate affinity to the enzymes, leading to a limited effect of ppGpp on adenylosuccinate synthesis.

  20. Application of Potential Phosphate-Solubilizing Bacteria and Organic Acids on Phosphate Solubilization from Phosphate Rock in Aerobic Rice

    OpenAIRE

    Qurban Ali Panhwar; Shamshuddin Jusop; Umme Aminun Naher; Radziah Othman; Mohd Ismail Razi

    2013-01-01

    A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB) and organic acids (oxalic & malic) on phosphate (P) solubilization from phosphate rock (PR) and growth of aerobic rice. Four rates of each organic acid (0, 10, 20, and 30 mM), and PSB strain (Bacillus sp.) were applied to aerobic rice. Total bacterial populations, amount of P solubilization, P uptake, soil pH, and root morphology were determined. The results of the study showed ...

  1. Dexamethasone enhances glutamine synthetase activity and reduces N-methyl-D-aspartate neurotoxicity in mixed cultures of neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Edith Debroas

    2015-05-01

    Full Text Available Astrocytes are claimed to protect neurons against excitotoxicity by clearing glutamate from the extracellular space and rapidly converting it into glutamine. Glutamine, is then released into the extracellular medium, taken up by neurons and transformed back into glutamate which is then stored into synaptic vesicles. Glutamine synthetase (GS, the key enzyme that governs this glutamate/glutamine cycle, is known to be upregulated by glucocorticoids. In the present work we have thus studied in parallel the effects of dexamethasone on glutamine synthetase activity and NMDA-induced neuronal death in cultures derived from the brain cortex of murine embryos. We showed that dexamethasone was able to markedly enhance GS activity in cultures of astrocytes but not in near pure neuronal cultures. The pharmacological characteristics of the dexamethasone action strongly suggest that it corresponds to a typical receptor-mediated effect. We also observed that long lasting incubation (72 h of mixed astrocyte-neuron cultures in the presence of 100 nM dexamethasone significantly reduced the toxicity of NMDA treatment. Furthermore we demonstrated that methionine sulfoximine, a selective inhibitor of GS, abolished the dexamethasone-induced increase in GS activity and also markedly potentiated NMDA toxicity. Altogether these results suggest that dexamethasone may promote neuroprotection through a stimulation of astrocyte glutamine synthetase.

  2. MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

    Directory of Open Access Journals (Sweden)

    Rongzhong Li

    2015-07-01

    Full Text Available While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes.

  3. Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement.

    Science.gov (United States)

    Zhang, Shaowei; Han, Yingkun; Kumar, Ashok; Gao, Haofeng; Liu, Ziduo; Hu, Nan

    2017-02-07

    A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

  4. Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

    Science.gov (United States)

    Boehlein, S K; Schuster, S M; Richards, N G

    1996-03-01

    Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

  5. Proteomic Analysis of the Response of Liangyoupeijiu (Super High-Yield Hybrid Rice) Seedlings to Cold Stress

    Institute of Scientific and Technical Information of China (English)

    Ping-Fang Yang; Xiao-Juan Li; Yu Liang; Yu-Xiang Jing; Shi-Hua Shen; Ting-Yun Kuang

    2006-01-01

    Liangyoupeijiu is a super high-yield hybrid rice. Despite its advantages with respect to yield and grain quality, it is sensitive to cold, which keeps it from being widely cultivated. We subjected Liangyoupeijiu seedlings to 4 ℃ cold treatment, then extracted the leaf proteins. After 2-D gel electrophoresis separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, a series of differentially displayed proteins were identified. Some metabolism-associated proteins were found among the downregulated proteins, such as carbamoyl phosphate synthetase, transketolase 1, dihydrolipoamide dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase. The upregulated proteins included both stress-resistance proteins such as nucleoside diphosphate kinase Ⅰ and proteins that are negative for rice growth, such as FtsH-like protein, plastid fusion and/or translocation factor (Pftf) and actin. Our results indicate that cold may inhibit Liangyoupeijiu growth through decreasing metabolic activity and damaging cell structure.

  6. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    Science.gov (United States)

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  7. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

    Science.gov (United States)

    Gudzera, Olga I; Golub, Andriy G; Bdzhola, Volodymyr G; Volynets, Galyna P; Lukashov, Sergiy S; Kovalenko, Oksana P; Kriklivyi, Ivan A; Yaremchuk, Anna D; Starosyla, Sergiy A; Yarmoluk, Sergiy M; Tukalo, Michail A

    2016-03-01

    Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6μM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27μM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01μM and IC90=13.53μM.

  8. Overexpression of a glutamine synthetase gene affects growth and development in sorghum.

    Science.gov (United States)

    Urriola, Jazmina; Rathore, Keerti S

    2015-06-01

    Nitrogen is a primary macronutrient in plants, and nitrogen fertilizers play a critical role in crop production and yield. In this study, we investigated the effects of overexpressing a glutamine synthetase (GS) gene on nitrogen metabolism, and plant growth and development in sorghum (Sorghum bicolor L., Moench). GS catalyzes the ATP dependent reaction between ammonia and glutamate to produce glutamine. A 1,071 bp long coding sequence of a sorghum cytosolic GS gene (Gln1) under the control of the maize ubiquitin (Ubq) promoter was introduced into sorghum immature embryos by Agrobacterium-mediated transformation. Progeny of the transformants exhibited higher accumulation of the Gln1 transcripts and up to 2.2-fold higher GS activity compared to the non-transgenic controls. When grown under optimal nitrogen conditions, these Gln1 transgenic lines showed greater tillering and up to 2.1-fold increase in shoot vegetative biomass. Interestingly, even under greenhouse conditions, we observed a seasonal component to both these parameters and the grain yield. Our results, showing that the growth and development of sorghum Gln1 transformants are also affected by N availability and other environmental factors, suggest complexity of the relationship between GS activity and plant growth and development. A better understanding of other control points and the ability to manipulate these will be needed to utilize the transgenic technology to improve nitrogen use efficiency of crop plants.

  9. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    Science.gov (United States)

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

  10. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase.

    Science.gov (United States)

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M; Vega, José M; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress.

  11. Carnosine modulates glutamine synthetase expression in senescent astrocytes exposed to oxygen-glucose deprivation/recovery.

    Science.gov (United States)

    Shi, Xiaojie; Wang, Bingyu; Liu, Yuan; Zhang, Jingjing; Huang, Yuyan; Cao, Pei; Shen, Yao; Lyu, Jianxin

    2017-01-20

    Carnosine is believed to be neuroprotective in cerebral ischemia. However, few reports concern its function on senescent astrocytes during cerebral ischemia. The aim of this study was to investigate the effects of carnosine on cell damage and glutamine synthetase (GS) expression in D-galactose-induced senescent astrocytes exposed to oxygen-glucose deprivation/recovery (OGD/R). The results showed that OGD/R caused massive cell damage and a significant decrease in GS expression both in the young and senescent astrocytes. The GS expression level was partly recovered whereas it continued to decline in the recovery stage in the young and senescent astrocytes, respectively. Decreased GS expression significantly inhibited glutamate uptake and glutamine production and release. Carnosine prevented the cell damage, rescued the expression of GS and reversed the glutamate uptake activity and glutamine production in the senescent astrocytes exposed to OGD/R. The modulatory effect of carnosine on GS expression was partly antagonized by pyrilamine, a selective histamine H1 receptors antagonist, but not bestatin. Bisindolylmaleimide II, a broad-spectrum inhibitor of PKC could also reverse the action of carnosine on GS expression. Thus, histamine H1 receptors and PKC pathway may be involved in the modulatory action of carnosine in GS expression in the senescent astrocytes exposed to OGD/R.

  12. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase

    Directory of Open Access Journals (Sweden)

    Margarita eGarcía-Calderón

    2015-09-01

    Full Text Available This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2 in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L .japonicus plants in response to stress.

  13. Influence of different host associations on glutamine synthetase activity and ammonium transporter in Santalumalbum L.

    Science.gov (United States)

    Deepa, P; Yusuf, A

    2016-07-01

    The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica-sandal association compared to other leguminous and non-leguminous host associations. The association of N2 fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the [Formula: see text] method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.

  14. Assessment of glutamine synthetase activity by [13N]ammonia uptake in living rat brain.

    Science.gov (United States)

    Momosaki, Sotaro; Ito, Miwa; Tonomura, Misato; Abe, Kohji

    2015-01-01

    Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

  15. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme.

    Science.gov (United States)

    Seabra, Ana R; Carvalho, Helena G

    2015-01-01

    Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  16. Lanthionine Synthetase C-Like 2 Modulates Immune Responses to Influenza Virus Infection

    Science.gov (United States)

    Leber, Andrew; Bassaganya-Riera, Josep; Tubau-Juni, Nuria; Zoccoli-Rodriguez, Victoria; Lu, Pinyi; Godfrey, Victoria; Kale, Shiv; Hontecillas, Raquel

    2017-01-01

    Broad-based, host-targeted therapeutics have the potential to ameliorate viral infections without inducing antiviral resistance. We identified lanthionine synthetase C-like 2 (LANCL2) as a new therapeutic target for immunoinflammatory diseases. To examine the therapeutic efficacy of oral NSC61610 administration on influenza, we infected C57BL/6 mice with influenza A H1N1pdm virus and evaluated influenza-related mortality, lung inflammatory profiles, and pulmonary histopathology. Oral treatment with NSC61610 ameliorates influenza virus infection by down-modulating pulmonary inflammation through the downregulation of TNF-α and MCP-1 and reduction in the infiltration of neutrophils. NSC61610 treatment increases IL10-producing CD8+ T cells and macrophages in the lungs during the resolution phase of disease. The loss of LANCL2 or neutralization of IL-10 in mice infected with influenza virus abrogates the ability of NSC61610 to accelerate recovery and induce IL-10-mediated regulatory responses. These studies validate that oral treatment with NSC61610 ameliorates morbidity and mortality and accelerates recovery during influenza virus infection through a mechanism mediated by activation of LANCL2 and subsequent induction of IL-10 responses by CD8+ T cells and macrophages in the lungs.

  17. Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

    Indian Academy of Sciences (India)

    MANGAL SINGH; SANDEEP CHAUDHARY; DIPTI SAREEN

    2017-03-01

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are themajor multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically importantantibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid,followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, theknowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of theever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thusdeciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is thesubstrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtHhomolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequencedgenome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPSgene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

  18. Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

    Science.gov (United States)

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Balish, Rebecca S; Meagher, Richard B

    2005-06-01

    Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed.

  19. Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

    Science.gov (United States)

    Sircar, Kanishka; Huang, Heng; Hu, Limei; Cogdell, David; Dhillon, Jasreman; Tzelepi, Vassiliki; Efstathiou, Eleni; Koumakpayi, Ismaël H.; Saad, Fred; Luo, Dijun; Bismar, Tarek A.; Aparicio, Ana; Troncoso, Patricia; Navone, Nora; Zhang, Wei

    2013-01-01

    The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells. PMID:22245216

  20. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats.

    Science.gov (United States)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S; Simonsen, Mette; Ott, Peter; Vilstrup, Hendrik; Sørensen, Michael

    2014-03-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

  1. Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles.

    Science.gov (United States)

    Ottesen, Elizabeth A; Leadbetter, Jared R

    2011-05-01

    In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.

  2. Effect of bromochloromethane and fumarate on phylogenetic diversity of the formyltetrahydrofolate synthetase gene in bovine rumen.

    Science.gov (United States)

    Mitsumori, Makoto; Matsui, Hiroki; Tajima, Kiyoshi; Shinkai, Takumi; Takenaka, Akio; Denman, Stuart E; McSweeney, Christopher S

    2014-01-01

    Effect of the methane inhibitor, bromochloromethane (BCM) and dietary substrate, fumarate, on microbial community structure of acetogen bacteria in the bovine rumen was investigated through analysis of the formyltetrahydrofolate synthetase gene (fhs). The fhs sequences obtained from BCM-untreated, BCM-treated, fumarate-untreated and fumarate-treated bovine rumen were categorized into homoacetogens and nonhomoacetogenic bacteria by homoacetogen similarity scores. Phylogenetic tree analysis indicated that most of the fhs sequences categorized into homoacetogens were divided into nine clusters, which were in close agreement with a result shown in a self-organizing map. The diversity of the fhs sequences from the BCM-treated rumen was significantly different from those from BCM-non-treated rumen. Principal component analysis also showed that addition of BCM to the rumen altered the population structure of acetogenic bacteria significantly but the effect of fumarate was comparatively minor. These results indicate that BCM affects diversity of actogens in the bovine rumen, and changes in acetogenic community structure in response to methane inhibitors may be caused by different mechanisms.

  3. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    Gabriela Beatriz Acosta; María Alejandra Fernández; Diego Martín Roselló; María Luján Tomaro; Karina Balestrasse; Abraham Lemberg

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into shamoperated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions.

  4. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

    Science.gov (United States)

    Tardito, Saverio; Oudin, Anaïs; Ahmed, Shafiq U; Fack, Fred; Keunen, Olivier; Zheng, Liang; Miletic, Hrvoje; Sakariassen, Per Øystein; Weinstock, Adam; Wagner, Allon; Lindsay, Susan L; Hock, Andreas K; Barnett, Susan C; Ruppin, Eytan; Mørkve, Svein Harald; Lund-Johansen, Morten; Chalmers, Anthony J; Bjerkvig, Rolf; Niclou, Simone P; Gottlieb, Eyal

    2015-12-01

    L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

  5. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    Science.gov (United States)

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  6. Identification of a phosphinothricin-resistant mutant of rice glutamine synthetase using DNA shuffling.

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Zhao, Wei; Xing, Xiao-Juan; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2015-10-23

    To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.

  7. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    Science.gov (United States)

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

  8. Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

    Directory of Open Access Journals (Sweden)

    Niran Roongsawang

    2010-12-01

    Full Text Available Lipopeptide biosurfactants (LPBSs consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive and Pseudomonas (Gram-negative have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs. Production of active‑form NRPSs requires not only transcriptional induction and translation but also post‑translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

  9. Cancer association study of aminoacyl-tRNA synthetase signaling network in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Yong-Wan Kim

    Full Text Available Aminoacyl-tRNA synthetases (ARSs and ARS-interacting multifunctional proteins (AIMPs exhibit remarkable functional versatility beyond their catalytic activities in protein synthesis. Their non-canonical functions have been pathologically linked to cancers. Here we described our integrative genome-wide analysis of ARSs to show cancer-associated activities in glioblastoma multiforme (GBM, the most aggressive malignant primary brain tumor. We first selected 23 ARS/AIMPs (together referred to as ARSN, 124 cancer-associated druggable target genes (DTGs and 404 protein-protein interactors (PPIs of ARSs using NCI's cancer gene index. 254 GBM affymetrix microarray data in The Cancer Genome Atlas (TCGA were used to identify the probe sets whose expression were most strongly correlated with survival (Kaplan-Meier plots versus survival times, log-rank t-test <0.05. The analysis identified 122 probe sets as survival signatures, including 5 of ARSN (VARS, QARS, CARS, NARS, FARS, and 115 of DTGs and PPIs (PARD3, RXRB, ATP5C1, HSP90AA1, CD44, THRA, TRAF2, KRT10, MED12, etc. Of note, 61 survival-related probes were differentially expressed in three different prognosis subgroups in GBM patients and showed correlation with established prognosis markers such as age and phenotypic molecular signatures. CARS and FARS also showed significantly higher association with different molecular networks in GBM patients. Taken together, our findings demonstrate evidence for an ARSN biology-dominant contribution in the biology of GBM.

  10. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    Science.gov (United States)

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

  11. Structural characterization of Helicobacter pylori dethiobiotin synthetase reveals differences between family members

    Energy Technology Data Exchange (ETDEWEB)

    Porebski, Przemyslaw J.; Klimecka, Maria; Chruszcz, Maksymilian; Nicholls, Robert A.; Murzyn, Krzysztof; Cuff, Marianne E.; Xu, Xiaohui; Cymborowski, Marcin; Murshudov, Garib N.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (MCSG); (UV); (MRC)

    2012-07-11

    Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS-ATP and hpDTBS-GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.

  12. Role of tRNAPro in pretransfer editing of alanine by prolyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Boyarshin K. S.

    2013-09-01

    Full Text Available Aim. To characterize the process of tRNA-dependent pretransfer edi- ting of alanine by prolyl-tRNA synthetase of bacteria Enterococcus faecalis (ProRSEf. Methods. Velocity of the editing processes in vitro was determined by ATP hydrolysis by ProRSEf. Pretransfer and posttransfer editing were experimentally separated by site-directed mutagenesis. Results. tRNA-dependent pretransfer editing is characterized by three-fold larger velocity then tRNA-independent editing. Effectivity of the process depends on the presence of 2'-hydroxyle group of A76 tRNAPro. In the absence of tRNAPro selective release of alanyl-AMP occurs simultaneously with tRNA-independent pretransfer editing. Released alanyl-AMP can be re-bound and hydrolyzed. Conclusions. tRNA-dependent pretransfer editing of alanine by ProRSEf is the catalytic mechanism, mediated by 2'-hydroxyl group of A76 tRNAPro. In the absence of tRNAPro tRNA-independent pretransfer editing and selective release of alanyl-AMP occur.

  13. Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase

    Science.gov (United States)

    Liu, Ru-Juan; Lukarska, Maria; Gut, Jiri; Bougdour, Alexandre; Touquet, Bastien; Wang, En-Duo; Li, Xianfeng; Alley, M. R. K.; Freund, Yvonne R.; Rosenthal, Philip J.; Hakimi, Mohamed-Ali

    2016-01-01

    The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNALeu in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents. PMID:27431220

  14. Chitin synthetases in Candida albicans: a review on their subcellular distribution and biological function.

    Science.gov (United States)

    Martínez, J P; Gozalbo, D

    1994-09-01

    In the light of recent genetic advances, some results regarding chitin biosynthetic activities are reviewed in this paper. Genes coding for distinct enzymes displaying chitin synthetase activities have been characterized in Saccharomyces cerevisiae as well as in other fungal species including Candida albicans. Several activities seem to exist in the cells: (i) one zymogenic, located in cytoplasmic vesicles called chitosomes, although the presence of other types of vesicles with zymogenic activity cannot be completely discarded, and (ii) plasma membrane associated activities (the active enzyme and probably two distinct pools of zymogenic activity). Possible relationships between these activities, if any, remain to be determined. These multiplicity of enzymes is not surprising taking into account that chitin biosynthesis is required during very well defined temporal and spatial events of the cell cycle. A general repair function for one of the chitin biosynthetic activities is proposed as a possible salvage mechanism to warrant cell survival after wall damage has been caused, since chitin appears to be the most suitable polymer to carry out this function due to its particular physico-chemical properties.

  15. An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

    Institute of Scientific and Technical Information of China (English)

    SUNJIANGE; XIAOLIYAO; 等

    1998-01-01

    In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

  16. The Relationship of Arginine Deprivation, Argininosuccinate Synthetase and Cell Death in Melanoma

    Directory of Open Access Journals (Sweden)

    Niramol Savaraj

    2007-01-01

    Full Text Available It has been shown that melanoma cells do not express argininosuccinate synthetase (ASS and therefore are unable to synthesize arginine from citrulline. Depleting arginine using pegylated arginine deiminase (ADI-PEG20 results in cell death in melanoma but not normal cells. This concept was translated into clinical trial and responses were seen. However, induction of ASS expression does occur which results in resistance to ADI -PEG20. We have used 4 melanoma cell lines to study factors which may govern ASS expression. Although these 4 melanoma cell lines do not express ASS protein or mRNA as detected by both immunoblot and northernblot analysis, ASS protein can be induced after these cells are grown in the presence of ADI-PEG20, but again repressed after replenishing arginine in the media. The levels of induction are different and one cell line could not be induced. Interestingly, a melanoma cell line with the highest level of induction could also be made resistant to ADI-PEG20. This resistant line possesses high levels of ASS mRNA and protein expression which cannot be repressed with arginine. Our study indicates that ASS expression in melanoma cells is complex and governed by biochemical parameters which are different among melanoma cells.

  17. Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening.

    Science.gov (United States)

    Loulakakis, K. A.; Roubelakis-Angelakis, K. A.; Kanellis, A. K.

    1994-09-01

    The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.

  18. Impaired novelty acquisition and synaptic plasticity in congenital hyperammonemia caused by hepatic glutamine synthetase deficiency

    Science.gov (United States)

    Chepkova, Aisa N.; Sergeeva, Olga A.; Görg, Boris; Haas, Helmut L.; Klöcker, Nikolaj; Häussinger, Dieter

    2017-01-01

    Genetic defects in ammonia metabolism can produce irreversible damage of the developing CNS causing an impairment of cognitive and motor functions. We investigated alterations in behavior, synaptic plasticity and gene expression in the hippocampus and dorsal striatum of transgenic mice with systemic hyperammonemia resulting from conditional knockout of hepatic glutamine synthetase (LGS-ko). These mice showed reduced exploratory activity and delayed habituation to a novel environment. Field potential recordings from LGS-ko brain slices revealed significantly reduced magnitude of electrically-induced long-term potentiation (LTP) in both CA3-CA1 hippocampal and corticostriatal synaptic transmission. Corticostriatal but not hippocampal slices from LGS-ko brains demonstrated also significant alterations in long-lasting effects evoked by pharmacological activation of glutamate receptors. Real-time RT-PCR revealed distinct patterns of dysregulated gene expression in the hippocampus and striatum of LGS-ko mice: LGS-ko hippocampus showed significantly modified expression of mRNAs for mGluR1, GluN2B subunit of NMDAR, and A1 adenosine receptors while altered expression of mRNAs for D1 dopamine receptors, the M1 cholinoreceptor and the acetylcholine-synthetizing enzyme choline-acetyltransferase was observed in LGS-ko striatum. Thus, inborn systemic hyperammonemia resulted in significant deficits in novelty acquisition and disturbed synaptic plasticity in corticostriatal and hippocampal pathways involved in learning and goal-directed behavior. PMID:28067279

  19. The Role of a Nonribosomal Peptide Synthetase in l-Lysine Lactamization During Capuramycin Biosynthesis.

    Science.gov (United States)

    Liu, Xiaodong; Jin, Yuanyuan; Cui, Zheng; Nonaka, Koichi; Baba, Satoshi; Funabashi, Masanori; Yang, Zhaoyong; Van Lanen, Steven G

    2016-05-03

    Capuramycins are one of several known classes of natural products that contain an l-Lys-derived l-α-amino-ɛ-caprolactam (l-ACL) unit. The α-amino group of l-ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP-independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP-dependent amide-bond-forming catalyst responsible for the biosynthesis of the remaining amide bond present in l-ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l-Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS-mediated mechanism is employed in the biosynthesis of other l-ACL-containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS-derived peptides that contain an unmodified l-Lys residue.

  20. Large-scale filament formation inhibits the activity of CTP synthetase.

    Science.gov (United States)

    Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

    2014-07-16

    CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

  1. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

    Science.gov (United States)

    Madiraju, Anila K; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2016-06-14

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism.

  2. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

  3. Cyclitols protect glutamine synthetase and malate dehydrogenase against heat induced deactivation and thermal denaturation.

    Science.gov (United States)

    Jaindl, Martina; Popp, Marianne

    2006-06-30

    The accumulation of cyclitols in plants is a widespread response that provides protection against various environmental stresses. The capacity of myo-Inositol, pinitol, quercitol, and other compatible solutes (i.e., sorbitol, proline, and glycinebetaine) to protect proteins against thermally induced denaturation and deactivation was examined. Enzymatic activity measurements of L-glutamine synthetase from Escherichia coli and Hordeum vulgare showed that the presence of cyclitols during heat treatment resulted in a significantly higher percentage of residual activity. CD spectroscopy experiments were used to study thermal stabilities of protein secondary structures upon the addition of myo-Inositol, pinitol, and glucose. 0.4 M myo-Inositol was observed to raise the melting temperature (Tm) of GS from E. coli by 3.9 degrees C and MDH from pig heart by 3.4 degrees C, respectively. Pinitol showed an increase in Tm of MDH by 3.8 degrees C, whereas glucose was not effective. Our results show a great potential of stabilizing proteins by the addition of cyclitols.

  4. Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

    Science.gov (United States)

    Mariotti, Marco; Santesmasses, Didac; Capella-Gutierrez, Salvador; Mateo, Andrea; Arnan, Carme; Johnson, Rory; D'Aniello, Salvatore; Yim, Sun Hee; Gladyshev, Vadim N; Serras, Florenci; Corominas, Montserrat; Gabaldón, Toni; Guigó, Roderic

    2015-09-01

    Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.

  5. Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

    OpenAIRE

    Lílian Estrela Borges Baldotto; Marihus Altoé Baldotto; Fábio Lopes Olivares; Adriane Nunes de Souza

    2014-01-01

    Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i) to select and characterize diazotrophs abl...

  6. Synthesis of Caged Bicyclic Phosphate Derivatives

    Institute of Scientific and Technical Information of China (English)

    FANG Xiao-min; OU Yu-xiang; LUO Rui-bin; WANG Yong; LIAN Dan-jun; LI Xin

    2008-01-01

    Seven caged bicyclic phosphate compounds were synthesized by using 1-oxo-4-hydroxymethy1-2,6,7-trioxa-1-pho-sphabicyclo[2.2.2] octane (PEPA) as starting material. Within them were three PEPA derivatives containing single caged bicyclic phosphate structure(1a,2a,3a), another three PEPA deviratives containing two caged bicyclic phosphate structures(1b,2b,3b) and one devirative(1c) containing three caged bicyclic phosphate structures. Structures of the products were characterized by FTIR, 1H NMR, elemental analysis and TG analysis. The reaction conditions were also discussed. Thermal analysis showed they had high thermal stability and excellent char-forming ability. Besides, these compounds had pentaerythritol bone and flame retardant elements of phosphorus, bromine or nitrogen simultaneously in their molecules, endowed them with good fire retardancy, and made them can be used as intumescent flame retardant.

  7. Pyridoxal Phosphate vs Pyridoxine for Intractable Seizures

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2005-05-01

    Full Text Available The efficacy of pyridoxal phosphate (PLP compared to pyridoxine (PN in the control of idiopathic intractable epilepsy was studied in 94 children, aged 8 months to 15 years, at the National Taiwan University Hospital, Taipei, Taiwan.

  8. Enzyme activity in dialkyl phosphate ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  9. Enzyme activity in dialkyl phosphate ionic liquids.

    Science.gov (United States)

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  10. Phosphate solubilizing bacteria around Indian peninsula

    Digital Repository Service at National Institute of Oceanography (India)

    DeSouza, M.J.B.D.; Nair, S.; Chandramohan, D.

    organic and combined inorganic compounds accumulated outside the cells as soluble reactive phosphorus (SRP), indicating an efficient regeneration of orthophosphate from various phosphorus compounds . The major forms of phosphorus are solubilized... to be the phosphate solubilizers. These solubilizers of inorganic phosphate were further screened for phosphatase production (mineralisa- tion)12 also. About 1 ml of culture suspension (1 cm cell O.D600 a = 0.1) was inoculated into 10 ml of 0.2 ?m filtered...

  11. Physical Properties of Acidic Calcium Phosphate Cements

    OpenAIRE

    2014-01-01

    The gold standard for bone replacement today, autologous bone, suffers from several disadvantages, such as the increased risk of infection due to the need for two surgeries. Degradable synthetic materials with properties similar to bone, such as calcium phosphate cements, are a promising alternative. Calcium phosphate cements are suited for a limited amount of applications and improving their physical properties could extend their use into areas previously not considered possible. For example...

  12. Using phosphate supplementation to reverse hypophosphatemia and phosphate depletion in neurological disease and disturbance.

    Science.gov (United States)

    Håglin, Lena

    2016-06-01

    Hypophosphatemia (HP) with or without intracellular depletion of inorganic phosphate (Pi) and adenosine triphosphate has been associated with central and peripheral nervous system complications and can be observed in various diseases and conditions related to respiratory alkalosis, alcoholism (alcohol withdrawal), diabetic ketoacidosis, malnutrition, obesity, and parenteral and enteral nutrition. In addition, HP may explain serious muscular, neurological, and haematological disorders and may cause peripheral neuropathy with paresthesias and metabolic encephalopathy, resulting in confusion and seizures. The neuropathy may be improved quickly after proper phosphate replacement. Phosphate depletion has been corrected using potassium-phosphate infusion, a treatment that can restore consciousness. In severe ataxia and tetra paresis, complete recovery can occur after adequate replacement of phosphate. Patients with multiple risk factors, often with a chronic disease and severe HP that contribute to phosphate depletion, are at risk for neurologic alterations. To predict both risk and optimal phosphate replenishment requires assessing the nutritional status and risk for re-feeding hypophosphatemia. The strategy for correcting HP depends on the severity of the underlying disease and the goal for re-establishing a phosphate balance to limit the consequences of phosphate depletion.

  13. Isolation of phosphate-solubilizing fungus and its application in solubilization of rock phosphates.

    Science.gov (United States)

    Wu, Yingben; He, Yuelin; Yin, Hongmei; Chen, Wei; Wang, Zhen; Xu, Lijuan; Zhang, Aiqun

    2012-12-01

    Microorganisms have been obtained to improve the agronomic value of rock phosphates (RPs), but the phosphorus solubilizing rate by these approaches is very slow. It is important to explore a high-efficient phosphate-solubilizing approach with a kind of microorganisms. This study aimed to isolate a high-efficient level of phosphate-solubilizing fungus from rhizosphere soil samples phosphate mines (Liuyang County, Hunan province, China) and apply it in solubilization of RPs. The experiments were carried out by the conventional methodology for morphological and biochemical fungus characterization and the analysis of 18s rRNA sequence. Then the effects of time, temperature, initial pH, phosphorus (P) sources, RPs concentration, shaking speed and silver ion on the content of soluble P released by this isolate were investigated. The results showed this isolate was identified as Galactomyces geotrichum P14 (P14) in GeneBank and the maximum amount of soluble P was 1252.13 mg L(-1) within 40 h in a modified phosphate growth agar's medium (without agar) where contained tricalcium phosphate (TCP) as sole phosphate source. At the same time, it could release phosphate and solubilize various rock phosphates. The isolated fungus can convert RPs from insoluble form into plant available form and therefore it hold great potential for biofertilizers to enhance soil fertility and promote plant growth.

  14. The localization within plant cells of enzymes involved in arginine biosynthesis.

    Science.gov (United States)

    Shargool, P D; Steeves, T; Weaver, M; Russell, M

    1978-04-01

    Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids. Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.

  15. The evolution of the marine phosphate reservoir.

    Science.gov (United States)

    Planavsky, Noah J; Rouxel, Olivier J; Bekker, Andrey; Lalonde, Stefan V; Konhauser, Kurt O; Reinhard, Christopher T; Lyons, Timothy W

    2010-10-28

    Phosphorus is a biolimiting nutrient that has an important role in regulating the burial of organic matter and the redox state of the ocean-atmosphere system. The ratio of phosphorus to iron in iron-oxide-rich sedimentary rocks can be used to track dissolved phosphate concentrations if the dissolved silica concentration of sea water is estimated. Here we present iron and phosphorus concentration ratios from distal hydrothermal sediments and iron formations through time to study the evolution of the marine phosphate reservoir. The data suggest that phosphate concentrations have been relatively constant over the Phanerozoic eon, the past 542 million years (Myr) of Earth's history. In contrast, phosphate concentrations seem to have been elevated in Precambrian oceans. Specifically, there is a peak in phosphorus-to-iron ratios in Neoproterozoic iron formations dating from ∼750 to ∼635 Myr ago, indicating unusually high dissolved phosphate concentrations in the aftermath of widespread, low-latitude 'snowball Earth' glaciations. An enhanced postglacial phosphate flux would have caused high rates of primary productivity and organic carbon burial and a transition to more oxidizing conditions in the ocean and atmosphere. The snowball Earth glaciations and Neoproterozoic oxidation are both suggested as triggers for the evolution and radiation of metazoans. We propose that these two factors are intimately linked; a glacially induced nutrient surplus could have led to an increase in atmospheric oxygen, paving the way for the rise of metazoan life.

  16. Capturing phosphates with iron enhanced sand filtration.

    Science.gov (United States)

    Erickson, Andrew J; Gulliver, John S; Weiss, Peter T

    2012-06-01

    Most treatment practices for urban runoff capture pollutants such as phosphorus by either settling or filtration while dissolved phosphorus, typically as phosphates, is untreated. Dissolved phosphorus, however, represents an average 45% of total phosphorus in stormwater runoff and can be more than 95%. In this study, a new stormwater treatment technology to capture phosphate, called the Minnesota Filter, is introduced. The filter comprises iron filings mixed with sand and is tested for phosphate removal from synthetic stormwater. Results indicate that sand mixed with 5% iron filings captures an average of 88% phosphate for at least 200 m of treated depth, which is significantly greater than a sand filter without iron filings. Neither incorporation of iron filings into a sand filter nor capture of phosphates onto iron filings in column experiments had a significant effect on the hydraulic conductivity of the filter at mixtures of 5% or less iron by weight. Field applications with up to 10.7% iron were operated over 1 year without detrimental effects upon hydraulic conductivity. A model is applied and fit to column studies to predict the field performance of iron-enhanced sand filters. The model predictions are verified through the predicted performance of the filters in removing phosphates in field applications. Practical applications of the technology, both existing and proposed, are presented so stormwater managers can begin implementation.

  17. Maize endophytic bacteria as mineral phosphate solubilizers.

    Science.gov (United States)

    de Abreu, C S; Figueiredo, J E F; Oliveira, C A; Dos Santos, V L; Gomes, E A; Ribeiro, V P; Barros, B A; Lana, U G P; Marriel, I E

    2017-02-16

    In the present study, we demonstrated the in vitro activity of endophytic phosphate-solubilizing bacteria (PSB). Fifty-five endophytic PSB that were isolated from sap, leaves, and roots of maize were tested for their ability to solubilize tricalcium phosphate and produce organic acid. Partial sequencing of the 16S rRNA-encoding gene showed that the isolates were from the genus Bacillus and different species of Enterobacteriaceae. The phosphate solubilization index on solid medium and phosphate solubilization in liquid medium varied significantly among the isolates. There was a statistically significant difference (P ≤ 0.05) for both, the values of phosphate-solubilizing activity and pH of the growth medium, among the isolates. Pearson correlation was statistically significant (P ≤ 0.05) between P-solubilization and pH (R = -0.38), and between the gluconic acid production and the lowering of the pH of the liquid medium at 6 (R = 0.28) and 9 days (R = 0.39). Gluconic acid production was prevalent in all the PSB studied, and Bacillus species were most efficient in solubilizing phosphate. This is the first report on the characterization of bacterial endophytes from maize and their use as potential biofertilizers. In addition, this may provide an alternative strategy for improving the phosphorus acquisition efficiency of crop plants in tropical soils.

  18. Effects of inositol phosphates on mineral utilization

    Energy Technology Data Exchange (ETDEWEB)

    Tao, S.H.; Fox, M.R.S.; Phillippy, B.Q.; Fry, B.E. Jr.; Johnson, M.L.; Johnston, M.R.

    1986-03-05

    The present study was designed to compare the effects of inositol tri-, tetra-, and pentaphosphate (IP3, IP4, and IP5) with those of phytic acid (IP6) on growth, development and mineral utilization of young quail. Day-old Japanese quail were fed a purified casein-gelatin control diet containing 20 ppm Zn with 0 or 8.33 mmoles/kg of each inositol phosphate, corresponding to 0.55% of IP6, for a week. As compared with controls, IP6 caused reduced body weight, poor feathering, severe perosis, decreased tibia Zn and ash, and decreased pancreas Zn and liver Mn. IP5 produced all the same adverse effects and tissue mineral changes as those by phytic acid, whereas birds fed IP3 or IP4 were normal. Moreover, IP3 and IP4 caused an increased tibia weight and ash. None of the above effects was produced by feeding inositol or inorganic phosphate. In a second experiment, the inositol phosphates were fed at either 8.33 or 16.66 mmoles/kg diet. Doubling inositol phosphate levels resulted in similar effects as those observed previously. Additionally, IP4 decreased pancreas Zn and IP3 increased tibia Zn. These results indicate that unlike IP6 and IP5, inositol phosphates with 4 or fewer phosphate groups, which can arise from hydrolysis of phytic acid during food processing, have very minor adverse effects but may be beneficial for bone mineralization.

  19. S-adenosyl-L-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Brown, James Mike [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Kuhlman, Christopher [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Terneus, Marcus V. [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Labenski, Matthew T. [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Lamyaithong, Andre Benja; Ball, John G. [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Lau, Serrine S. [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Valentovic, Monica A., E-mail: Valentov@marshall.edu [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States)

    2014-12-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n = 5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S + A). APAP toxicity was confirmed by an increase (p < 0.05) in plasma ALT (U/l) and liver weight/10 g body weight relative to the Veh, SAMe and S + A groups 4 h following APAP treatment. SAMe administered 1 h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S + A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S + A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. - Highlights: • Acetaminophen (APAP) toxicity protected by S-adenosylmethionine (SAMe) • 4-Hydroxynonenal adducted to sarcosine dehydrogenase • 4-Hydroxynonenal adducted to carbamoyl phosphate synthetase-1 • SAMe reduced APAP mediated CPS-1 mitochondrial leakage.

  20. Effect of the calcium to phosphate ratio of tetracalcium phosphate on the properties of calcium phosphate bone cement.

    Science.gov (United States)

    Burguera, Elena F; Guitian, Francisco; Chow, Laurence C

    2008-06-01

    Six different tetracalcium phosphate (TTCP) products were synthesized by solid state reaction at high temperature by varying the overall calcium to phosphate ratio of the synthesis mixture. The objective was to evaluate the effect of the calcium to phosphate ratio on a TTCP-dicalcium phosphate dihydrate (DCPD) cement. The resulting six TTCP-DCPD cement mixtures were characterized using X-ray diffraction analysis, scanning electron microscopy, and pH measurements. Setting times and compressive strength (CS) were also measured. Using the TTCP product with a Ca/P ratio of 2.0 resulted in low strength values (25.61 MPa) when distilled water was used as the setting liquid, even though conversion to hydroxyapatite was not prevented, as confirmed by X-ray diffraction. The suspected CaO presence in this TTCP may have affected the cohesiveness of the cement mixture but not the cement setting reaction, however no direct evidence of CaO presence was found. Lower Ca/P ratio products yielded cements with CS values ranging from 46.7 MPa for Ca/P ratio of 1.90 to 38.32 MPa for Ca/P ratio of 1.85. When a dilute sodium phosphate solution was used as the setting liquid, CS values were 15.3% lower than those obtained with water as the setting liquid. Setting times ranged from 18 to 22 min when water was the cement liquid and from 7 to 8 min when sodium phosphate solution was used, and the calcium to phosphate ratio did not have a marked effect on this property.

  1. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

    Directory of Open Access Journals (Sweden)

    Benedikt Frieg

    2016-02-01

    Full Text Available Glutamine synthetase (GS catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

  2. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    Science.gov (United States)

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  3. Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli.

    Science.gov (United States)

    Okano, Hiroyuki; Hwa, Terence; Lenz, Peter; Yan, Dalai

    2010-12-03

    Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR(-)/AT(+) mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR(-) mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.

  4. MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES(1).

    Science.gov (United States)

    Ghoshroy, Sohini; Robertson, Deborah L

    2012-06-01

    Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum-likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well-supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile-haptophyte-cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences

  5. The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

    Science.gov (United States)

    Hauf, Ksenia; Kayumov, Airat; Gloge, Felix; Forchhammer, Karl

    2016-02-12

    TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

  6. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    Science.gov (United States)

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  7. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M

    2015-11-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

  8. Peptide markers of aminoacyl tRNA synthetases facilitate taxa counting in metagenomic data

    Directory of Open Access Journals (Sweden)

    Persi Erez

    2012-02-01

    Full Text Available Abstract Background Taxa counting is a major problem faced by analysis of metagenomic data. The most popular method relies on analysis of 16S rRNA sequences, but some studies employ also protein based analyses. It would be advantageous to have a method that is applicable directly to short sequences, of the kind extracted from samples in modern metagenomic research. This is achieved by the technique proposed here. Results We employ specific peptides, deduced from aminoacyl tRNA synthetases, as markers for the occurrence of single genes in data. Sequences carrying these markers are aligned and compared with each other to provide a lower limit for taxa counts in metagenomic data. The method is compared with 16S rRNA searches on a set of known genomes. The taxa counting problem is analyzed mathematically and a heuristic algorithm is proposed. When applied to genomic contigs of a recent human gut microbiome study, the taxa counting method provides information on numbers of different species and strains. We then apply our method to short read data and demonstrate how it can be calibrated to cope with errors. Comparison to known databases leads to estimates of the percentage of novelties, and the type of phyla involved. Conclusions A major advantage of our method is its simplicity: it relies on searching sequences for the occurrence of just 4000 specific peptides belonging to the S61 subgroup of aaRS enzymes. When compared to other methods, it provides additional insight into the taxonomic contents of metagenomic data. Furthermore, it can be directly applied to short read data, avoiding the need for genomic contig reconstruction, and taking into account short reads that are otherwise discarded as singletons. Hence it is very suitable for a fast analysis of next generation sequencing data.

  9. Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India.

    Science.gov (United States)

    Lumb, Vanshika; Das, Manoj K; Singh, Neeru; Dev, Vas; Khan, Wajihullah; Sharma, Yagya D

    2011-06-01

    With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine

  10. Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots.

    Science.gov (United States)

    Konishi, Noriyuki; Ishiyama, Keiki; Beier, Marcel Pascal; Inoue, Eri; Kanno, Keiichi; Yamaya, Tomoyuki; Takahashi, Hideki; Kojima, Soichi

    2016-12-21

    Glutamine synthetase (GS) catalyzes a reaction that incorporates ammonium into glutamate and yields glutamine in the cytosol and chloroplasts. Although the enzymatic characteristics of the GS1 isozymes are well known, their physiological functions in ammonium assimilation and regulation in roots remain unclear. In this study we show evidence that two cytosolic GS1 isozymes (GLN1;2 and GLN1;3) contribute to ammonium assimilation in Arabidopsis roots. Arabidopsis T-DNA insertion lines for GLN1;2 and GLN1;3 (i.e. gln1;2 and gln1;3 single-mutants), the gln1;2:gln1;3 double-mutant, and the wild-type accession (Col-0) were grown in hydroponic culture with variable concentrations of ammonium to compare their growth, and their content of nitrogen, carbon, ammonium, and amino acids. GLN1;2 and GLN1;3 promoter-dependent green fluorescent protein was observed under conditions with or without ammonium supply. Loss of GLN1;2 caused significant suppression of plant growth and glutamine biosynthesis under ammonium-replete conditions. In contrast, loss of GLN1;3 caused slight defects in growth and Gln biosynthesis that were only visible based on a comparison of the gln1;2 single- and gln1;2:gln1;3 double-mutants. GLN1;2, being the most abundantly expressed GS1 isozyme, markedly increased following ammonium supply and its promoter activity was localized at the cortex and epidermis, while GLN1;3 showed only low expression at the pericycle, suggesting their different physiological contributions to ammonium assimilation in roots. The GLN1;2 promoter-deletion analysis identified regulatory sequences required for controlling ammonium-responsive gene expression of GLN1;2 in Arabidopsis roots. These results shed light on GLN1 isozyme-specific regulatory mechanisms in Arabidopsis that allow adaptation to an ammonium-replete environment.

  11. Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

    Directory of Open Access Journals (Sweden)

    Diego Benítez

    2016-04-01

    Full Text Available The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS, catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate.A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively, and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl diamine derivatives and an N5-substituted paullone (MOL2008 halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range and Leishmania infantum promastigotes (EC50 = 12 μM, respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds.Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent

  12. Functional role of C-terminal domain of Thermus thermophilus leucyl-tRNA synthetase

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    Tukalo M. A.

    2010-11-01

    Full Text Available Aim. To study a role of C-terminal domain of T. thermophilus leucyl-tRNA synthetase (LeuRSTT in the reactions of aminoacylation and editing. Methods. A mutant of LeuRSTT without C- terminal domain (ΔС was obtained by the method of mutagenesis. The kinetic constants in aminoacylation reaction catalyzed by LeuRS and its mutant (ΔС were determined by the methods of equilibrium enzyme kinetics. To evaluate the contribution of C-terminal domain to interaction of the enzyme with tRNALeu, Kd of a complex between tRNA and LeuRSTT and its mutant ΔС was determined by fluorescence titration. Results. The C-terminal domain is shown to play a significant role in the aminoacylation and editing reactions of LeuRSTT and not essential for the activity in the reaction of amino acid activation. The kinetic parameters of aminoacylation of tRNALeu and tRNATyr by LeuRS and ΔС mutant were also determined, their analysis suggests that the C-domain is not critical for the manifestation of specificity of the enzyme in the recognition of homologous RNAs. At the same time a significant influence of the C-terminal domain on the value of catalytic constant was shown. At the domain deletion the kcat value is lower by 152-fold. Conclusion. The C-terminal domain of LeuRSTT is evolutionarily acquired to enhance the rate of catalysis in the aminoacylation and editing reactions, and makes no significant contribution to the specificity of the enzyme in the recognition of tRNA.

  13. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

    Science.gov (United States)

    Xia, Mengna; Malkaram, Sridhar A; Zempleni, Janos

    2013-11-01

    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

  14. Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe.

    Science.gov (United States)

    Perysinakis, A; Kinghorn, J R; Drainas, C

    1995-06-01

    Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

  15. Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen

    2008-01-01

    BACKGROUND:Currently, one of the tough problems for the application of bioartiifcial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxiifcation. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS:hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and ampliifed. Then hGS mRNA was measured by semi-quantitative RT-PCR;hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS:The expression of hGS mRNA in HepG2/pLNChGS cells (8.306±0.336) and HepG2/pLNAFhGS cells (21.358±1.716) was much stronger than in control cells (P CONCLUSION:The constructed hepatocytes (HepG2 cells) with speciifc high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

  16. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

    Science.gov (United States)

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-02-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

  17. Effect of nitrate on activities and transcript levels of nitrate reductase and glutamine synthetase in rice

    Institute of Scientific and Technical Information of China (English)

    CAO Yun; FAN Xiao-Rong; SUN Shu-Bin; XU Guo-Hua; HU Jiang; SHEN Qi-Rong

    2008-01-01

    Real-time polymerase chain reaction analysis was used to compare the effect of NO-3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS),and the transcript levels of two NR genes,OsNia1 and OsNia2,two cytceolic GS1 genes,OsGln1;1 and OsGln1;2,and one plastid GS2 gene OsGln2,in two rice (Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ).Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+ nutrition.Supply of NO-3 increased NR activity in both leaves and roots.Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1.NO-3 also caused an increase in GS activity,but had a complex effect on the expression of the three GS genes.In roots,the OsGln1;1 transcript increased,but OsGln1;2 decreased.In leaves,NO-3 had no effect on the GS1 expression,but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N.These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes.In addition,the NO-3 induced increase of biomass,NR activity,GS activity,and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ,indicating a stronger response of NG to NO-3 nutrition than YJ.

  18. Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats.

    Science.gov (United States)

    van der Hel, W Saskia; Hessel, Ellen V S; Bos, Ineke W M; Mulder, Sandra D; Verlinde, Suzanne A M W; van Eijsden, Pieter; de Graan, Pierre N E

    2014-12-01

    Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.

  19. Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

    Science.gov (United States)

    Guerreiro, Juliano R.; Lameu, Claudiana; Oliveira, Eduardo F.; Klitzke, Clécio F.; Melo, Robson L.; Linares, Edlaine; Augusto, Ohara; Fox, Jay W.; Lebrun, Ivo; Serrano, Solange M. T.; Camargo, Antonio C. M.

    2009-01-01

    Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases. PMID:19491403

  20. Poly(Adp-ribose) synthetase inhibition prevents lipopolysaccharide-induced peroxynitrite mediated damage in diaphragm.

    Science.gov (United States)

    Ozdülger, Ali; Cinel, Ismail; Unlü, Ali; Cinel, Leyla; Mavioglu, Ilhan; Tamer, Lülüfer; Atik, Ugur; Oral, Ugur

    2002-07-01

    Although the precise mechanism by which sepsis causes impairment of respiratory muscle contractility has not been fully elucidated, oxygen-derived free radicals are thought to play an important role. In our experimental study, the effects of poly(ADP-ribose) synthetase (PARS) inhibition on the diaphragmatic Ca(2+)-ATPase, malondialdehyde (MDA), and 3-nitrotyrosine (3-NT) levels and additionally histopathology of the diaphragm in lipopolysaccharide (LPS)-induced endotoxemia are investigated.Thirty-two male Wistar rats, weighing between 180-200 g were randomly divided into four groups. The first group (control; n=8) received saline solution and the second (LPS group; n=8) 10 mgkg(-1) LPS i.p. 3-Aminobenzamide (3-AB) as a PARS inhibitor; was given to the third group (C+3-AB, n=8) 20 min before administration of saline solution while the fourth group (LPS+3-AB, n=8) received 3-AB 20 min before LPS injection. Six hours later, under ketamin/xylasine anesthesia diapraghmatic specimens were obtained and the rats were decapitated. Diaphragmatic specimens were divided into four parts, three for biochemical analyses and one for histopathologic assessment. In the LPS group, tissue Ca(2+)-ATPase levels were found to be decreased and tissue MDA and 3-NT levels were found to be increased (P<0.05). In the LPS+3-AB group, 3-AB pretreatment inhibited the increase in MDA and 3-NT levels and Ca(2+)-ATPase activity remained similar to those in the control group (P<0.05). Histopathologic examination of diaphragm showed edema between muscle fibers only in LPS group. PARS inhibition with 3-AB prevented not only lipid peroxidation but also the decrease of Ca(2+)-ATPase activity in endotoxemia. These results highlights the importance of nitric oxide (NO)-peroxynitrite (ONOO(-))-PARS pathway in preventing free radical mediated injury. PARS inhibitors should further be investigated as a new thearapetic alternative in sepsis treatment.

  1. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase.

    Science.gov (United States)

    Bowles, Tawnya L; Kim, Randie; Galante, Joseph; Parsons, Colin M; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J

    2008-10-15

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by approximately 50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

  2. Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Ru Li; Jing Gu; Peng Chen; Zhiping Zhang; Jiaoyu Deng; XianEn Zhang

    2011-01-01

    Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA,which is involved in many catabolic and anabolic pathways.Although this enzyme has been studied for many years in many organisms,the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown.Here,the putative acs gene of M.tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli.The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed.The optimal pH and temperature,and the kinetic parameters of Mt-Acs were determined.To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs,its mutant K617R was also generated.Determination of the enzymatic activity suggests that Lys-617 is critical for its function.We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor,which resulted in the decrease of its activity.CoA,the substrate for AcCoA formation,inhibited the auto-acetylation.Furthermore,the silent information regulator (Sir2) of M.tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation,which resulted in activation of Acs.These results may provide more insights into the physiological roles of Mt-Acs in M.tuberculosis central metabolism.

  3. Arabidopsis plastidial folylpolyglutamate synthetase is required for seed reserve accumulation and seedling establishment in darkness.

    Directory of Open Access Journals (Sweden)

    Hongyan Meng

    Full Text Available Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3 of the plastidial folylpolyglutamate synthetase gene (AtDFB was defective in seed reserves and skotomorphogenesis. Lower carbon (C and higher nitrogen (N content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3-. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3- conditions, and further enhanced under NO3- limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3- during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3- as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis.

  4. Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus.

    Science.gov (United States)

    Miao, G H; Hirel, B; Marsolier, M C; Ridge, R W; Verma, D P

    1991-01-01

    A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

  5. Selenophosphate Synthetase 1 is an Essential Protein with Roles in Regulation of Redox Homeostasis in Mammals

    Science.gov (United States)

    Tobe, Ryuta; Carlson, Bradley A.; Huh, Jang Hoe; Castro, Nadia P.; Xu, Xue-Ming; Tsuji, Petra A.; Lee, Sang-Goo; Bang, Jeyoung; Na, Ji-Woon; Kong, Young-Yun; Beaglehole, Daniel; Southon, Eileen; Seifried, Harold; Tessarollo, Lino; Salomon, David S.; Schweizer, Ulrich; Gladyshev, Vadim N.; Hatfield, Dolph L.; Lee, Byeong Jae

    2016-01-01

    Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogs, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyzes the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1 knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by E8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development, and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione-S-transferase omega 1. To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Further, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homeostasis and controlling cell growth. PMID:27208177

  6. Metabolic indicators of drought stress tolerance in wheat: glutamine synthetase isoenzymes and Rubisco.

    Science.gov (United States)

    Nagy, Zoltán; Németh, Edit; Guóth, Adrienn; Bona, Lajos; Wodala, Barnabás; Pécsváradi, Attila

    2013-06-01

    Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance.

  7. Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase.

    Science.gov (United States)

    Ortiz, Jorge G Muñiz; Opoka, Robert; Kane, Daniel; Cartwright, Iain L

    2009-02-01

    Chronic exposure to arsenic-contaminated drinking water can lead to a variety of serious pathological outcomes. However, differential responsiveness within human populations suggests that interindividual genetic variation plays an important role. We are using Drosophila to study toxic metal response pathways because of unrivalled access to varied genetic approaches and significant demonstrable overlap with many aspects of mammalian physiology and disease phenotypes. Genetic analysis (via chromosomal segregation and microsatellite marker-based recombination) of various wild-type strains exhibiting relative susceptibility or tolerance to the lethal toxic effects of arsenite identified a limited X-chromosomal region (16D-F) able to confer a differential response phenotype. Using an FRT-based recombination approach, we created lines harboring small, overlapping deficiencies within this region and found that relative arsenite sensitivity arose when the dose of the glutathione synthetase (GS) gene (located at 16F1) was reduced by half. Knockdown of GS expression by RNA interference (RNAi) in cultured S2 cells led to enhanced arsenite sensitivity, while GS RNAi applied to intact organisms dramatically reduced the concentration of food-borne arsenite compatible with successful growth and development. Our analyses, initially guided by observations on naturally occurring variants, provide genetic proof that an optimally functioning two-step glutathione (GSH) biosynthetic pathway is required in vivo for a robust defense against arsenite; the enzymatic implications of this are discussed in the context of GSH supply and demand under arsenite-induced stress. Given an identical pathway for human GSH biosynthesis, we suggest that polymorphisms in GSH biosynthetic genes may be an important contributor to differential arsenic sensitivity and exposure risk in human populations.

  8. Astrocyte glutamine synthetase: importance in hyperammonemic syndromes and potential target for therapy.

    Science.gov (United States)

    Brusilow, Saul W; Koehler, Raymond C; Traystman, Richard J; Cooper, Arthur J L

    2010-10-01

    Many theories have been advanced to explain the encephalopathy associated with chronic liver disease and with the less common acute form. A major factor contributing to hepatic encephalopathy is hyperammonemia resulting from portacaval shunting and/or liver damage. However, an increasing number of causes of hyperammonemic encephalopathy have been discovered that present with the same clinical and laboratory features found in acute liver failure, but without liver failure. Here, we critically review the physiology, pathology, and biochemistry of ammonia (i.e., NH3 plus NH4+) and show how these elements interact to constitute a syndrome that clinicians refer to as hyperammonemic encephalopathy (i.e., acute liver failure, fulminant hepatic failure, chronic liver disease). Included will be a brief history of the status of ammonia and the centrality of the astrocyte in brain nitrogen metabolism. Ammonia is normally detoxified in the liver and extrahepatic tissues by conversion to urea and glutamine, respectively. In the brain, glutamine synthesis is largely confined to astrocytes, and it is generally accepted that in hyperammonemia excess glutamine compromises astrocyte morphology and function. Mechanisms postulated to account for this toxicity will be examined with emphasis on the osmotic effects of excess glutamine (the osmotic gliopathy theory). Because hyperammonemia causes osmotic stress and encephalopathy in patients with normal or abnormal liver function alike, the term "hyperammonemic encephalopathy" can be broadly applied to encephalopathy resulting from liver disease and from various other diseases that produce hyperammonemia. Finally, the possibility that a brain glutamine synthetase inhibitor may be of therapeutic benefit, especially in the acute form of liver disease, is discussed.

  9. Interstitial lung disease in anti-synthetase syndrome: Initial and follow-up CT findings

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    Debray, Marie-Pierre, E-mail: marie-pierre.debray@bch.aphp.fr [AP-HP, Bichat-Claude Bernard Hospital, Department of Radiology, 46, rue Henri Huchard, 75877 Paris Cedex 18 (France); Borie, Raphael, E-mail: raphael.borie@bch.aphp.fr [AP-HP, Bichat-Claude Bernard Hospital, Department of Pneumology A and Centre de Compétence Maladies Pulmonaires rares, DHU Fire 46, rue Henri Huchard, 75877 Paris Cedex 18 (France); Inserm, U1152, Paris (France); Revel, Marie-Pierre, E-mail: marie-pierre.revel@htd.aphp.fr [AP-HP, Cochin Hospital, Department of Radiology, 27, Rue du Fg Saint Jacques, 75679 Paris Cedex 14 (France); Naccache, Jean-Marc, E-mail: jean-marc.naccache@tnn.aphp.fr [AP-HP, Avicenne Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, Bobigny (France); AP-HP, Tenon Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, 4, rue de la Chine, 75020 Paris (France); Khalil, Antoine, E-mail: antoine.khalil@tnn.aphp.fr [AP-HP, Tenon Hospital, Department of Radiology, 4, rue de la Chine, 75020 Paris (France); Toper, Cécile, E-mail: cecile.toper@gmail.com [AP-HP, Tenon Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, 4, rue de la Chine, 75020 Paris (France); Israel-Biet, Dominique, E-mail: dominique.israel-biet@egp.aphp.fr [Université Paris Descartes and AP-HP, Department of Pneumology, Georges Pompidou European Hospital, 20, rue Leblanc, 75015 Paris (France); and others

    2015-03-15

    Purpose: To describe the initial and follow-up CT features of interstitial lung disease associated with anti-synthetase syndrome (AS-ILD). Materials and methods: Two independent thoracic radiologists retrospectively analysed thin-section CT images obtained at diagnosis of AS-ILD in 33 patients (17 positive for anti-Jo1, 13 for anti-PL12, and three for anti-PL7 antibodies). They evaluated the pattern, distribution and extent of the CT abnormalities. They also evaluated the change in findings during follow-up (median 27 months; range 13–167 months) in 26 patients. Results: At diagnosis, ground-glass opacities (100%), reticulations (87%) and traction bronchiectasis (76%) were the most common CT findings. Consolidations were present in 45% of patients. A non-specific interstitial pneumonia (NSIP), organizing pneumonia (OP) or mixed NSIP-OP CT pattern were observed in 15 out of 33 (45%), seven out of 33 (21%) and eight out of 33 (24%) patients, respectively, whereas the CT pattern was indeterminate in three patients. During follow-up, consolidations decreased or disappeared in 11 out of 12 patients (92%), among which seven within the first 6 months, but honeycombing progressed or appeared in ten out of 26 patients (38%) and overall disease extent increased in nine out of 26 patients (35%). Conclusion: CT features at diagnosis of AS-ILD mainly suggest NSIP and OP, isolated or in combination. Consolidations decrease or disappear in most cases but the disease may progress to fibrosis in more than one third of patients.

  10. Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli

    NARCIS (Netherlands)

    van Veen, H.W; Abee, T.; Kortstee, G.J J; Konings, W.N; Zehnder, A.J B

    1994-01-01

    P-i transport via the phosphate inorganic transport system (Pit) of Escherichia coil was studied in natural and artificial membranes. P-i uptake via Pit is dependent on the presence of divalent cations, like Mg2+, Ca2+, Co2+, or Mn2+, which form a soluble, neutral metal phosphate (MeHPO(4)) complex.

  11. Biological Removal of Phosphate Using Phosphate Solubilizing Bacterial Consortium from Synthetic Wastewater: A Laboratory Scale

    Directory of Open Access Journals (Sweden)

    Dipak Paul

    2015-01-01

    Full Text Available Biological phosphate removal is an important process having gained worldwide attention and widely used for removing phosphorus from wastewater. The present investigation was aimed to screen the efficient phosphate solubilizing bacterial isolates and used to remove phosphate from synthetic wastewater under shaking flasks conditions. Pseudomonas sp. JPSB12, Enterobacter sp. TPSB20, Flavobacterium sp. TPSB23 and mixed bacterial consortium (Pseudomonas sp. JPSB12+Enterobacter sp. TPSB20+Flavobacterium sp. TPSB23 were used for the removal of phosphate. Among the individual strains, Enterobacter sp. TPSB20 was removed maximum phosphate (61.75% from synthetic wastewater in presence of glucose as a carbon source. The consortium was effectively removed phosphate (74.15-82.50% in the synthetic wastewater when compared to individual strains. The pH changes in culture medium with time and extracellular phosphatase activity (acid and alkaline were also investigated. The efficient removal of phosphate by the consortium may be due to the synergistic activity among the individual strains and phosphatase enzyme activity. The use of bacterial consortium in the remediation of phosphate contaminated aquatic environments has been discussed.

  12. Cloning and characterization of a glucose 6-phosphate/phosphate translocator from Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    姜华武; 佃蔚敏; 刘非燕; 吴平

    2003-01-01

    Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy, and glucose 6-phosphate is the preferred hexose phosphate taken up by nongreen plastids. A cDNA clone encoding glucose 6-phosphate/phosphate translocator (GPT) was isolated from a cDNA library of immature seeds of rice and named as OsGPT. The cDNA has one uninterrupted open reading frame encoding a 42 kDa polypeptide possessing transit peptide consisting of 70 amino acid residues. The OsGPT gene maps on chromosome 8 of rice and is linked to the quantitative trait locus for 1000-grain weight. The expression of OsGPT is mainly restricted to heterotrophic tissues. These results suggest that glucose 6-phosphate imported via GPT can be used for starch biosynthesis in rice nongreen plastids.

  13. Solid state 31NMR studies of the conversion of amorphous tricalcium phosphate to apatitic tricalcium phosphate.

    Science.gov (United States)

    Roberts, J E; Heughebaert, M; Heughebaert, J C; Bonar, L C; Glimcher, M J; Griffin, R G

    1991-12-01

    The hydrolytic conversion of a solid amorphous calcium phosphate of empirical formula Ca9 (PO4)6 to a poorly crystalline apatitic phase, under conditions where Ca2+ and PO4(3-) were conserved, was studied by means of solid-state magic-angle sample spinning 31P-NMR (nuclear magnetic resonance). Results showed a gradual decrease in hydrated amorphous calcium phosphate and the formation of two new PO4(3-)-containing components: an apatitic component similar to poorly crystalline hydroxyapatite and a protonated PO4(3-), probably HPO4(2-) in a dicalcium phosphate dihydrate (DCPD) brushite-like configuration. This latter component resembles the brushite-like HPO4(2-) component previously observed by 31P-NMR in apatitic calcium phosphates of biological origin. Results were consistent with previous studies by Heughebaert and Montel [18] of the kinetics of the conversion of amorphous calcium phosphate to hydroxyapatite under the same conditions.

  14. A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

    Directory of Open Access Journals (Sweden)

    Nederbragt Alexander J

    2009-08-01

    Full Text Available Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS. Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454" and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully

  15. The effect of gamma irradiation on astaxanthin synthetase encoding gene in two mutant strains of Phaffia rhodozyma.

    Directory of Open Access Journals (Sweden)

    Naeimeh Najafi

    2013-09-01

    Full Text Available Astaxanthin, an orange-red carotenoid pigment, acts as a protective agent against oxidative damage to cells in vivo. The astaxanthin synthetase gene (crtS size consists of 3995 bp. This gene has been suggested to catalyse β-carotene to astaxanthin in Phaffia rhodozyma. The aim of this research was to find any possible changes in this gene in two mutant strains, Gam1 and Gam2 (with high astaxanthin pigment production, previously created by gamma irradiation.The astaxanthin synthetase gene sequence of Phaffia rhodozyma in the NCBI Gene bank was used to design primer. In Gam1, this gene was amplified using primers Asta F1, Asta R2, Asta F3, Asta R4. In Gam2, primers asta F1, asta R4 were used to amplify the gene. The amplified fragments were 8 sequenced using primers Asta F1, Asta R1, Asta F2, Asta R2, Asta F3, Asta R3 and Asta F4, Asta R4. Astaxanthin synthetase gene from two mutant strains, Gam1 and Gam2 were amplified using PCR. The amplified products were sequenced and aligned using the ClustalW software.The comparison of this gene showed 98% and 99% similarities between the reference sequence and Gam1 and Gam2 mutant strains, respectively, whereas the comparison of this gene in Gam1 and Gam2 mutant strains showed 97% similarity. However, the deduced proteins showed 78% and 83% between the reference protein obtained from the wild type and Gam1 and Gam2, respectively. This similarity was 75% between the mutant strains.

  16. Phosphoribosylpyrophosphate synthetase superactivity and recurrent infections is caused by a p.Val142Leu mutation in PRS-I.

    Science.gov (United States)

    Moran, Rocio; Kuilenburg, André B P; Duley, John; Nabuurs, Sander B; Retno-Fitri, Aditia; Christodoulou, John; Roelofsen, Jeroen; Yntema, Helger G; Friedman, Neil R; van Bokhoven, Hans; de Brouwer, Arjan P M

    2012-02-01

    We identified a novel missense mutation, c.424G>C (p.Val142Leu) in PRPS1 in a patient with uric acid overproduction without gout but with developmental delay, hypotonia, hearing loss, and recurrent respiratory infections. The uric acid overproduction accompanying this combination of symptoms suggests that the patient presented with phosphoribosylpyrophosphate (PRPP) synthetase superactivity, but recurrent infections have not been associated with superactivity until now. However, recurrent infections are a prominent feature of patients with Arts syndrome, which is caused by PRPS1 loss-of-function mutations, indicating that the patient reported here has an intermediate phenotype. Molecular modeling predicts that the p.Val142Leu change affects both allosteric sites that are involved in inhibition of PRPS1 and the ATP-binding site, which suggests that this substitution can result both in a gain-of-function and loss-of-function of PRPP synthetase. This finding is in line with the normal PRPP synthetase activity in fibroblasts and the absence of activity in erythrocytes of the present patient. We postulate that the overall effect of the p.Val142Leu change on protein activity is determined by the cell type, being a gain-of-function in proliferating cells and a loss-of-function in postmitotic cells. Our results show that missense mutations in PRPS1 can cause a continuous spectrum of features ranging from progressive non-syndromic postlingual hearing impairment to uric acid overproduction, neuropathy, and recurrent infections depending on the functional sites that are affected.

  17. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

    Directory of Open Access Journals (Sweden)

    Siliakus Melvin

    2010-04-01

    Full Text Available Abstract Background Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6. Results Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms. Conclusion The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.

  18. Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

    Science.gov (United States)

    Apfel, C M; Takács, B; Fountoulakis, M; Stieger, M; Keck, W

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

  19. MS_RHII-RSD, a dual-function RNase HII-(p)ppGpp synthetase from Mycobacterium smegmatis.

    Science.gov (United States)

    Murdeshwar, Maya S; Chatterji, Dipankar

    2012-08-01

    In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

  20. Solvothermal synthesis of strontium phosphate chloride nanowire

    Science.gov (United States)

    Lam, W. M.; Wong, C. T.; Li, Z. Y.; Luk, K. D. K.; Chan, W. K.; Yang, C.; Chiu, K. Y.; Xu, B.; Lu, W. W.

    2007-08-01

    Strontium phosphate chloride nanowire was synthesized via a solvothermal treatment of strontium tri-polyphosphate and Collin salt in 1,4-dioxane at 150 °C. The effects of 1,4-dioxane concentration on particle morphology, crystallinity and phase purity were investigated in this study. The specimen morphology was analyzed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). When the concentration of 1,4-dioxane was below 10%, micron-sized whisker was the dominant form. At 20-25% concentration of 1,4-dioxane, strontium phosphate chloride single-crystalline nanowire was 31±12 nm in diameter and 1.43±0.6 μm in length with an aspect ratio of 52.28±29.41. X-ray diffraction (XRD) pattern of this nanowire matched with that of strontium phosphate chloride (JCPDS #083-0973). When 1,4-dioxane concentration exceeded 25%, nanorod aggregate was the dominant form instead of nanowire. At 20-25% 1,4-dioxane concentration suitable strontium concentration combine with high chemical potential environment favors the formation of nanowires. By adding 1,4-dioxane impure phase such as β-strontium hydrogen phosphate, nanorod formation was suppressed. This method provides an efficient way to synthesize high aspect ratio strontium phosphate chloride nanowire. It has potential bioactive nanocomposite, high mechanical performance bioactive bone cement filler and fluorescent material applications.

  1. Sodium phosphate as an ergogenic aid.

    Science.gov (United States)

    Buck, Christopher L; Wallman, Karen E; Dawson, Brian; Guelfi, Kym J

    2013-06-01

    Legal nutritional ergogenic aids can offer athletes an additional avenue to enhance their performance beyond what they can achieve through training. Consequently, the investigation of new nutritional ergogenic aids is constantly being undertaken. One emerging nutritional supplement that has shown some positive benefits for sporting performance is sodium phosphate. For ergogenic purposes, sodium phosphate is supplemented orally in capsule form, at a dose of 3-5 g/day for a period of between 3 and 6 days. A number of exercise performance-enhancing alterations have been reported to occur with sodium phosphate supplementation, which include an increased aerobic capacity, increased peak power output, increased anaerobic threshold and improved myocardial and cardiovascular responses to exercise. A range of mechanisms have been posited to account for these ergogenic effects. These include enhancements in 2,3-Diphosphoglycerate (2,3-DPG) concentrations, myocardial efficiency, buffering capacity and adenosine triphosphate/phosphocreatine synthesis. Whilst there is evidence to support the ergogenic benefits of sodium phosphate, many studies researching this substance differ in terms of the administered dose and dosing protocol, the washout period employed and the fitness level of the participants recruited. Additionally, the effect of gender has received very little attention in the literature. Therefore, the purpose of this review is to critically examine the use of sodium phosphate as an ergogenic aid, with a focus on identifying relevant further research.

  2. Sodium ascorbyl phosphate in topical microemulsions.

    Science.gov (United States)

    Spiclin, Polona; Homar, Miha; Zupancic-Valant, Andreja; Gasperlin, Mirjana

    2003-04-30

    Sodium ascorbyl phosphate is a hydrophilic derivative of ascorbic acid, which has improved stability arising from its chemical structure. It is used in cosmetic and pharmaceutical preparations since it has many favorable effects in the skin, the most important being antioxidant action. In order to achieve this, it has to be converted into free ascorbic acid by enzymatic degradation in the skin. In the present work, o/w and w/o microemulsions composed of the same ingredients, were selected as carrier systems for topical delivery of sodium ascorbyl phosphate. We showed that sodium ascorbyl phosphate was stable in both types of microemulsion with no significant influence of its location in the carrier system. To obtain liquid microemulsions appropriate for topical application, their viscosity was increased by adding thickening agents. On the basis of rheological characterization, 4.00% (m/m) colloidal silica was chosen as a suitable thickening agent for w/o microemulsions and 0.50% (m/m) xanthan gum for the o/w type. The presence of thickening agent and the location of sodium ascorbyl phosphate in the microemulsion influenced the in vitro drug release profiles. When incorporated in the internal aqueous phase, sustained release profiles were observed. This study confirmed microemulsions as suitable carrier systems for topical application of sodium ascorbyl phosphate.

  3. Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

    Directory of Open Access Journals (Sweden)

    Miller Andrew D

    2003-06-01

    Full Text Available Abstract Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common

  4. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Buddha,M.; Crane, B.

    2005-01-01

    The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNATrp with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.

  5. Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma.

    Science.gov (United States)

    Joseph, Nancy M; Ferrell, Linda D; Jain, Dhanpat; Torbenson, Michael S; Wu, Tsung-Teh; Yeh, Matthew M; Kakar, Sanjay

    2014-01-01

    Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant

  6. Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17.

    Science.gov (United States)

    Milne, J C; Roy, R S; Eliot, A C; Kelleher, N L; Wokhlu, A; Nickels, B; Walsh, C T

    1999-04-13

    In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.

  7. [Phosphate solubilization of Aureobasidium pullulan F4 and its mechanism].

    Science.gov (United States)

    Wang, Dan; Zhan, Jing; Sun, Qing-Ye

    2014-07-01

    The Aureobasidium pullulans F4 was isolated from the rhizosphere of Hippochaete ramosissimum in Tongguanshan mine wasteland in Tongling City, Anhui Province. Liquid culture was conducted with four kinds of phosphorus sources, calcium phosphate, aluminum phosphate, ferric phosphate and rock phosphate to determine the pH, dissolved phosphorus, phosphorus in the bacteria and organic acid in the solution. The results showed that the phosphate solubilization by A. pullulans F4 varied with phosphorus sources, which decreased in order of aluminum phosphate > ferric phosphate, calcium phosphate > rock phosphate. The amounts of dissolved phosphorus in the different treatments were all higher than 200 mg x L(-1). The pH of the medium dropped immediately in 48 h, and the aluminum phosphate and ferric phosphate treatments showed a greater decrease in pH than the calcium phosphate and rock phosphate treatments. The organic acid synthesized by A. pullulans F4 included oxalic acid, citric acid and tartaric acid, and oxalic acid, among which oxalic acid was the dominated component. The phosphate dissolving capacity of A. pullulans F4 showed no significant correlation with organic acid, but significantly correlated with the pH. The available phosphorus was significantly improved with the combined application of A. pullulans F4 and glucose, suggesting A. pullulans F4 was a potent candidate for remediation of copper mine wastelands.

  8. Phosphate Phosphors for Solid-State Lighting

    CERN Document Server

    Shinde, Kartik N; Swart, H C; Park, Kyeongsoon

    2012-01-01

    The idea for this book arose out of the realization that, although excellent surveys and a phosphor handbook are available, there is no single source covering the area of phosphate based phosphors especially for lamp industry. Moreover, as this field gets only limited attention in most general books on luminescence, there is a clear need for a book in which attention is specifically directed toward this rapidly growing field of solid state lighting and its many applications. This book is aimed at providing a sound introduction to the synthesis and optical characterization of phosphate phosphor for undergraduate and graduate students as well as teachers and researchers. The book provides guidance through the multidisciplinary field of solid state lighting specially phosphate phosphors for beginners, scientists and engineers from universities, research organizations, and especially industry. In order to make it useful for a wide audience, both fundamentals and applications are discussed, together.

  9. Computational Design of Biomimetic Phosphate Scavengers

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Wood, Elizabeth Baker; Truelsen, Sigurd Friis

    2015-01-01

    for phosphorus recovery, as well as improving existing techniques, has increased. In this study we apply a hybrid simulation approach of molecular dynamics and quantum mechanics to investigate the binding modes of phosphate anions by a small intrinsically disordered peptide. Our results confirm...... that the conformational ensemble of the peptide is significantly changed, or stabilized, by the binding of phosphate anions and that binding does not take place purely as a result of a stable P-loop binding nest, but rather that multiple binding modes may be involved. Such small synthetic peptides capable of binding...... phosphate could be the starting point of new novel technological approaches toward phosphorus recovery, and they represent an excellent model system for investigating the nature and dynamics of functional de novo designed intrinsically disordered proteins....

  10. Solid state NMR study calcium phosphate ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Miquel, J.L.; Facchini, L.; Legrand, A.P. (Laboratoire de Physique Quantique, Paris (France). CNRS, URA421, ESPCI); Rey, C. (CNRS, Toulouse (France). ENSC. Laboratoire de Physico-chimie des Solides); Lemaitre, J. (EPF Lausanne (France). Laboratoire de Technologie des Poudres)

    1990-04-01

    High-resolution {sup 31}P and {sup 1}H NMR spectra at 40 and 121 MHz {sup 31}P and 300 MHz {sup 1}H of synthetic and biological samples of calcium phosphates have been obtained by magic angle spinning (MAS) at spinning speeds up to 6.5 kHz, and high power proton decoupling. The samples include crystalline hydroxyapatite, a deficient hydroxyapatite characterized by a Ca/P atomic ratio of 1.5, a poorly crystallized hydroxyapatite, monetite, brushite, octacalcium phosphate, {beta}-tricalcium phosphate and rabbit femoral bone. The interactions between nuclei in unlike structures and the mobility of acid protons are discussed. (author). 11 refs.; 2 figs.; 1 tab.

  11. Dicalcium phosphate cements: brushite and monetite.

    Science.gov (United States)

    Tamimi, Faleh; Sheikh, Zeeshan; Barralet, Jake

    2012-02-01

    Dicalcium phosphate cements were developed two decades ago and ever since there has been a substantial growth in research into improving their properties in order to satisfy the requirements needed for several clinical applications. The present paper presents an overview of the rapidly expanding research field of the two main dicalcium phosphate bioceramics: brushite and monetite. This review begins with a summary of all the different formulae developed to prepare dicalcium phosphate cements, and their setting reaction, in order to set the scene for the key cement physical and chemical properties, such as compressive and tensile strength, cohesion, injectability and shelf-life. We address the issue of brushite conversion into either monetite or apatite. Moreover, we discuss the in vivo behavior of the cements, including their ability to promote bone formation, biodegradation and potential clinical applications in drug delivery, orthopedics, craniofacial surgery, cancer therapy and biosensors.

  12. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

    OpenAIRE

    Mérida, A; Candau, P; Florencio, F J

    1991-01-01

    Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases...

  13. Removal of Phosphate from Aqueous Solution with Modified Bentonite

    Institute of Scientific and Technical Information of China (English)

    唐艳葵; 童张法; 魏光涛; 李仲民; 梁达文

    2006-01-01

    Bentonite combined with sawdust and other metallic compounds was used to remove phosphate from aqueous solutions in this study. The adsorption characteristics of phosphate on the modified bentonite were investigated, including the effects of temperature, adsorbent dosage, initial concentration of phosphate and pH on removal of phosphate by conducting a series of batch adsorption experiments. The results showed that 98% of phosphate removal rate was obtained since sawdust and bentonite used in this investigation were abundantly and locally available. It is concluded that modified bentonite is a relatively efficient, low cost and easily available adsorbent for the removal of phosphate from aqueous solutions.

  14. Phosphate valorization by dry chlorination route

    OpenAIRE

    Kanari N.; Menad N.; Diot F.; Allain E.; Yvon J.

    2016-01-01

    International audience; This work deals with the extraction of phosphorus chlorinated compounds from phosphate materials using chlorination with gaseous chlorine. An industrial sample of dicalcium phosphate dihydrate, after transformation into calcium pyrophosphate (Ca 2 P 2 O 7), is subjected to reactions with Cl 2 +CO+N 2 and Cl 2 +C+N 2 at temperatures ranging from 625 to 950 °C using boat experiments. Gathering results of the thermodynamic predictions and TG/DT analysis with those of SEM ...

  15. Dual mechanism of ion permeation through VDAC revealed with inorganic phosphate ions and phosphate metabolites.

    Science.gov (United States)

    Krammer, Eva-Maria; Vu, Giang Thi; Homblé, Fabrice; Prévost, Martine

    2015-01-01

    In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a "charged brush" which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.

  16. Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

    Directory of Open Access Journals (Sweden)

    Lílian Estrela Borges Baldotto

    2014-06-01

    Full Text Available Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i to select and characterize diazotrophs able to solubilize phosphates in vitro and (ii evaluate the initial performance of the pineapple cultivars Imperial and Pérola in response to inoculation with selected bacteria in combination with rock phosphate. The experiments were conducted at Universidade Estadual do Norte Fluminense Darcy Ribeiro, in 2009. In the treatments with bacteria the leaf contents of N, P and K were higher than those of the controls, followed by an increase in plant growth. These results indicate that the combined application of diazotrophic phosphate-solubilizing bacteria Burkholderia together with Araxá rock phosphate can be used to improve the initial performance of pineapple slips.

  17. Evaluation of intestinal phosphate binding to improve the safety profile of oral sodium phosphate bowel cleansing.

    Directory of Open Access Journals (Sweden)

    Stef Robijn

    Full Text Available Prior to colonoscopy, bowel cleansing is performed for which frequently oral sodium phosphate (OSP is used. OSP results in significant hyperphosphatemia and cases of acute kidney injury (AKI referred to as acute phosphate nephropathy (APN; characterized by nephrocalcinosis are reported after OSP use, which led to a US-FDA warning. To improve the safety profile of OSP, it was evaluated whether the side-effects of OSP could be prevented with intestinal phosphate binders. Hereto a Wistar rat model of APN was developed. OSP administration (2 times 1.2 g phosphate by gavage with a 12h time interval induced bowel cleansing (severe diarrhea and significant hyperphosphatemia (21.79 ± 5.07 mg/dl 6h after the second OSP dose versus 8.44 ± 0.97 mg/dl at baseline. Concomitantly, serum PTH levels increased fivefold and FGF-23 levels showed a threefold increase, while serum calcium levels significantly decreased from 11.29 ± 0.53 mg/dl at baseline to 8.68 ± 0.79 mg/dl after OSP. OSP administration induced weaker NaPi-2a staining along the apical proximal tubular membrane. APN was induced: serum creatinine increased (1.5 times baseline and nephrocalcinosis developed (increased renal calcium and phosphate content and calcium phosphate deposits on Von Kossa stained kidney sections. Intestinal phosphate binding (lanthanum carbonate or aluminum hydroxide was not able to attenuate the OSP induced side-effects. In conclusion, a clinically relevant rat model of APN was developed. Animals showed increased serum phosphate levels similar to those reported in humans and developed APN. No evidence was found for an improved safety profile of OSP by using intestinal phosphate binders.

  18. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

    Science.gov (United States)

    Palencia, Andrés; Li, Xianfeng; Bu, Wei; Choi, Wai; Ding, Charles Z.; Easom, Eric E.; Feng, Lisa; Hernandez, Vincent; Houston, Paul; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Rock, Fernando L.; Sexton, Holly; Zhang, Suoming; Zhou, Yasheen; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G.; Woolhiser, Lisa; Gruppo, Veronica; Lenaerts, Anne J.; O'Malley, Theresa; Parish, Tanya; Cooper, Christopher B.; Waters, M. Gerard; Ma, Zhenkun; Ioerger, Thomas R.; Sacchettini, James C.; Rullas, Joaquín; Angulo-Barturen, Iñigo; Pérez-Herrán, Esther; Mendoza, Alfonso; Barros, David; Cusack, Stephen; Plattner, Jacob J.

    2016-01-01

    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid. PMID:27503647

  19. Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase

    Directory of Open Access Journals (Sweden)

    Liron Klipcan

    2010-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS—bacterial (αβ2, eukaryotic/archaeal cytosolic (αβ2, and mitochondrial α—is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNAPhe anticodon recognition.

  20. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600

    Directory of Open Access Journals (Sweden)

    Biaosheng Lin

    2016-01-01

    Full Text Available To clone and express the γ-polyglutamic acid (γ-PGA synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.