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Sample records for 8-bromo-cyclic inosine diphosphoribose

  1. 8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

    Science.gov (United States)

    Kirchberger, Tanja; Moreau, Christelle; Wagner, Gerd K.; Fliegert, Ralf; Siebrands, Cornelia C.; Nebel, Merle; Schmid, Frederike; Harneit, Angelika; Odoardi, Francesca; Flügel, Alexander; Potter, Barry V. L.; Guse, Andreas H.

    2009-01-01

    cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2. PMID:19492987

  2. Endonuclease V cleaves at inosines in RNA.

    Science.gov (United States)

    Vik, Erik Sebastian; Nawaz, Meh Sameen; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  3. Inosine protects from oxidative damage induced by irradiation in rats

    International Nuclear Information System (INIS)

    Inosine is a non-toxic purine, abundant in meat and sugar beets, has been shown to exert a potent anti- inflammatory and immunomodulating actions. the present work was designed to evaluate the role of inosine in modulating the changes induced by irradiation in blood picture; red blood cell (RBCs). white blood cell counts (WBCs), haematocrit value (Hct) and haemoglobin content (Hb) and antioxidant status (blood reduced glutathione (GSH). advanced protein oxidation products (AOPP), lipid peroxidation (MDA), ascorbyl radical, (ASR) and protein-carbonyl value (PCO) as well as xanthine oxidoreductase (XOR) system: xanthine oxidase (XO) and xanthine dehydrogenase (XDH). female rats were exposed to whole body gamma radiation at the dose level of 6 Gy. inosine (200 mg kg -1 day-1) was administered by gavages, starting 7 days before irradiation and 14 day post irradiation until the end of experiment (21 day). animals were divided into four groups , control, irradiated group (6 Gy) , inosine treated group and irradiated inosine treated group . animals were sacrificed at two time intervals 10 and 15 days post- irradiation. the results obtained revealed that the prolonged administration of inosine before and after irradiation induced significant amelioration on values of blood parameter (RBCs, WBCs HB and Hct) when compared with the corresponding values in irradiated rats. significant improvements were observed in the level of uric acid, AOPP. MDA, ASR and PCO. in addition to remarkable amelioration in the the activity of XDH and GSH concentration were observed. it could be postulated that inosine as a multi- functional dietary supplement could exert a modulatory role in the radiation-induced oxidative damage and serum biochemical changes through its antioxidant properties

  4. Inosine improves functional recovery after experimental traumatic brain injury.

    Science.gov (United States)

    Dachir, Shlomit; Shabashov, Dalia; Trembovler, Victoria; Alexandrovich, Alexander G; Benowitz, Larry I; Shohami, Esther

    2014-03-25

    Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, pcognitive performance (object recognition, peffect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI. PMID:24502983

  5. Voltammetric and impedance studies of inosine-5'-monophosphate and hypoxanthine

    OpenAIRE

    Oliveira-Brett, Ana Maria; Silva, Luís A.; Farace, Giosi; Vadgama, Pankaj; Christopher M. A. Brett

    2003-01-01

    The oxidation mechanism and adsorption of inosine 5'-monophosphate and hypoxanthine were investigated in solutions of different pH using voltammetric and impedance methods at glassy carbon electrodes. For both compounds, the pH dependence from differential pulse voltammetry showed that the same number of electrons and protons are involved in the rate-determining step of the electrochemical reaction. In the case of hypoxanthine, it was also possible to study the effect of different concentrati...

  6. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    Science.gov (United States)

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  7. [Isolation of inosine-5'-monophosphate from fish muscles].

    Science.gov (United States)

    Tugaĭ, V A; Akulin, V N; Epshteĭn, L M

    1987-01-01

    Conditions for transformation of tissue adenosine-5'-monophosphate (AMP) into inosine-5'-monophosphate (IMP) with the aid of endogenic AMP-aminohydrolase are developed resting on the studied properties of AMP-aminohydrolase (EC 3.5.4.6) from saltwater fish muscles (one of the enzymes participating in the nucleotide metabolism). Sorption of the nucleotide is performed on the activated charcoals A gamma-3 A gamma-5 which eluate IMP from acid solutions. It reduces the process of isolation, permits application of the acid wash solutions to remove salts; the alkaline ethyl alcohol-aid elution at the subsequent stages accelerates the process of nucleotide concentration by means of vacuum evaporation. The suggested approaches allow developing a simple method of IMP production from fish tissues which diminishes the cost of preparation.

  8. The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer.

    Science.gov (United States)

    Ledesma-Amaro, Rodrigo; Buey, Rubén M; Revuelta, José Luis

    2016-09-01

    Inosine is a nucleoside with growing biotechnological interest due to its recently attributed beneficial health effects and as a convenient precursor of the umami flavor. At present, most of the industrial inosine production relies on bacterial fermentations. In this work, we have metabolically engineered the filamentous fungus Ashbya gossypii to obtain strains able to excrete high amounts of inosine to the culture medium. We report that the disruption of only two key genes of the purine biosynthetic pathway efficiently redirect the metabolic flux, increasing 200-fold the excretion of inosine with respect to the wild type, up to 2.2 g/L. These results allow us to propose A. gossypii as a convenient candidate for large-scale nucleoside production, especially in view of the several advantages that Ashbya has with respect to the bacterial systems used at present for the industrial production of this food additive. Biotechnol. Bioeng. 2016;113: 2060-2063. © 2016 Wiley Periodicals, Inc.

  9. The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias.

    Science.gov (United States)

    Welihinda, Ajith A; Kaur, Manmeet; Greene, Kelly; Zhai, Yongjiao; Amento, Edward P

    2016-06-01

    Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo. PMID:26903141

  10. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    Science.gov (United States)

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  11. The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer.

    Science.gov (United States)

    Ledesma-Amaro, Rodrigo; Buey, Rubén M; Revuelta, José Luis

    2016-09-01

    Inosine is a nucleoside with growing biotechnological interest due to its recently attributed beneficial health effects and as a convenient precursor of the umami flavor. At present, most of the industrial inosine production relies on bacterial fermentations. In this work, we have metabolically engineered the filamentous fungus Ashbya gossypii to obtain strains able to excrete high amounts of inosine to the culture medium. We report that the disruption of only two key genes of the purine biosynthetic pathway efficiently redirect the metabolic flux, increasing 200-fold the excretion of inosine with respect to the wild type, up to 2.2 g/L. These results allow us to propose A. gossypii as a convenient candidate for large-scale nucleoside production, especially in view of the several advantages that Ashbya has with respect to the bacterial systems used at present for the industrial production of this food additive. Biotechnol. Bioeng. 2016;113: 2060-2063. © 2016 Wiley Periodicals, Inc. PMID:26927228

  12. Inosine-mediated modulation of RNA sensing by Toll-like receptor 7 (TLR7) and TLR8.

    Science.gov (United States)

    Sarvestani, Soroush T; Tate, Michelle D; Moffat, Jessica M; Jacobi, Ashley M; Behlke, Mark A; Miller, Alistair R; Beckham, Simone A; McCoy, Claire E; Chen, Weisan; Mintern, Justine D; O'Keeffe, Meredith; John, Matthias; Williams, Bryan R G; Gantier, Michael P

    2014-01-01

    RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA. PMID:24227841

  13. Influence of concomitant infusion of thymidine and inosine on methotrexate activity in normal and P388-bearing mice

    NARCIS (Netherlands)

    Uitendaal, Martin P.; Schornagel, J.H.; Leyva, A.; Pinedo, H.M.

    1984-01-01

    Combinations of thymidine and inosine (ranging from 0 to 7.5 mg/hr) were co-administered during a 72-hr continuous i.v. infusion of 3 μg/hr methotrexate in normal and P388 solid tumor-bearing DBA/2 mice. Methotrexate alone was lethal to all normal mice. Inosine at 1.0–7.5 mg/hr could reverse toxicit

  14. Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

    Science.gov (United States)

    Buey, Rubén M; Ledesma-Amaro, Rodrigo; Balsera, Mónica; de Pereda, José María; Revuelta, José Luis

    2015-11-01

    Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii. PMID:26150243

  15. Increased riboflavin production by manipulation of inosine 5′-monophosphate dehydrogenase in Ashbya gossypii

    OpenAIRE

    Buey, Ruben M.; Ledesma Amaro, Rodrigo; Balsera, Mónica; Revuelta Doval, José Luis

    2015-01-01

    Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Her...

  16. Targeted Disruption of the Inosine 5′-Monophosphate Dehydrogenase Type I Gene in Mice

    OpenAIRE

    Gu, Jing Jin; Tolin, Amy K.; Jain, Jugnu; Huang, Hai; Santiago, Lalaine; Mitchell, Beverly S.

    2003-01-01

    Inosine 5′-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal b...

  17. The CBS subdomain of inosine 5’-monophosphate dehydrogenase regulates purine nucleotide turnover†

    OpenAIRE

    Pimkin, Maxim; Markham, George D.

    2008-01-01

    Inosine 5’-monophosphate dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP...

  18. Extended-gate field-effect transistor (EG-FET) with molecularly imprinted polymer (MIP) film for selective inosine determination.

    Science.gov (United States)

    Iskierko, Zofia; Sosnowska, Marta; Sharma, Piyush Sindhu; Benincori, Tiziana; D'Souza, Francis; Kaminska, Izabela; Fronc, Krzysztof; Noworyta, Krzysztof

    2015-12-15

    A novel recognition unit of chemical sensor for selective determination of the inosine, renal disfunction biomarker, was devised and prepared. For that purpose, inosine-templated molecularly imprinted polymer (MIP) film was deposited on an extended-gate field-effect transistor (EG-FET) signal transducing unit. The MIP film was prepared by electrochemical polymerization of bis(bithiophene) derivatives bearing cytosine and boronic acid substituents, in the presence of the inosine template and a thiophene cross-linker. After MIP film deposition, the template was removed, and was confirmed by UV-visible spectroscopy. Subsequently, the film composition was characterized by spectroscopic techniques, and its morphology and thickness were determined by AFM. The finally MIP film-coated extended-gate field-effect transistor (EG-FET) was used for signal transduction. This combination is not widely studied in the literature, despite the fact that it allows for facile integration of electrodeposited MIP film with FET transducer. The linear dynamic concentration range of the chemosensor was 0.5-50 μM with inosine detectability of 0.62 μM. The obtained detectability compares well to the levels of the inosine in body fluids which are in the range 0-2.9 µM for patients with diagnosed diabetic nephropathy, gout or hyperuricemia, and can reach 25 µM in certain cases. The imprinting factor for inosine, determined from piezomicrogravimetric experiments with use of the MIP film-coated quartz crystal resonator, was found to be 5.5. Higher selectivity for inosine with respect to common interferents was also achieved with the present molecularly engineered sensing element. The obtained analytical parameters of the devised chemosensor allow for its use for practical sample measurements.

  19. Crystal structure and MD simulation of mouse EndoV reveal wedge motif plasticity in this inosine-specific endonuclease

    Science.gov (United States)

    Nawaz, Meh Sameen; Vik, Erik Sebastian; Ronander, Mia Elise; Solvoll, Anne Marthe; Blicher, Pernille; Bjørås, Magnar; Alseth, Ingrun; Dalhus, Bjørn

    2016-04-01

    Endonuclease V (EndoV) is an enzyme with specificity for deaminated adenosine (inosine) in nucleic acids. EndoV from Escherichia coli (EcEndoV) acts both on inosines in DNA and RNA, whereas the human homolog cleaves only at inosines in RNA. Inosines in DNA are mutagenic and the role of EndoV in DNA repair is well established. In contrast, the biological function of EndoV in RNA processing is largely unexplored. Here we have characterized a second mammalian EndoV homolog, mouse EndoV (mEndoV), and show that mEndoV shares the same RNA selectivity as human EndoV (hEndoV). Mouse EndoV cleaves the same inosine-containing substrates as hEndoV, but with reduced efficiencies. The crystal structure of mEndoV reveals a conformation different from the hEndoV and prokaryotic EndoV structures, particularly for the conserved tyrosine in the wedge motif, suggesting that this strand separating element has some flexibility. Molecular dynamics simulations of mouse and human EndoV reveal alternative conformations for the invariant tyrosine. The configuration of the active site, on the other hand, is very similar between the prokaryotic and mammalian versions of EndoV.

  20. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    Science.gov (United States)

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

  1. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    Science.gov (United States)

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  2. Effects of intragastric infusion of inosine monophosphate and l-glutamate on vagal gastric afferent activity and subsequent autonomic reflexes

    OpenAIRE

    Kitamura, Akihiko; Sato, Wataru; Uneyama, Hisayuki; Torii, Kunio; NIIJIMA, Akira

    2010-01-01

    In this study we investigated the effects of intragastric infusion of palatable basic taste substances (umami, sweet, and salty) on the activity of the vagal gastric afferent nerve (VGA), the vagal celiac efferent nerve (VCE), and the splanchnic adrenal efferent nerve (SAE) in anesthetized rats. To test the three selected taste groups, rats were infused with inosine monophosphate (IMP) and l-glutamate (GLU) for umami, with glucose and sucrose for sweet, and with sodium chloride (NaCl) for sal...

  3. Induction of inosine containing mRNA during the inflammatory stress in C57BL/6 mouse

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A-to-I RNA editing is a recently discovered process of post-transcription modification of pre-mRNA by adenosine deamination that results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the acute inflammation. It was found that A-to-I edtiting activities of RNA editases, ADARs, were upregulated in lung, spleen, thymus and lymph node of mouse during endotoxin stimulation. Importantly, the number of inosine in poly(A) RNA isolated from mouse lung and spleen was significantly increased in correlating with the induction of ADARs' editing activity. The in vitro synthesized RNA which did not contain inosine was edited by thymus extracts and the generation of inosine was greatly increased after editing in LPS treated thymus extract. Take together, these data suggest that A-to-I RNA editing by ADARs may play an important role in pathogenesis of inflammation. The existence of high level of I-mRNA also suggests that more protein isoforms might be generated from a single gene via adenosine deamination by ADARs during inflammatory stress.

  4. Preparation and characterization of host-guest system between inosine and β-cyclodextrin through inclusion mode

    Science.gov (United States)

    Prabu, Samikannu; Sivakumar, Krishnamurty; Swaminathan, Meenakshisundaram; Rajamohan, Rajaram

    2015-08-01

    Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a β-N9-glycosidic bond. Inosine is commonly found in tRNAs. Inosine (INS) has been used widely as an antiviral drug. The inclusion complex of INS with β-CDx in solution phase is studied by ground and excited state with UV-visible and fluorescence spectroscopy, respectively. A binding constant and stoichiometric ratio between INS and β-CDx are calculated by BH equation. The lifetime and relative amplitude of INS is increases with increasing the concentrations of β-CDx, confirms the formation of inclusion complex in liquid state. The solid complexes are prepared by kneading method (KM) and co-precipitation method (CP). The solid complex is characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (XRD) and differential scanning colorimetry (DSC). CP method gives the solid product with good yield than that of physical mixture and KM method. The structure of complex is proposed based on the study of Patch - Dock server.

  5. PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS OF INOSINE MONOPHOSPHATE DEHYDROGENASE (IMPDH) ACTIVITY IN MMF-TREATED HCT RECIPIENTS

    Science.gov (United States)

    Li, Hong; Mager, Donald E.; Sandmaier, Brenda M.; Storer, Barry E.; Boeckh, Michael J.; Bemer, Meagan J.; Phillips, Brian R.; Risler, Linda J.; McCune, Jeannine S.

    2014-01-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplant (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNC) at five time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in the pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic/dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory Emax model with an IC50 = 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, non-relapse mortality, and overall mortality. In conclusion, a pharmacokinetic/dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. PMID:24727337

  6. Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer(®) peptides.

    Science.gov (United States)

    Jefferies, R; Yang, R; Woh, C K; Weldt, T; Milech, N; Estcourt, A; Armstrong, T; Hopkins, R; Watt, P; Reid, S; Armson, A; Ryan, U M

    2015-01-01

    Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function. PMID:25447124

  7. Myricetin is a novel inhibitor of human inosine 5'-monophosphate dehydrogenase with anti-leukemia activity.

    Science.gov (United States)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

    2016-09-01

    Human inosine 5'-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC50 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. PMID:27378425

  8. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  9. Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and γ-Cyclodextrin-Enhanced Fluorescence Detection.

    Science.gov (United States)

    Stephen, Terilyn K L; Guillemette, Katherine L; Green, Thomas K

    2016-08-01

    Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit ∼25-fold fluorescence enhancement upon the formation of inclusion complexes with γ-cyclodextrin (γ-CD). Association constants were determined as 4600 M(-1) for Ado and 1000 M(-1) for Ino by CE-LIF. The structure of the TNP-Ado:γ-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 μM for Ado and 4 μM for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence γ-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively. PMID:27314490

  10. Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

    Directory of Open Access Journals (Sweden)

    S. Yeswanth

    2013-10-01

    Full Text Available Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH from Staphylococcus aureus ATCC 12600. Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH was overexpressed with 1 mM isopropyl beta-D-1- thiogalactopyranoside (IPTG; Michaelis constant (Km, maximum enzyme velocity (Vmax and catalytic constant (Kcat of expressed IMPDH were determined. Results: The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 µM, Vmax of 0.247±0.84/µM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ µM, Vmax of 0.378±0.036 µM/mg/min and Kcat of 4.78±0.021 /min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa. Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis.

  11. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

    Science.gov (United States)

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D

    2013-05-23

    Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5'-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD(+). The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative 15a demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.

  12. Inosine inhibits apoptosis and cytochrome C mRNA expression in rat neurons after cerebral ischemia/reperfusion

    Institute of Scientific and Technical Information of China (English)

    Jinrong Wang; Mingjun Bi; Qin Li

    2006-01-01

    BACKGROUND: It has been demonstrated that adenosine can induce glial cell to release cytochrome C,enhance expression of apoptotic gene bax,inhibit anti-apoptotic gene bcl-2,and activate caspase-3 to apoptosis;Whereas inosine can inhibit neuronal apoptosis which is similar to bil-2.OBJECTIVE: To observe the affects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemia/reperfusion,and analyze the pathway of its neuroprotective effect.DESIGN: A randomised controlled animal trial.SETTINGS: Department of Neurology,Rongcheng Second People's Hospital;Department of Neruology,Affiliated Union Hospital,Tongji Medical College,Huazhong University of Science and Technology.MATERIALS: Sixty-eight rats,weighing 230-280 g and clean grade,were used.TdT-mediated dUTP-biotin nick end labeling(TUNEL)and cytochrome C mRNA in situ hybridization kits and DAB staining kit were purchased from Wuhan Boster Biological Co.,Ltd;Inosine injection[200mg(2ml)each] from Qingdao First Pharmaceutical Factory.METHODS:The experiment was accomplished in the animal experimental center in Tongji Medical College of Huazhong University of Science and Technology from December 2003 to June 2005.①Sixty-four rats were made into focal ischemia by middle cerebral artery occlusion(MCAO)with a nylon monofilament suture.The successfully induced rats were assigned to inosine group(n=32)and model group(n=32)at random.Rats in the inosine group were intraperitoneally administrated with inosine in dose of 100mg/kg preoperatively.twice a day,7 days in all.The rats in the control group were injected with the same dose of saline solution by the similar way preoperatively.Each group was randomized into ischemia/reperfusion 2,6,12,24 hours,2,3,7 and 14 days subgroups consisted of 4 rats.The other 4 rats were taken as the sham-operated group,the rats were given the same treatment except for not introduced the filament into the external carotid artery stump.and brain

  13. Pharmacokinetic and pharmacodynamic analysis of inosine monophosphate dehydrogenase activity in hematopoietic cell transplantation recipients treated with mycophenolate mofetil.

    Science.gov (United States)

    Li, Hong; Mager, Donald E; Sandmaier, Brenda M; Storer, Barry E; Boeckh, Michael J; Bemer, Meagan J; Phillips, Brian R; Risler, Linda J; McCune, Jeannine S

    2014-08-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplantation (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNCs) at 5 time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic-dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory maximum effect model with an IC50 of 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, nonrelapse mortality, and overall mortality. In conclusion, a pharmacokinetic-dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker.

  14. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    Science.gov (United States)

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  15. Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor*

    Science.gov (United States)

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Öhman, Marie

    2011-01-01

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABAA receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABAA receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  16. Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter E.; Møllegaard, Niels Erik

    2008-01-01

    random library of DNA-binding sites containing inosine (I) and 2,6-diaminopurine (D) instead of guanine and adenine, respectively. Accordingly, the DNA helix minor groove is structurally altered due to the 'transfer' of the 2-amino group of guanine (now I) to adenine (now D), whereas the major groove is...... functionally intact. The majority of the selected sites contain the natural consensus sequence TGTGAN(6)TCACA (i.e. TITIDN(6)TCDCD). Thus, direct readout of the consensus sequence is independent of minor groove conformation. Consequently, the indirect readout known to occur in the TG/CA base pair step (primary...

  17. Targeting the immune system to fight cancer using chemical receptor homing vectors carrying Poly Inosine/Cytosine (PolyIC

    Directory of Open Access Journals (Sweden)

    Alexander eLevitzki

    2012-02-01

    Full Text Available Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, poly Inosine/Cytosine (polyIC, into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA binding proteins, such as dsRNA dependent protein kinase (PKR, Toll-3 receptor (TLR3, retinoic acid–inducible gene I (RIG-1 and melanoma differentiation–associated gene 5 (MDA5. The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1 Recruitment of the immune system is localized to the tumor. (2 The response is rapid, leading to fast tumor eradication. (3 The bystander effects lead to the eradication of tumor cells not harboring the target. (4 The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which be a small molecule, a single chain antibody or an

  18. Effect of Protein Levels on Beef Inosine Acid Content%蛋白质水平对牛肉肌苷酸含量的影响

    Institute of Scientific and Technical Information of China (English)

    徐英; 李石友; 李琦华; 段刚; 杨国荣; 梁应海

    2011-01-01

    探讨不同蛋白质水平对牛肉肌苷酸含量的影响,设计5个营养水平的精料补充科配方,分别饲喂5个试验组肉牛,并与不添加精料补充料的对照组进行比较.结果表明,精料补充料粗蛋白质水平的提高对牛肉肌苷酸含量没有明显影响.%To study on effect of feed different level proteina on beef inosine acide content , 5 snupplement dietary treatments ( no supplement as control) were fed 5 groups ' cattle and it was compared the differences between the fed protein groupa and control ones. The result showed there were no much difference between The fed protein groupa and control group that the beef inosine acid content.

  19. Pharmacogenetic research progress of inosine triphosphate pyrophosphatase%三磷酸肌苷焦磷酸酶药理遗传学的研究进展

    Institute of Scientific and Technical Information of China (English)

    李兴军

    2013-01-01

    嘌呤类药物主要用于治疗自身免疫性疾病、器官移植、急性淋巴细胞白血病等,其不良反应的发生率为15% ~28%,严重影响了药物在临床中的应用.三磷酸肌苷焦磷酸酶(inosine triphosphate pyrophosphatase,ITPA)存在个体间的差异,ITPA缺陷的患者在应用嘌呤类药物时会发生一定的不良反应,因此,有必要了解ITPA对嘌呤类药物临床应用的影响,该文综述了ⅡPA的药理遗传学方面的研究进展.%Purine drugs are for the treatment of autoimmune diseases,organ transplantation,acute lymphoblastic leukemia.The adverse reaction rate is 15% ~ 28%,which impacts on the clinical application in recent years.Studies have shown that inosine triphosphate pyrophosphatase (ITPA) are different between individuals,and there are adverse reactions in patients with defects of ITPA when purine drugs are used.This paper reviewes the ITPA pharmacogenetic research progresses.

  20. Scientific Opinion on the safety and efficacy of disodium 5?-ribonucleotides, disodium 5?-guanylate, disodium 5?-inosinate for all animal species and categories

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP

    2014-03-01

    Full Text Available The flavours included in this assessment are widely present in nature as the building blocks of DNA and RNA. In the absence of any information on the microbial strains or substrates used for the production of the additives, and with little information on the manufacturing process, the FEEDAP Panel is unable to ascertain whether the manufacturing process introduces any safety concerns. Disodium 5′-guanylate and disodium 5′-inosinate and their mixture are considered to be safe for the target animals and the consumer. However, considering the lack of information on the production process, these conclusions apply only to the compounds ‘per se’ and their extrapolation to any feed additive containing these compounds is not possible. In the absence of any data related to hazard to the user, it would be prudent to regard disodium 5′-guanylate and disodium 5′-inosinate and their mixture as potentially hazardous to workers by skin or inhalation exposure. The compounds under assessment are naturally present in feed materials; therefore, no risk to the safety for the environment is foreseen. Since these compounds are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary.

  1. Studying of Biosynthetic Pathways of 2H-labeled Purine Ribonucleoside Inosine in a Chemoheterotrophic Bacterium Bacillus subtilis B-3157 by FAB Mass-Spectrometry

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-09-01

    Full Text Available This paper deals with studying biosynthetic pathways of 2H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW medium with 2% (v/v hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2H-labeled growth substrates. Isolation of 2H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammonium–formate buffer (pH = 8,9, crystallization in 80% (v/v EtOH, and ion exchange chromatography (IEC on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammonium–formate buffer and 0,045 M NH4Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65,5 atom% 2H with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to the preservation in this bacterium the minor pathways of de novo glucose biosynthesis in 2H2O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from HMP shunt reactions, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [2H]amino acids, primarily glutamine and glycine, that originated from deuterated hydrolysate. A glycoside proton at -N9-glycosidic bond could be replaced with deuterium via the reaction of СО2 elimination at the stage of ribulose-5-monophosphate formation from 3-keto-6-phosphogluconic acid with subsequent proton (deuteron attachment at the С1-position of ribulose-5-monophosphate. Two other protons at C2(C3 and C4 positions in ribose residue could be

  2. Variants of the inosine triphosphate pyrophosphatase gene are associated with reduced relapse risk following treatment for HCV genotype 2/3

    DEFF Research Database (Denmark)

    Rembeck, Karolina; Waldenström, Jesper; Hellstrand, Kristoffer;

    2014-01-01

    The present study evaluated the impact of variations in the inosine triphosphate pyrophosphatase (ITPase) gene (ITPA) on treatment outcome in patients with hepatitis C virus (HCV) genotype 2/3 infection receiving peginterferon-α2a and lower, conventional 800 mg daily dose of ribavirin. Previous...... naïve HCV genotype 2/3 infected patients, enrolled in a phase III trial (NORDynamIC), were genotyped for ITPA (rs1127354 and rs7270101). Homo- or heterozygosity at Ars1127354 or Crs7270101, entailing reduced ITPase activity, was observed in 37% of patients and was associated with increased likelihood...... duration arms, HCV genotype, fibrosis stage and IL28B genotype, and was not secondary to improved adherence to therapy or less pronounced anemia. Gene variants predicting reduced predicted ITPase activity also were associated with decreased risk of anemia (P

  3. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  4. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

    Science.gov (United States)

    Delvaux, Nathália; da Costa, Vanessa Duarte; da Costa, Maristella Matos; Villar, Livia Melo; Coelho, Henrique Sérgio Moraes; Esberard, Eliane Bordalo Cathalá; Flores, Priscila Pollo; Brandão-Mello, Carlos Eduardo; Villela-Nogueira, Cristiane Alves; de Almeida, Adilson José; Lampe, Elisabeth

    2015-01-01

    Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are strongly associated with protection against ribavirin (RBV)-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354) frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354), respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475). In women, there was no influence of genotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women. PMID:26154744

  5. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

    Directory of Open Access Journals (Sweden)

    Nathália Delvaux

    2015-08-01

    Full Text Available Inosine triphosphatase (ITPA single nucleotide polymorphisms (SNPs are strongly associated with protection against ribavirin (RBV-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354 frequency in healthy and hepatitis C virus (HCV-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101 and CC genotypes (rs1127354, respectively. Men with AA (rs7270101 showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475. In women, there was no influence of genotype (p = 0.5295. For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women.

  6. Infrared Spectrum Analysis of the Unknown Floss of in Inosine Glucose Injection%2例肌苷葡萄糖注射液配药后不明絮状物的红外光谱分析

    Institute of Scientific and Technical Information of China (English)

    李湘晖; 田甜; 许世伟; 杜智敏

    2011-01-01

    目的:分析临床配药过程中出现的2例与肌苷葡萄糖注射液有关的絮状物的成分.方法:将临床配药过程中肌苷葡萄糖注射液与阿昔洛韦注射液混合及肌苷葡萄糖注射液与还原型谷胱甘肽混合时出现的不明絮状物分离出,低温干燥后进行红外光谱扫描,扫描结果通过与SDBS(Spectral Data Base System)谱图数据库中各组分红外图谱峰位、峰形及相对强度进行对比,确定是否为单一物质,分析原因.结果:该絮状物既不是肌苷,也不是阿昔洛韦或还原型谷胱甘肽,为茵类污染的可能性极大.结论:对于肌苷葡萄糖注射液类生物药品,临床使用时应先仔细观察外观后再行配药.%OBJECTIVE: To analyze the unknown floss of Inosine glucose injection in 2 cases during drug dispensing. METHODS: The unknown floss was isolated from mixture of lnosine glucose injection with Acyclovir injection and mixture of Inosine glucose injection with reduced glutathione. Isolated unknown floss was analyzed by infrared spectroscopy (IR) after cold drying. Results of IR were compared with SDBS (Spectral Data Base System) in terms of IR peak, peak shape and relative peak density to confirm whether the unknown floss was homogenous material or not and analyze its reason. RESULTS: The floss was not inosine or reduced glutathione or acyclovir. It was most likely a kind of bacteria. CONCLUSION: For such biological drugs of Inosine glucose injection, appearance should be observed carefully before clinical utilization.

  7. 肌苷减少高浓度锌损伤的PC12细胞坏死而不是凋亡%Inosine attenuates necrosis, but not apoptosis, of zinc-injured PC12 cells

    Institute of Scientific and Technical Information of China (English)

    史明; 郑春霞; 游思维

    2005-01-01

    Objective To explore the death types of PC12 cells injured by a high concentration of zinc, and effects of inosine on the types of zinc-induced cell death. Methods MTT assay was used to assess the viability of PC12 cells treated with different concentrations of zinc chloride (50, 100,200,400 μmoL/L) or inosine (0.1,0.5, 1.0, 2.0 mmol/L) for 12 h. Hoechst 33342 / PI double staining, Annexin-V binding assay and DNA agarose gel electrophoresis were employed to investigate the death forms of PC12 cells with treatment of 200 μmol/L zinc chloride or 2.0 mmol/L inosine for 12 h. Results Zinc at 100 μ mol/L and more reduced cell viability significantly. After treatment with 200 μmoL/L zinc,56.5, 24.4 and 19.1% of total PC12 cells were necrotic, survival and apoptotic. Inosine, from the concentration of 0.5 mmol/L, markedly increased cell viability of zinc-induced PC12 cells. However, additional exposure to 2.0 mmol/L inosine, necrotic, survival and apoptotic cells were 27.9, 33.8 and 38.4% of the total PC12 cells that were injured by zinc.Conclusion The viability of PC12 cells decreases when the concentration of zinc increases, and inosine protects zinc-induced PC12 cells at a dose-dependent manner. A high concentration of zinc causes both necrosis and apoptosis, and inosine attenuates necrosis, but not apoptosis, of zinc-injured PC12 cells.%目的探讨高浓度锌损伤后PC12细胞的死亡类型和肌苷对该死亡类型的影响.方法用MTT比色法测定不同浓度氯化锌(50、100、200、400 μmol/L)或肌苷(0.1、0.5、1.0、2.0 mmol/L)作用PC12细胞12 h后的存活率;用Hoechst33342/PI荧光双染色、Annexin-V结合实验及DNA琼脂糖凝胶电泳等方法检测200μmol/L氯化锌、2.0 mmol/L肌苷作用PC12细胞12 h后细胞死亡类型的变化.结果锌从100 μmol/L作用浓度开始可明显降低细胞存活率;200 μmol/L锌可引起(56.5±7.2)%坏死、(24.4±2.5)%的正常和(19.1±7.6)%的细胞凋亡.肌苷从0.5 mmol/L作用浓度开

  8. 枯草芽孢杆菌二步发酵法生产5'-肌苷酸%Production of inosine 5'-monophosphate by Bacillus subtilis using a novel two-step fermentation method

    Institute of Scientific and Technical Information of China (English)

    何菊华; 吴雪娇; 谢希贤; 徐庆阳; 张成林; 陈宁

    2015-01-01

    5'-Monophosphate (5'-IMP) plays an important role in food additive industry since it is an indispensable component of flavor enhancer.To shorten the period and lower the cost of 5 '-IMP production,characteristic of acid phosphotranferase (AP/PTase) from Morganella morganii was studied and its encoding gene was cloned to inosine-producing strain Bacillus subtilis JG to obtain Bacillus subtilis JAB and Bacillus subtilis JAF.Then according to the character of inosine production and phosphotranferase expression by the two strains,5'-IMP production by twostep fermentation combined with inosine accumulation and enzyme catalysis was achieved.The final production of 5'-IMP by the two strains was 2.4 g/L and 3.0 g/L,respectively.This study provided new insights into 5'-IMP production that used fermentation products as substrates.%5'-肌苷酸作为新一代增味剂的重要组成成分,在调味品行业具有十分重要的地位.为进一步缩短5'-肌苷酸生产周期,降低生产成本,在研究来源于摩氏摩根菌Morganella morganii的酸性磷酸酶AP/PTaseM催化条件基础上,将该酶编码基因phoCYM克隆至肌苷生产菌株Bacillus subtilis JG,获得B.subtilis JAB和B.subtilis JAF,并根据重组菌株合成肌苷及表达酸性磷酸酶的特性,通过调控发酵条件实现了肌苷发酵和酶催化相偶联的二步发酵法生产5'-肌苷酸.经摇瓶发酵实验验证,两菌株5'-肌苷酸产量分别为2.4 g/L和3.0 g/L.

  9. Effect of Inosine on Dopamine D2 Receptor and Dopamine Transporter in Brain of Rats with Tourette Syndrome%肌苷对Tourette综合征大鼠脑组织多巴胺D2受体和多巴胺转运蛋白的影响

    Institute of Scientific and Technical Information of China (English)

    郝伟红; 欧阳颖

    2011-01-01

    目的 研究肌苷对Tourette综合征(TS)大鼠脑组织多巴胺D2受体(DRD2)和多巴胺转运蛋白(DAT)的影响,探讨肌苷治疗TS可能的作用机制.方法 将40只SPF级雄性SD大鼠随机分为正常对照组、模型组、阳性对照组(氟哌啶醇0.5 mg·kg-1)、联合用药组(氟哌啶醇0.25 mg·kg-1+肌苷320 mg·kg-1)、肌苷组(肌苷320 mg·kg-1),每组8只.采用亚氨基二丙腈(IDNP)腹腔注射法建立TS大鼠模型.氟哌啶醇和(或)肌苷给药28 d后通过大鼠刻板行为评分、酶联免疫吸附法、原位杂交法,研究肌苷对TS模型大鼠刻板行为、大鼠脑组织DRD2、DAT水平的影响以及DRD2 mRNA表达情况.结果 1.肌苷组大鼠刻板行为评分低于模型组(P0.05).2.DRD2阳性细胞广泛分布于大脑皮质、海马、纹状体等处,以模型组DRD2阳性细胞分布最为密集.3.肌苷组DRD2水平低于模型组(P0.05).4.肌苷组DAT水平高于正常对照组、模型组及阳性对照组(Pa0.05).结论 肌苷改善TS模型大鼠的刻板行为的作用机制可能是通过促进多巴胺释放和转运,起到类似于DRD2拮抗剂的作用.%Objective To study the effect of inosine on dopamine D2 receptor ( DRD2 ) and dopamine transporter( DAT ) in the brain of rat model with Tourette syndrome (TS) and to explore the mechanism of action in treating TS with inosine. Methods Forty SPF male SD rats were divided into normal control group, model group, positive control group (haloperidol 0. 5mg · kg-1) ,chug combination group (haloperidol 0.25 tag · kg - 1 and inosine 320 mg· kg - 1 ), inosine group( inosine 320 ng · kg - 1 ) averagely and randomly. Rat model of TS was established by intraperitoneal injection with β, β' - iminodipropiomtrile. The effect of inosine on stereotyped behavior of rats, the levels of DRD2 and DAT,the expression of DRD2 mRNA in brain of rats were researched by the scores of stereotyped behavior on rats,enzye - linked immunosorbent assay and hybridization in situ

  10. Progress of Inosine Mono-phosphate(IMP) and Guanosine Mono-phosphate(GMP) Acid Production by Microbial Technology%利用微生物技术生产肌苷酸和鸟苷酸的进展

    Institute of Scientific and Technical Information of China (English)

    王美玲

    2014-01-01

    肌苷酸( IMP)和鸟苷酸( GMP)是非常有效的风味增强剂。它们和谷氨酸钠(味精)一起被广泛用作食品添加剂,共同发挥增强食物鲜味的作用。近年来,由于具有抗氧化性、神经保护作用、强心剂作用和免疫调节等有利作用,嘌呤类核苷酸都展现出了重要性。本综述回顾了利用微生物技术生产IMP和GMP的进展,包括其合成的代谢途径和调控网络,以及为获得这些嘌呤化合物所采用的生物技术流程和所用微生物菌种。%Inosine mono -phosphate ( IMP ) and guanosine mono -phosphate ( GMP ) are very effective flavor enhancers.They and mono-sodium glutamate (MSG) are widely used as food additives, working together to enhance the role of food flavors .In recent years ,with antioxidant activity ,neuro protective car-diac function and other favorable immunomodulatory effects of purine nucleotides ,they are showing further importance .The progress of microbial technology used to produce IMP and GMP are reviewed in this pa-per,including the synthesis of metabolic pathways and regulatory networks , as well as the biotechnology processes used to accept these purine compounds and microbial strains used .

  11. Stereoselective Synthesis of N 1-Lyxitol Inosine Derivative

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    1,4-anhydro-2-triflyl-3,5-O-benzenylidene-L-xylitol (5) was constructed in six steps from protected D-xylose. Substitution of 5 with protected 8-bromoinosine 6 gave the key intermediate 5′-O-TBS-2′,3′-di-O-acetyl-N'-(2"-deoxy- 1",4"-anhydro-3",5"-O-benzenylidene-Llyxitol-2"-yl)-8-bromoinosine (14). Selective removal of 5′-O-TBS-group gave the corresponding 5′-O-phosphorodianilidate 4 though phosphorylation, which was characterized by X-ray crystallographic analysis.

  12. Changes and the relationship of inosine-5'-monophosphate and biogenic amine of chilled pork during storage%冷藏期内猪肉肌苷酸与生物胺两者变化关系的研究

    Institute of Scientific and Technical Information of China (English)

    姚振乐; 刘国庆; 严伟民; 谢科; 高潮; 朱明

    2012-01-01

    采用高效液相色谱法测定生鲜猪肉的背最长肌在4℃温度条件下肌苷酸(IMP)和腐胺、尸胺、组胺、酪胺、亚精胺、精胺这六种生物胺含量的变化情况,从而进一步分析它们之间的相关性。结果表明,随着货架期的延长,IMP含量呈先上升后降低的趋势,并在第2d达到最高;精胺含量基本保持在4.0mg/kg左右,组胺的含量始终很低,其它的胺类物质都有所增加,尸胺的变化最为突出;虽然腐胺和亚精胺的含量比较低,但是仍然有明显的变化;酪胺变化也非常明显。从Person积差相关系数可以看出,IMP与其他指标相关系数呈负相关显著,有的指标是不显著的;在0.01水平上,IMP与亚精胺之间的负相关性最强,达到了-0.981;其次是尸胺与IMP之间,相关系数是-0.960,呈显著负相关;酪胺也与IMP显著负相关;IMP与腐胺、组胺、精胺的相关系数都不显著。因此,通过测定IMP含量变化可以预测猪肉新鲜度,且可作为猪肉保藏与加工过程中品质控制的重要指标之一。%Changes of inosine-5'-monophosphate(IMP),putrescine,cadaverine,histamine,tyramine,spermidine and spermine contents in longissimus dorsi at 4℃ were analyzed by HPLC,and the relationship was further discussed.The results showed that storage time affected the concentration of IMP,concentration of IMP increased until reached the top on the next day,then decreased gradually;The content of spermine basically maintained at about 4.0mg/kg.The content of histamine kept low during the storage.Others all increased,especially cadaverine.Although the contents of putrescine and spermidine were low,but the changes were significant,so as tyramine.From the part of the person correlation coefficient,IMP had remarkable negative relationship with spermidine,cadaverine and the correlation coefficient were-0.981 and-0.960 respectively,which was the highest between spermidine and IMP on the level of 0.01.IMP also had

  13. Study on Stability of Monosodium Glutamate and Disodium Inosine-5'-monophosphate +Disodium Guanosine-5'-monophosphate (I+G) Added to Food%谷氨酸钠及5’-肌苷酸钠+5'-鸟核酸钠(I+G)添加到食品中的稳定性研究

    Institute of Scientific and Technical Information of China (English)

    王向阳; 金菲; 李刚

    2012-01-01

    谷氨酸钠和5’-肌苷酸钠+5’-鸟苷酸钠(I+G)常被添加到食品中来提高鲜味.为了有效利用谷氨酸钠和I+G,将其添加到新鲜萝卜、腌制蔬菜、面粉中,并做热处理实验.谷氨酸钠和I+G含量采用HPLC法测定.结果表明,将谷氨酸钠和I+G添加到新鲜萝卜中,经过85℃和100℃热处理,放置3d,谷氨酸钠保存率分别为76.0%和76.3%,I+G保存率分别为95.6%和95.8%.而先放置3d,再分别85℃和100℃热处理,谷氨酸钠保存率分别为49.0%和41.8%,I+G保存率分别为8.7%和6.9%.将谷氨酸钠和I+G添加到腌制榨菜和腌制雪菜中,经过85℃热处理5 min并放置7d,榨菜和雪菜中谷氨酸钠保存率分别为62.5%和67.5%,I+G保存率分别为86.2%和87.2%.而先放置7d,再85℃热处理5 min,榨菜和雪菜中谷氨酸钠保存率分别为7.5%和7.5%,I+G保存率分别为38.7%和38.4%.将谷氨酸钠和I+G加到面粉中,180℃高温油炸处理3 min,谷氨酸钠保存率为18.8%,I+G保存率为7.9%.在新鲜切割蔬菜和腌制蔬菜中添加谷氨酸钠和I+G,需进行85℃以上热处理,油炸食品的油炸温度不宜过高.%Monosodium glutamate (MSC), disodium inosine-S'-monophosphate + disodium Cuanosine-S'-monophos-phate (I+G) were always added to food to enhance flavor. To make effective use of added monosodium glutamate and I+ G,they were added to flesh radish, pickled vegetables and flour to measure its content change by HPLC after heat treatment. The resuLts showed that when monosodium glutamate and I+G were added to fresh radish with 85 ℃ and 100 ℃ heat treatment, monosodium glutamate reserved 76.0% and 76.3%, I+G reserved 95.6% and 95.8% after 3 days. However, monosodium glutamate reserved 49.0% and 41.8%, I+G reserved 8.7% and 6.9% with 85℃and 100 ℃ heat treatment after 3 days. When monosodium glutamate and I+G were added to pickled tuber mustard and pickled yukina with 85 ℃ heat treatment for 5 min, monosodium

  14. Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

    OpenAIRE

    Sharmeen, L; Bass, B.; Sonenberg, N; Weintraub, H; Groudine, M

    1991-01-01

    Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs...

  15. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, R.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R.

    1999-01-01

    IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K{sub m} for NAD (1180 {mu}M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 {angstrom} with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione {beta}-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

  16. Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl.

    Science.gov (United States)

    Carsia, R V; Scanes, C G; Malamed, S

    1985-07-01

    Maturation of domestic fowl corticosteroidogenesis was evaluated using purified adrenocortical cells. Basal corticosterone production decreased steadily from 2 days to 26 weeks after hatching. However, maximally stimulated corticosterone production was not changed. In contrast, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH analogs increased approximately 40 times by 26 weeks, but the ED50 values of 8-bromo-cyclic AMP and pregnenolone were not changed. This suggests that adrenocortical cell sensitivity to ACTH decreases with maturation of the domestic fowl.

  17. 日粮中添加百里香对滩羊肉中肌苷酸和肌苷含量的影响%Effect of dietary thyme on muscle concentrations of inosine acid and inosine in Tan Sheep

    Institute of Scientific and Technical Information of China (English)

    康艳梅; 李爱华; 杨志峰

    2015-01-01

    为探究日粮中添加百里香对滩羊肌肉中肌苷酸和肌苷含量的影响,本试验分别在全混合日粮中添加5%百里香组、10%百里香组和对照组(不加百里香)对宁夏滩羊进行饲养,屠宰后利用高效液相色谱分析法,对滩羊背最长肌肉中肌苷酸和肌苷进行了分析.结果表明:日粮中添加百里香可从不同程度上提高肌肉中肌苷酸的含量,影响效果与添加百里香的剂量有关;5%百里香组极显著(P<0.01)提高了肌苷酸的含量,且可在一定时间范围内延缓肌苷酸降解生成肌苷,提高了IMP/ (IMP+I)新鲜度值,提高羊肉鲜味物质的含量,进而提高羊肉的风味;10%百里香组对肌苷酸和肌苷的影响不大,甚至略有下降的趋势.

  18. 酱油中肌苷和肌苷酸的高效液相色谱法分析%Quantitative analysis of inosine and inosinic acid in soy sauce by HPLC

    Institute of Scientific and Technical Information of China (English)

    吴波; 张寒俊

    2008-01-01

    采用高效液相色谱法对酱油中的肌苷和肌苷酸进行了定量分析,使用Polaris NH2色谱柱(200mm×4.6mm,5μm)、Phenomenex NH2预柱(4mm×3.0mm),以水(含0.6%KH2PO4,pH6.4)-乙醇为流动相,进行梯度淋洗,检测波长248nm.肌苷、肌苷酸分别在3~90 mg/L呈线性关系,样品加标平均回收率分别为98.9%、94.2%,相对标准偏差为1.1%~1.9%.实验证明,该方法适用于酱油中肌苷、肌苷酸的分析.

  19. 风味酱中肌苷、肌苷酸的高效液相色谱分析%Quantitative determination of inosine and inosinic acid in flavor paste by HPLC

    Institute of Scientific and Technical Information of China (English)

    吴波; 张寒俊

    2007-01-01

    采用高效液相色谱对风味酱中的肌苷、肌苷酸进行定量分析.Polaris C118-A色谱柱以水(含0.5%H3PO4)、异丙醇为流动相,梯度淋洗,检测波长248nm.结果表明,肌苷、肌苷酸分别在2mg/L~32mg/L呈线性关系,样品加样平均回收率分别为92.6%、95.7%,相对平均偏差1.4%~2.0%.

  20. Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

    Science.gov (United States)

    Huberman, Eliezer; Baccam, Mekhine J.

    2007-02-27

    The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

  1. Sensory Evaluation Test on Umami of Inosine-5'-Monophosphate%肌苷酸的鲜味评价试验

    Institute of Scientific and Technical Information of China (English)

    陈继兰; 文杰; 赵桂苹; 郑麦青; 杨宁

    2004-01-01

    试验对肌苷酸(IMP)及其与谷氨酸钠(MSG)的协同鲜味呈味作用进行了感官验证.试验结果表明,IMP的鲜味呈味作用显著,肉品IMP含量的差异会对肉品总体风味产生一定作用,作用大小与IMP和MSG等其它风味物的协同作用有关.

  2. Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration

    DEFF Research Database (Denmark)

    Christensen, S T; Chemnitz, J; Straarup, E M;

    1998-01-01

    phosphorylation of the PKC-specific substrate, myelin basic protein fragment 4-14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented......Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro...... by 8-bromo-cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine-induced cell death is an active process, associated with and/or requiring de novo RNA synthesis....

  3. Screening strains with high yield of inosine mutated by ions implantation%离子注入诱变选育肌苷高产菌株

    Institute of Scientific and Technical Information of China (English)

    刘国生; 侯瑛; 吴飞燕; 赵婷; 王秀强; 李宗义

    2009-01-01

    以肌苷产生菌--枯草芽孢杆菌(Bacillus subtilis HSD06)为出发菌株,经不同剂量的N+离子束注入处理,定向选育磺胺胍抗性提高的突变株.获得磺胺胍突变株的最佳照射剂量为3×1015cm-2.从528个抗性提高的菌落中筛选获得B11和B13肌苷高产菌株,其摇瓶发酵水平肌苷产量为14.83 g/L和14.38 g/L,分别比出发菌株提高了16.3%和12.8%.

  4. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579

    NARCIS (Netherlands)

    Hornstra, L.M.; Vries, de Y.P.; Vos, de W.M.; Abee, T.; Wells-Bennik, M.H.J.

    2005-01-01

    Bacillus cereus endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the gerA family of operons. Analysis of the Bacillus cereus ATCC 14579 genome revealed seven gerA family homologues. Using a transposon Tn917-

  5. Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Dandanell, Gert

    2005-01-01

    as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 k...

  6. Effect of Zhiqi Fungal Substance on Performance, Immunity and Inosine Monophosphate of Macrobrachium rosenbergii%芝芪菌质对罗氏沼虾生长和免疫及肌苷酸含量的影响

    Institute of Scientific and Technical Information of China (English)

    霍光明; 张李阳; 张永江; 阮鸣; 饶玉鹏

    2009-01-01

    以罗氏沼虾(Macrobrachium rosenbergii)为研究对象,研究芝芪菌质对其生长性能、机体免疫水平和肌苷酸含量的影响.将罗氏沼虾随机分成4组,每组3个平行,每个平行约70尾,第1组为对照组,常规投喂;另外3组为试验组,在基础日粮中分别添加0.25%、0.5%、1%的芝芪菌质,饲养35 d后测定其生长、血清和肝胰腺溶菌酶、SOD、碱性磷酸酶和酸性磷酸酶活力及肌肉中肌苷酸含量.试验表明,与对照组相比,试验组罗氏沼虾的增重率提高、死亡率降低.血清溶菌酶和SOD活性明显升高,0.5%、1%组中肝胰脏碱性磷酸酶和酸性磷酸酶活力明显增高(P0.05).饲喂适量的芝芪菌质能促进罗氏沼虾的生长和免疫力的提高.

  7. Comparative Study on Inosinic Acid and Related Nucleotides Contents of Muscles in Wuding Chicken%武定鸡肌肉肌苷酸及相关核苷酸含量的比较

    Institute of Scientific and Technical Information of China (English)

    朱仁俊; 唐臻睿; 李清; 黄启超; 李天祥; 冷静

    2014-01-01

    试验选取40、70、150和230日龄健康、体重接近的武定鸡公、母鸡各12只,比较不同性别、日龄和屠体部位肌肉肌苷酸(IMP)及IMP前体物、IMP降解物和ATP总代谢物含量,探讨肌苷酸的沉积规律。结果表明:日龄对武定鸡肌肉IMP及其相关核苷酸含量影响显著(P<0.05),随着日龄的增加IMP含量呈先增后降的趋势,150日龄时IMP含量显著高于其他日龄(P<0.05);性别及屠体部位对武定鸡肌肉IMP含量有显著影响(P<0.05),但对其相关核苷酸含量影响不明显,150日龄时武定鸡母鸡肌肉IMP含量显著高于公鸡(P<0.05),40和230日龄时其胸部肌肉的IMP含量显著高于腿部肌肉(P<0.05)。%The experiments through compared the contents of Wuding chicken's muscle IMP and its related nucleotides (ATP,ADP,AMP,Hx,HxR) in different gender, age and carcass parts to explore the deposition rule of IMP. The results showed that the age having significant effects to the contents of IMP and its related nucleotides (P<0.05), with the age increasing, the contents of IMP showed a tendency that first increased and then dropped, and the contents of IMP were significant higher in 150-day-age than other ages (P<0.05). The gender and carcass parts also having significant effects to the contents of IMP (P<0.05), but no significant effects to the contents of IMP related nucleotides;The contents of IMP in the hen muscles in 150-day-age were significant higher than the cock (P<0.05), and the IMP contents of chest muscles in 40-day-age and 230-day-age were significant higher than the leg muscles(P<0.05).

  8. 不同贮藏条件下鸡肉肌苷酸生成与降解规律的研究%Research on the Formation and Degradation of Inosinic 5'-Monophosphate in Chicken Muscle under Different Storage Temperature

    Institute of Scientific and Technical Information of China (English)

    陈继兰; 文杰; 李建军; 王述柏; 赵桂苹; 郑麦青

    2004-01-01

    研究了在室温(21~22℃)和4℃不同贮藏温度下,屠宰后1~24 h之内鸡胸肉肌苷酸(IMP)及其相关物含量的变化规律.结果显示,4℃下放血后24 h内IMP及其相关物浓度变化不大;12 h后的IMP含量与屠宰后1 h相当,24 h后仅降低4.9%.室温(21~22℃)下4 h后IMP浓度迅速下降,IMP降解产物浓度明显上升;12 h后鸡胸肉IMP含量损失16%(P<0.01),24 h后损失55%(P<0.01).经模拟计算,得回归方程y=-0.1282x2+0.4733x+2.0587(R2=0.9542),求得在本试验条件下,室温为21~22℃时,屠宰后110 min鸡胸肉IMP含量达到最高峰,其值为2.916 mg/g.4℃下4 h和8 h IMP的含量分别比室温高5.6%(P<0.05)和8.2%(P<0.05),12 h和24 h分别高19.4%(P<0.01)和105%(P<0.01).结果表明,鸡肉中生成IMP的反应很快完成,基本不受时间和温度的影响;但贮藏时间和温度对鸡肉IMP的降解速率有显著影响,温度越高,时间越长,IMP含量越低.

  9. 产氨短杆菌GMA-2802 1.2L罐肌苷发酵试验%The fermentation tests of inosine in 1.2L scale by Brevibacterium ammoniagenes mutant GMA-2802

    Institute of Scientific and Technical Information of China (English)

    施庆珊; 李良秋; 林小平; 邱玉棠

    2002-01-01

    采用诱变得来的产氨短杆菌GMA-2802,在1.2L自控发酵罐上进行5批发酵肌苷试验,发酵周期54小时,平均产肌苷20.4g/L.结果表明该菌株是一株具有较多优良特性的肌苷产生菌.

  10. Signal transduction triggered by iron to induce the nuclear importation of a Myb3 transcription factor in the parasitic protozoan Trichomonas vaginalis.

    Science.gov (United States)

    Hsu, Hong-Ming; Lee, Yu; Hsu, Pang-Hung; Liu, Hsing-Wei; Chu, Chien-Hsin; Chou, Ya-Wen; Chen, Yet-Ran; Chen, Shu-Hui; Tai, Jung-Hsiang

    2014-10-17

    Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.

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

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

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

  12. Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels.

    Science.gov (United States)

    Kolisek, Martin; Beck, Andreas; Fleig, Andrea; Penner, Reinhold

    2005-04-01

    The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzymatic Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2), but their mechanisms of action remain unknown. Here, we identify cyclic adenosine diphosphoribose (cADPR) as an agonist of TRPM2 with dual activity: at concentrations above 100 microM, cADPR can gate the channel by itself, whereas lower concentrations of 10 microM have a potentiating effect that enables ADPR to gate the channel at nanomolar concentrations. ADPR's breakdown product adenosine monophosphate (AMP) specifically inhibits ADPR, but not cADPR-mediated gating of TRPM2, whereas the cADPR antagonist 8-Br-cADPR exhibits the reverse block specificity. Our results establish TRPM2 as a coincidence detector for ADPR and cADPR signaling and provide a functional context for cADPR as a second messenger for Ca2+ influx.

  13. Two nucleoside uptake systems in Lactococcus lactis: Competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

    DEFF Research Database (Denmark)

    Martinussen, Jan; Wadskov-Hansen, Steen Lyders Lerche; Hammer, Karin

    2003-01-01

    in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K. for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition...

  14. Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared...

  15. Structural bioinformatics study of PNP from Schistosoma mansoni

    International Nuclear Information System (INIS)

    The parasite Schistosoma mansoni lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. Schistosomiasis is endemic in 76 countries and territories and amongst the parasitic diseases ranks second after malaria in terms of social and economic impact and public health importance. The PNP is an attractive target for drug design and it has been submitted to extensive structure-based design. The atomic coordinates of the complex of human PNP with inosine were used as template for starting the modeling of PNP from S. mansoni complexed with inosine. Here we describe the model for the complex SmPNP-inosine and correlate the structure with differences in the affinity for inosine presented by human and S. mansoni PNPs

  16. Taste receptors for umami: the case for multiple receptors1234

    OpenAIRE

    Chaudhari, Nirupa; Pereira, Elizabeth; Roper, Stephen D.

    2009-01-01

    Umami taste is elicited by many small molecules, including amino acids (glutamate and aspartate) and nucleotides (monophosphates of inosinate or guanylate, inosine 5′-monophosphate and guanosine-5′-monophosphate). Mammalian taste buds respond to these diverse compounds via membrane receptors that bind the umami tastants. Over the past 15 y, several receptors have been proposed to underlie umami detection in taste buds. These receptors include 2 glutamate-selective G protein–coupled receptors,...

  17. New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

    Directory of Open Access Journals (Sweden)

    Niamh Mannion

    2015-09-01

    Full Text Available The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases.

  18. A-to-I editing of protein coding and noncoding RNAs.

    Science.gov (United States)

    Mallela, Arka; Nishikura, Kazuko

    2012-01-01

    Adenosine deaminase acting on RNA (ADAR) catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) substrates. Inosine pairs preferentially with cytidine, as opposed to uridine; therefore, ADAR editing alters the sequence and base pairing properties of both protein-coding and non-coding RNA. Editing can directly alter the sequence of protein-coding transcripts and modify splicing, or affect a variety of non-coding targets, including microRNA, small interfering RNA, viral transcripts, and repeat elements such as Alu and LINE. Such editing has a wide range of physiological effects, including modification of targets in the brain and in disease states.

  19. A distant cis acting intronic element induces site-selective RNA editing

    DEFF Research Database (Denmark)

    Daniel, Chammiran; Venø, Morten Trillingsgaard; Ekdahl, Ylva;

    2012-01-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated th...

  20. Sequence Classification: 892238 [

    Lifescience Database Archive (English)

    Full Text Available nthase, an enzyme that catalyzes the second step in the biosynthesis of GMP from inosine 5'-phosphate (IMP); transcription is not sub...ject to regulation by guanine but is negatively regulated by nutrient starvation; Gua1p || http://www.ncbi.nlm.nih.gov/protein/6323873 ...

  1. 21 CFR 170.60 - Nitrites and/or nitrates in curing premixes.

    Science.gov (United States)

    2010-04-01

    ... oils, disodium inosinate, disodium guanylate, hydrolysates of animal or plant origin (such as hydrolyzed vegetable protein), oleoresins of spices, soy products, and spice extractives. Such food additives... Section 170.60 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

  2. Flavour formation in pork semimembranosus: Combination of pan-temperature and raw meat quality

    DEFF Research Database (Denmark)

    Meinert, Lene; Tikk, Kaja; Tikk, Meelis;

    2008-01-01

    /240 degrees C). It was further investigated whether the precursor levels of glycogen, IMP, inosine, and hypoxanthine in the raw meat were correlated to the raw meat quality and fried/grilled attributes. Pork schnitzels were fried on a pan (155 degrees C) or grill-pan (240-250 degrees C) to a core...

  3. Taste Perception with Age: Generic or Specific Losses in Supra-threshold Intensities of Five Taste Qualities?

    NARCIS (Netherlands)

    Mojet, J.; Heidema, J.; Christ-Hazelhof, E.

    2003-01-01

    The influence of ageing on supra-threshold intensity perception of NaCl, KCl, sucrose, aspartame, acetic acid, citric acid, caffeine, quinine HCl, monosodium glutamate (MSG) and inosine 5'-monophosphate (IMP) dissolved in water and in `regular' product was studied in 21 young (19¿33 years) and 21 el

  4. Taste perception with age: pleasantness and its relationships with threshold sensitivity and supra-threshold intensity of five taste qualities

    NARCIS (Netherlands)

    Mojet, J.; Christ-Hazelhof, E.; Heidema, J.

    2005-01-01

    The relationships between threshold sensitivity, supra-threshold intensity of NaCl, KCl, sucrose, aspartame, acetic acid, citric acid, caffeine, quinine HCl, monosodium glutamate (MSG) and inosine 5¿-monophosphate (IMP), and the pleasantness of these stimuli in products, were studied in 21 young sub

  5. Brain hypoxanthine concentration correlates to lactate/pyruvate ratio but not intracranial pressure in patients with acute liver failure

    DEFF Research Database (Denmark)

    Bjerring, Peter Nissen; Hauerberg, John; Jørgensen, Linda;

    2010-01-01

    hypoxanthine, inosine, and lactate/pyruvate (LP) ratio are increased and correlated in patients with acute liver failure. Furthermore, we expect the purines and L/P ratio to correlate with intracranial pressure (ICP) (positively), and cerebral perfusion pressure (CPP) (negatively)....

  6. Sequence Classification: 183954 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17988432|ref|NP_541065.1| INOSINE-URIDINE PREFERRING NUC...LEOSIDE HYDROLASE || http://www.ncbi.nlm.nih.gov/protein/17988432 ...

  7. Sequence Classification: 246812 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|62391672|ref|YP_227074.1| INOSINE-URIDINE PREFERRING NUC...LEOSIDE HYDROLASE || http://www.ncbi.nlm.nih.gov/protein/62391672 ...

  8. IMP metabolism in human skeletal muscle after exhaustive exercise

    DEFF Research Database (Denmark)

    Tullson, P. C.; Bangsbo, Jens; Hellsten, Ylva;

    1995-01-01

    inosine 5'-monophosphate (IMP) formation. After exercise, blood flow to one leg was occluded. Muscle biopsies (vastus lateralis) were taken before and 3.6 +/- 0.2 min after exercise from the occluded leg and 0.7 +/- 0.0, 1.1 +/- 0.0, and 2.9 +/- 0.1 min postexercise in the nonoccluded leg. Exercise...... activated AMPD; at exhaustion IMP was 3.5 +/- 0.4 mmol/kg dry muscle. Before exercise, 16.0 +/- 1.6% of AMPD cosedimented with the myosin fraction; the extent of AMPD:myosin binding was unchanged by exercise. Inosine content increased about threefold during exercise and twofold more during recovery; by 2.......9 min postexercise it was 0.43 +/- 0.02 mmol/kg dry muscle. IMP decreased 2.1 +/- 0.3 mmol/kg dry muscle with no change in total adenylates. Total purines declined significantly (P

  9. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity.

    Science.gov (United States)

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R; Cuny, Gregory D; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-05-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications. PMID:25945705

  10. [Metformin impact on purine metabolism in breast cancer].

    Science.gov (United States)

    Shatova, O P; Butenko, Eu V; Khomutov, Eu V; Kaplun, D S; Sedakov, I Eu; Zinkovych, I I

    2016-03-01

    Large-scale epidemiological and clinical studies have demonstrated the efficacy of metformin in oncology practice. However, the mechanisms of implementation of the anti-tumor effect of this drug there is still need understanding. In this study we have investigated the effect of metformin on the activity of adenosine deaminase and respectively adenosinergic immunosuppression in tumors and their microenvironment. The material of the study was taken during surgery of breast cacer patients receiveing metformin, and also patients which did not take this drug. The adenosine deaminase activity and substrate (adenosine) and products (inosine, hypoxanthine) concentrations were determined by HPLC. Results of this study suggest that metformin significantly alters catabolism of purine nucleotides in the node breast adenocarcinoma tisue. However, the metformin-induced increase in the adenosine deaminase activity is not sufficient to reduce the level of adenosine in cancer tissue. Thus, in metformin treated patients the adenosine concentration remained unchanged, and inosine and hypoxanthine concentration significantly increased. PMID:27420623

  11. Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1989-01-01

    Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated b...... a purF, a purL or a purM mutation. A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE....

  12. Mycophenolic acid:a novel immunosuppressive drug%霉吩酸:一种新型免疫抑制剂

    Institute of Scientific and Technical Information of China (English)

    李红良; 任先达; 叶开和

    2001-01-01

    Mycophenolate acid is a novel immunosuppressive drug. Its target of action is the isomerⅡof inosine 5'-monophosphate dehydrogenase(IMPDH). It inhibits de nove purine synthesis and also decreases expression of adhesive molecule. It inhibits selectively the proliferation of lymphocyte, so that it has strong immunosuppressive effects on various rejections to allograft or xenograft, and on autoimmune diseases, and has the features of higher potency and lower toxicity.

  13. Mycophenolic acid: a drug with a potential beyond renal transplantation

    Directory of Open Access Journals (Sweden)

    Jammula P. Patro

    2016-09-01

    Full Text Available Mycophenolic acid is an anti-metabolite immunosuppressant. It inhibits the enzyme inosine monophosphate dehydrogenase, which is essential for purine synthesis. It is indicated in prevention of rejection after renal transplantation. It is one of the few drugs, which were discovered more than a century ago and still in active use. This review discusses the other current and potential uses of the drug. [Int J Res Med Sci 2016; 4(9.000: 3666-3669

  14. Experiencia con micofenolato mofetil en el trasplante renal

    OpenAIRE

    Esteban de la Rosa, Rafael Jos??

    2001-01-01

    Micofenolato mofetil (MMF) es un potente inmunosupresor, libre de efecto nefrot??xico, eficaz en la prevencion de rechazo agudo y en la nefropatia cronica del trasplante renal. Bloquea la fase de sintesis sobre la estirpe linfocitaria a trav??s de inhibici??n selectiva, reversible y no competitiva de la enzima inosin-monofosfato-deshidrogenasa(IMPDH), clave en la biosintesis de novo de las purinas. OBJETIVOS:Se evaluaron tres grupos de trasplantados renales (TR) seg??n la terapia inmunosupres...

  15. Protein expression in response to folate stress in Escherichia coli.

    OpenAIRE

    Huang, E Y; Mohler, A M; Rohlman, C E

    1997-01-01

    Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the ...

  16. Possible Processes for Origin of First Chemoheterotrophic Microorganisms with Modeling of Physiological Processes of Bacterium Bacillus subtilis as a Model System in 2H2O

    OpenAIRE

    Ignat Ignatov; Oleg Mosin

    2015-01-01

    We studied possible processes for origin of first chemoheterotrophic microorganisms with modeling of physiological processes of a Gram-positive chemoheterotrophic bacterium Bacillus subtilis, producer of purine ribonucleoside inosine as a model system in heavy water. The physiological influence of deuterium on the chemoheterotrophic bacterium B. subtilis was studied on a heavy water (HW) medium with a maximal concentration of 2H2O (89–90 atom% 2H). Also various suitable samples of hot mineral...

  17. Inhibition of T lymphocyte activation in mice heterozygous for loss of the IMPDH II gene

    OpenAIRE

    Gu, Jing Jin; Stegmann, Sander; Gathy, Karen; Murray, Robert; Laliberte, Josee; Ayscue, Lanier; Mitchell, Beverly S.

    2000-01-01

    Inosine 5′-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome–linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of th...

  18. Synthesis and biological evaluation of 2,4-diaminopteridine derivatives as nitric oxide synthase inhibitor

    Institute of Scientific and Technical Information of China (English)

    Fei Ma; Gang Lü; Wei Fen Zhou; Qiu Juan Wang; Yi Hua Zhang; Qi Zheng Yao

    2009-01-01

    A series of novel 2,4-diamino-pteridines(9a-1)were synthesized and evaluated as inhibitors of inducible nitric oxide synthase (iNOS)in vitro.It was found that 9a,9d,9e,9h,9i and 91 showed potent inhibitory activities similar to that of methotrexate(MTX),while the activities of 9b,9c,9f,9g,9j and 9k ale stronger than MTX.

  19. REMAXOL: MECHANISMS OF ACTION AND APPLICATION IN REAL CLINICAL PRACTICE

    Directory of Open Access Journals (Sweden)

    L. Yu. Ilchenko

    2016-01-01

    Full Text Available The main pathogenic effects of the original nativedrug — remaxol combining properties of balanced polyionic solution (methionine, inosine, nicotinamide and succinic acid were introduced additionally, antioxidant, antihypoxant and hepatotropic agent are considered in review. The results of its application in clinical practice among patients with alcoholic fatty liver disease, metabolic disorders, viral hepatitis, drug hepatotoxicity and in the perioperative period are presented.

  20. A-Z of nutritional supplements: dietary supplements, sports nutrition foods and ergogenic aids for health and performance-Part 20

    OpenAIRE

    Currell, K; Derave, Wim; Everaert, Inge; McNaughton, L; Slater, G.; Burke, LM; Stear, SJ; Castell, LM

    2011-01-01

    As usual, the alphabet throws together a mixture of supplements with different levels of popularity and scientific support. Part 20 covers some rarely reported, studied and/or little used supplements in sport: glycine, histidine and inosine. The majority of human studies of supplementation with the essential amino acid histidine has involved clinical work. In terms of athletic performance, there is current interest in supplementation strategies to increase muscle content of the histidine-cont...

  1. CHARACTERIZATION OF UMAMI TASTE SENSITIVITY IN CHILDREN WITH AND WITHOUT CANCER

    OpenAIRE

    Elman Ilana; Geraldo Ana Paula Gines; Karcher Cristiane; Pinto-e-Silva Maria Elisabeth Machado

    2014-01-01

    INTRODUCTION: The umami taste comes from glutamate and 5 ribonucleotides including inosinate and guanylate, which appear naturally in many foods. It can be identified by monosodium glutamate, being considered as a subtle taste, but blending well with other tastes, expands and enhances the flavor. OBJECTIVE: to identify umami taste thresholds in children with ALL or NHL and in healthy school children and to correlate taste sensitivity with nutritional status, age and gender.&nbs...

  2. イワシ調味液製造に関する研究

    OpenAIRE

    坪内, 一夫; 久松, 眞; 山田, 哲也; Tsubouchi, Kazuo; Hisamatsu, Makoto; Yamada, Tetsuya

    1991-01-01

    Production of fish sauces from sardines with proteases was investigated. To prevent sardine putrefaction during processing and obtain a good yield regarding the umami component,the hydrdysis condition was discussed, and the results obtained are following: 1. Eleven commercial proteases were examined, and the three proteases were selected from them on the data of hydrolysis ratio of sardine, contents of soluble nitrogen components and amounts ofliberated glutamic acid and inosinic acid. 2. ...

  3. Understanding the role of Umami in appetite control: a protein-specific effect?

    OpenAIRE

    Masic, Una

    2014-01-01

    The fifth basic taste, ‘umami’, is the flavour function elicited by amino acids like monosodium glutamate (MSG) in foods. This taste is recognized for its flavour enhancing properties but little is known about its effects on appetite and intake. Thus the experiments in this thesis aimed to understand how umami influences pleasantness, appetite stimulation, satiation and satiety using MSG, with some additional focus on its associated ribonucleotide inosine 5’-monophosphate (IMP). Chapter 2...

  4. Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to bitter, sweet, and umami taste stimuli

    OpenAIRE

    DeSimone, John A.; Phan, Tam-Hao T.; Ren, Zuojun; Mummalaneni, Shobha; Lyall, Vijay

    2012-01-01

    The relationship between taste receptor cell (TRC) intracellular Ca2+ ([Ca2+]i) and rat chorda tympani (CT) nerve responses to bitter (quinine and denatonium), sweet (sucrose, glycine, and erythritol), and umami [monosodium glutamate (MSG) and MSG + inosine 5′-monophosphate (IMP)] taste stimuli was investigated before and after lingual application of ionomycin (Ca2+ ionophore) + Ca2+, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Ca2+ chelator), U7312...

  5. Purine metabolism in Toxoplasma gondii

    Energy Technology Data Exchange (ETDEWEB)

    Krug, E.C.; Marr, J.J.; Berens, R.L.

    1989-06-25

    We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.

  6. The Epitranscriptome and Innate Immunity.

    Directory of Open Access Journals (Sweden)

    Mary A O'Connell

    2015-12-01

    Full Text Available Our knowledge of the variety and abundances of RNA base modifications is rapidly increasing. Modified bases have critical roles in tRNAs, rRNAs, translation, splicing, RNA interference, and other RNA processes, and are now increasingly detected in all types of transcripts. Can new biological principles associated with this diversity of RNA modifications, particularly in mRNAs and long non-coding RNAs, be identified? This review will explore this question by focusing primarily on adenosine to inosine (A-to-I RNA editing by the adenine deaminase acting on RNA (ADAR enzymes that have been intensively studied for the past 20 years and have a wide range of effects. Over 100 million adenosine to inosine editing sites have been identified in the human transcriptome, mostly in embedded Alu sequences that form potentially innate immune-stimulating dsRNA hairpins in transcripts. Recent research has demonstrated that inosine in the epitranscriptome and ADAR1 protein establish innate immune tolerance for host dsRNA formed by endogenous sequences. Innate immune sensors that detect viral nucleic acids are among the readers of epitranscriptome RNA modifications, though this does preclude a wide range of other modification effects.

  7. Modeling of DNA zipper reaction rates

    Science.gov (United States)

    Landon, Preston; Sanchez, Casey; Mo, Alexander; Lal, Ratnesh

    2012-02-01

    DNA zippers are a thermodynamically driven system consisting of three DNA oligonucleotides. Two of the strands are designed to create a small helix the third is designed to invade and separated the helix. A zipper system consisting of a normal strand (N), a weak strand (W), and an opening strand (O). N is made up of normal DNA bases, while W is engineered with inosine bases substituted for guanine. Inosine forms one less hydrogen bond with cytosine than guanine. By varying the number and order of inosine, W is engineered to provide less than natural bonding affinities to N in forming the [N:W] helix. When O is introduced (a natural complement of N), it competitively displaces W from [N:W] and forms [N:O]. DNA zippers have been used to create new DNA devices such as springs and tweezers and to create functionalized DNA origami structures. Currently, The basic principles and interactions of DNA zippers are not well understood. Here we will report the results on an investigation of several different DNA zipper constructs designed to aid in the creation of a mathematical prediction of the reaction rate for DNA zippers.

  8. Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

    Science.gov (United States)

    Wang, Xiaoyue; Wang, Guanglu; Li, Xinli; Fu, Jing; Chen, Tao; Wang, Zhiwen; Zhao, Xueming

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis. PMID:27234879

  9. Umami the Fifth Basic Taste: History of Studies on Receptor Mechanisms and Role as a Food Flavor

    Directory of Open Access Journals (Sweden)

    Kenzo Kurihara

    2015-01-01

    Full Text Available Three umami substances (glutamate, 5′-inosinate, and 5′-guanylate were found by Japanese scientists, but umami has not been recognized in Europe and America for a long time. In the late 1900s, umami was internationally recognized as the fifth basic taste based on psychophysical, electrophysiological, and biochemical studies. Three umami receptors (T1R1 + T1R3, mGluR4, and mGluR1 were identified. There is a synergism between glutamate and the 5′-nucleotides. Among the above receptors, only T1R1 + T1R3 receptor exhibits the synergism. In rats, the response to a mixture of glutamate and 5′-inosinate is about 1.7 times larger than that to glutamate alone. In human, the response to the mixture is about 8 times larger than that to glutamate alone. Since glutamate and 5′-inosinate are contained in various foods, we taste umami induced by the synergism in daily eating. Hence umami taste induced by the synergism is a main umami taste in human.

  10. Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus).

    Science.gov (United States)

    del Castillo Velasco-Martínez, Iris; Hernández-Camacho, Claudia J; Méndez-Rodríguez, Lía C; Zenteno-Savín, Tania

    2016-01-01

    In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD(+)), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

  11. Early Cellular Responses of Purine Nucleoside-mediated Protection of Hypoxia-induced Injuries of Neuronal PC12 Cells

    Directory of Open Access Journals (Sweden)

    Bettina Tomaselli

    2005-01-01

    Full Text Available Hypoxia in brain may lead to cell death by apoptosis and necrosis. In parallel adenosine, a powerful endogenous neuroprotectant is formed. We wanted to investigate the effect of adenosine and its purine nucleoside relatives, inosine and guanosine on early cellular responses to hypoxia. O2-sensitive neuronal PC12-cells were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Loss of viability after hypoxic insult was impressively rescued by adenosine, guanosine and inosine. PC12-cells mainly express the A2A adenosine receptor. Its inhibition with a specific antagonist (CSC induced cell death of PC12-cells, which could be salvaged by adenosine but not with guanosine or inosine. We have previously demonstrated the important role of mitogen activated protein kinases 1/2 (p42/44 MAPK in purine-mediated rescue. In this study we were interested in the involvement of protein kinases whose activities mediate these processes, including protein kinase A (PKA, phosphoinositide 3-kinase (PI3-K and protein kinase C-related kinases (PRK 1/2. Pharmacological inhibition of PKA and PI3-K increased hypoxia-induced toxicity and likewise also affected the rescue by purine nucleosides. Nerve growth factor (NGF and purine nucleosides induced an activation of PRK 1/2, which to our knowledge indicates for the first time that these kinases are potentially involved in purine nucleoside-mediated rescue of hypoxic neuronal cells. Results suggest that A2A receptor expressing cells are mainly dependent on the purine nucleoside adenosine for their rescue after hypoxic insult. In addition to PKA, PI3-K is an important effector molecule in A2A-mediated signaling and for the rescue of PC12-cells after hypoxic insult.

  12. Identification of feeding stimulants for shrimp%摄食促进剂对对虾生长的影响

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two experiments were conducted to screen 5'-inosine- monophosphate,betaine,fish hydrolysate,TMA-O and DMPT as feeding stimulants for shrimp.Feeding stimulants carrier was non-attractive diet.The first experiment was conducted to observe the attractant response.The maximum attractant response was attained when the diet contained DMPT.The second experiment was to observe the effect of attractant on growth and FCR of shrimp.The maximum weight gain rate and the minimum FCR was attained when the diet contained DMPT.

  13. Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine from Octopus cyanea.

    Science.gov (United States)

    Shaker, Kamel H; Göhl, Matthias; Müller, Tobias; Seifert, Karlheinz

    2015-11-01

    The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1), 2-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and auramine (3) have been isolated from the sea anemone Heteractis aurora. Both indole alkaloids were synthesized for the confirmation of the structures. Homarine (4), along with uracil (5), hypoxanthine (6), and inosine (7) have been obtained from Octopus cyanea.

  14. Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

    OpenAIRE

    Macbeth, Mark R.; Lingam, Arunth T.; Bass, Brenda L.

    2004-01-01

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of on...

  15. The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers | Office of Cancer Genomics

    Science.gov (United States)

    Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of 6,236 patient samples of 17 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions.

  16. Methods and materials relating to IMPDH and GMP production

    Science.gov (United States)

    Collart, Frank R.; Huberman, Eliezer

    1997-01-01

    Disclosed are purified and isolated DNA sequences encoding eukaryotic proteins possessing biological properties of inosine 5'-monophosphate dehydrogenase ("IMPDH"). Illustratively, mammalian (e.g., human) IMPDH-encoding DNA sequences are useful in transformation or transfection of host cells for the large scale recombinant production of the enzymatically active expression products and/or products (e.g., GMP) resulting from IMPDH catalyzed synthesis in cells. Vectors including IMPDH-encoding DNA sequences are useful in gene amplification procedures. Recombinant proteins and synthetic peptides provided by the invention are useful as immunological reagents and in the preparation of antibodies (including polyclonal and monoclonal antibodies) for quantitative detection of IMPDH.

  17. Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

    OpenAIRE

    Smoot, L A; Pierson, M. D.

    1981-01-01

    The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cystein...

  18. Kidney Disease and the Nexus of Chronic Kidney Disease and Acute Kidney Injury: The Role of Novel Biomarkers as Early and Accurate Diagnostics.

    Science.gov (United States)

    Yerramilli, Murthy; Farace, Giosi; Quinn, John; Yerramilli, Maha

    2016-11-01

    Chronic kidney disease (CKD) and acute kidney injury (AKI) are interconnected and the presence of one is a risk for the other. CKD is an important predictor of AKI after exposure to nephrotoxic drugs or major surgery, whereas persistent or repetitive injury could result in the progression of CKD. This brings new perspectives to the diagnosis and monitoring of kidney diseases highlighting the need for a panel of kidney-specific biomarkers that reflect functional as well as structural damage and recovery, predict potential risk and provide prognosis. This article discusses the kidney-specific biomarkers, symmetric dimethylarginine (SDMA), clusterin, cystatin B, and inosine. PMID:27485279

  19. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  20. [Studies on chemical protectors against radiation. XXXIII. Protective mechanisms of various compounds against skin injury induced by radiation].

    Science.gov (United States)

    Sato, Y; Kumazawa, N; Suzuki, M; Wang, C M; Ohta, S; Shinoda, M

    1991-01-01

    The radiation protective mechanisms on skin injury induced by soft X-irradiation were investigated by use of various radiation protective agents such as sulfur compounds (MEA, MEG, thiourea), nucleic acid constitutional compounds (adenosine, inosine), antioxidative compounds (sesamol, ferulic acid, ascorbic acid), crude drugs (Rosae Fructus, Anemarrhenae Rhizoma, Trapae Fructus, Forsythiae Fructus, Aloe arborescens). Scavenge action of activated oxygen, inhibitory effect of lipid peroxidation, induction of antioxidative protein and protective effect against damage of deoxyribonucleic acid and superoxide dismutase by X-irradiation were evaluated as the radiation protective mechanisms, and relationship between these results and protective effect of skin injury induced by radiation was studied. PMID:1905349

  1. Studies on chemical protectors against radiation, 33; Protective mechanisms of various compounds against skin injury induced by radiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Yushi; Kumazawa, Noriko; Suzuki, Makoto; Wang Cheng-Ming; Ohta, Setsuko; Shinoda, Masato (Hoshi Univ., Tokyo (Japan))

    1991-01-01

    The radiation protective mechanisms on skin injury induced by soft X-irradiation were investigated by use of various radiation protective agents such as sulfur compounds (MEA, MEG, thiourea), nucleic acid constitutional compounds (adenosine, inosine), antioxidative compounds (sesamol, ferulic acid, ascorbic acid), crude drugs (Rosae Fructus, Anemarrhenae Rhizoma, Trapae Fructus, Forsythiae Fructus, Aloe arborescens). Scavenge action of activated oxygen, inhibitory effect of lipid peroxidation, induction of antioxidative protein and protective effect against damage of deoxyribonucleic acid and superoxide dismutase by X-irradiation were evaluated as the radiation protective mechanisms, and relationship between these results and protective effect of skin injury induced by radiation was studied. (author).

  2. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    OpenAIRE

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent ...

  3. Variation in umami perception and in candidate genes for the umami receptor in mice and humans1234

    OpenAIRE

    Shigemura, Noriatsu; Shirosaki, Shinya; Ohkuri, Tadahiro; Sanematsu, Keisuke; Islam, AA Shahidul; Ogiwara, Yoko; Kawai, Misako; Yoshida, Ryusuke; Ninomiya, Yuzo

    2009-01-01

    The unique taste induced by monosodium glutamate is referred to as umami taste. The umami taste is also elicited by the purine nucleotides inosine 5′-monophosphate and guanosine 5′-monophosphate. There is evidence that a heterodimeric G protein–coupled receptor, which consists of the T1R1 (taste receptor type 1, member 1, Tas1r1) and the T1R3 (taste receptor type 1, member 3, Tas1r3) proteins, functions as an umami taste receptor for rodents and humans. Splice variants of metabotropic glutama...

  4. 老化に伴うラットの味覚嗜好性の変化

    OpenAIRE

    原田, 秀逸; 三浦, 裕仁; ハラダ, シュウイツ; ミウラ, ヒロヒト; Harada, Shuitsu; MIURA, Hirohito

    2015-01-01

    The effect of aging on taste were compared between the preference and neural responses from the greater superficial petrosal nerve (GSP) innervating the soft palate and the chorda tympani nerve (CT) innervating the fungiform papillae in the young (5-12 weeks) and aged (19-22 months) Sprague Dawley rat. A two-bottle preference test revealed that younger rats significantly preferred 0.001 M 5'-inosine monophosphate (IMP), 0.01 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + ...

  5. ロウカ ニ ト モナウ ラット ノ ミカク シコウセイ ノ ヘンカ

    OpenAIRE

    原田, 秀逸; 三浦, 裕仁; ハラダ, シュウイツ; ミウラ, ヒロヒト; Harada, Shuitsu; MIURA, Hirohito

    2015-01-01

    The effect of aging on taste were compared between the preference and neural responses from the greater superficial petrosal nerve (GSP) innervating the soft palate and the chorda tympani nerve (CT) innervating the fungiform papillae in the young (5-12 weeks) and aged (19-22 months) Sprague Dawley rat. A two-bottle preference test revealed that younger rats significantly preferred 0.001 M 5'-inosine monophosphate (IMP), 0.01 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + ...

  6. Genetics of intake of umami-tasting solutions by mice

    OpenAIRE

    Bachmanov, Alexander A.; Gary K Beauchamp

    2001-01-01

    Inbred strains of mice provide a powerful tool for genetic dissection of quantitative behavioral traits. We have investigated intake of the umami-tasting substances monosodium glutamate (MSG) and inosine 5′-monophosphate (IMP) in inbred mice. Studies with two inbred strains, C57BL/6ByJ and I29P3/J have revealed strain differences in voluntary consumption of 300 mM MSG which depend, at least partially, on postingestive effects of solution consumption, as well as on strain differences in prefer...

  7. Umami the Fifth Basic Taste: History of Studies on Receptor Mechanisms and Role as a Food Flavor

    OpenAIRE

    Kenzo Kurihara

    2015-01-01

    Three umami substances (glutamate, 5′-inosinate, and 5′-guanylate) were found by Japanese scientists, but umami has not been recognized in Europe and America for a long time. In the late 1900s, umami was internationally recognized as the fifth basic taste based on psychophysical, electrophysiological, and biochemical studies. Three umami receptors (T1R1 + T1R3, mGluR4, and mGluR1) were identified. There is a synergism between glutamate and the 5′-nucleotides. Among the above receptors, only T...

  8. Umami taste transduction mechanisms1234

    OpenAIRE

    Kinnamon, Sue C.

    2009-01-01

    l-Glutamate elicits the umami taste sensation, now recognized as a fifth distinct taste quality. A characteristic feature of umami taste is its potentiation by 5′-ribonucleotides such as guanosine-5'-monophosphate and inosine 5′-monophosphate, which also elicit the umami taste on their own. Recent data suggest that multiple G protein–coupled receptors contribute to umami taste. This review will focus on events downstream of the umami taste receptors. Ligand binding leads to Gβγ activation of ...

  9. Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/J mice

    OpenAIRE

    INOUE, MASASHI; Gary K Beauchamp; Bachmanov, Alexander A.

    2004-01-01

    In long-term two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more monosodium L-glutamate (MSG) and inosine-5′-monophosphate (IMP) compared with mice from the 129P3/J (129) strain. The goal of this study was to assess the role of afferent gustatory input in these strain differences. We measured integrated responses of the mouse chorda tympani and glossopharyngeal nerves to lingual application of compounds that evoke umami taste in humans: MSG, monoammonium L-glutamate (NH4 glutama...

  10. 留兰香水溶性部分化学成分的分离与鉴定%Chemical constituents of water-soluble part of Mentha spicata L.

    Institute of Scientific and Technical Information of China (English)

    宋妍; 陈广通; 孙博航; 黄健; 李铣; 吴立军

    2008-01-01

    目的 对唇形科薄荷属植物留兰香(Mentha spicata L.)的水溶性部分的化学成分进行研究.方法 采用甲基化的方法处理水溶性部分样品,采用硅胶柱色谱等方法进行分离纯化,根据理化性质和NMR波谱数据进行结构鉴定.结果 分离得到5个化合物,分别鉴定为柠檬酸三甲酯(trimethyl citrate,1)、原儿茶酸甲酯(methyl protocatechuate,2)、香草酸甲酯(methyl vanillate,3)、次黄嘌呤核苷(inosine,4)、尿苷(uridine,5).将它们还原为甲基化前的结构,依次为柠檬酸(citricacid,1')、原儿茶酸(protocatechuic acid,2')、香草酸(vanillic acid,3')、次黄嘌呤核苷(inosine,4)、尿苷(uridine,5).结论 化合物4、5为首次从薄荷属植物中分离得到,化合物1'为首次从留兰香中分离得到.

  11. The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7.

    Science.gov (United States)

    Kusada, Hiroyuki; Hanada, Satoshi; Kamagata, Yoichi; Kimura, Nobutada

    2014-07-01

    Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

  12. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    Science.gov (United States)

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures. PMID:26983735

  13. Purinergic nerves and purinoceptors: early perspectives.

    Science.gov (United States)

    Satchell, D

    2000-07-01

    I have had the pleasure and privilege of being involved in one facet of Geoffrey Burnstock's early career. I have reviewed this work together with more recent developments in the area. In 1968, the presence of non-adrenergic, non-cholinergic inhibitory nerves had been established but the identity of their neurotransmitter was unknown. Stimulation of these nerves in recycled perfused toad and guinea-pig stomachs caused release of adenosine and inosine. When ATP was added to recycled perfusates, it was broken down to adenosine and inosine. These findings together with information that AMP was released from stimulated, isolated turkey Auerbach's plexus which was known to contain the nerves, suggested that ATP could be the neurotransmitter. This was supported by observations that ATP elicited responses similar to that of nerve stimulation in a variety of tissues. Developments from the early purinergic nerve hypothesis are considered including independence of extracellular actions of ATP from its intracellular actions, identification and cloning of purinoceptors and cotransmission of ATP with other substances.

  14. Identification and regional localization of a human IMP dehydrogenase-like locus (IMPHDL1) at 16p13. 13

    Energy Technology Data Exchange (ETDEWEB)

    Doggett, N.A.; Tesmer, J.G.; Duesing, L.A. (Los Alamos National Lab., NM (United States)); Callen, D.F.; Chen, Z.L.; Moore, S. (Adelaide Children' s Hospital, North Adelaide (Australia)); Stallings, R.L. (Univ. of Pittsburgh, PA (United States))

    1993-12-01

    Sequence-tagged sites (STS)s are versatile chromosomal markers for a variety of genome mapping efforts. In this report, the authors describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5[prime]-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5[prime]-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 bp of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.3 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. The authors conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13. 11 refs., 2 figs.

  15. Biochemical brain markers and purinergic parameters in rat CSF after seizure induced by pentylenetetrazol.

    Science.gov (United States)

    Oses, Jean Pierre; Leke, Renata; Portela, Luis Valmor; Lara, Diogo R; Schmidt, André P; Casali, Emerson André; Wofchuk, Susana; Souza, Diogo O; Sarkis, João José Freitas

    2004-09-30

    Cellular and molecular mechanisms involved in the generation of seizures and the magnitude of neural cells injury are not fully understood. We evaluated astrocyte and/or neuronal injury in rats in the pentylenetetrazol model of acute seizures by measuring S100B and NSE levels in cerebrospinal fluid. Additionally, we determined ADP and GDP hydrolysis by soluble nucleoside triphosphate diphosphohydrolase in the cerebrospinal fluid, and the concentration of nucleosides adenosine, inosine and guanosine as putative markers of brain injury. After pentylenetetrazol-induced seizures: (i) S100B values increased from 10 to 30 min, returning to control levels at 24 h; NSE levels presented a biphasic increase: an increase at 10 to 30 min returning to control levels, and again at 240 min followed by a decline at 24 h; (ii) nucleotidase activities increased from 10 min, returning to control levels at 240 min; (iii) guanosine and inosine levels increased exclusively after 30 min. In summary, this study showed biochemical changes in the cerebrospinal fluid occurring after seizures induced by pentylenetetrazol. Such events may have a modulating effect upon seizure expression, particularly nucleoside triphosphate diphosphohydrolase activities and nucleoside concentrations, but are nevertheless followed by neural death as evidenced by the increase in NSE and S100B levels.

  16. 不同促摄食物质对哲罗鲑生长、体成分、消化酶和血液生化指标的影响%=Effects of feeding attractants on growth and body composition, digestive enzyme and serum indices of Hucho taimen

    Institute of Scientific and Technical Information of China (English)

    王常安; 徐奇友; 畅雅萍; 许红; 尹家胜

    2011-01-01

    研究二甲基-β-丙酸噻亭(DMPT)、氧化三甲胺(TMAO)、甜菜碱和肌苷酸钠(IMP)对初重 9.39±0.26 g 哲罗鲑(Hucho taimen)摄食、生长、体成分、消化酶和血清生化指标的影响.哲罗鲑在室内玻璃钢水族箱中流水饲养,试验设1个对照组,4个处理组,每处理组3个重复,每重复50 尾,养殖周期56 d.试验期间水温9.3~14.2℃,溶氧>8.0 mg·L-1.试验结果表明,在低鱼粉饲料中添加0.2% DMPT、0.2% TMAO和0.2%甜菜碱后,哲罗鲑增重率和特定生长率显著提高(P0.05);添加0.2% DMPT后哲罗鲑鱼体粗蛋白水平升高(P0.05);添加0.2% DMPT和0.2% TMAO后消化道消化酶活性提高(P<0.05);添加0.2%甜菜碱后,消化道脂肪酶活性显著升高(P<0.05);添加0.2% DMPT后血清总蛋白、白蛋白和球蛋白水平显著升高(P<0.05);添加0.2% TMAO和0.2%甜菜碱后,高密度脂蛋白含量显著升高(P<0.05),胆固醇、甘油三酯、低密度脂蛋白含量显著下降(P<0.05);添加0.05% IMP后,血清补体C3和C4水平显著提高(P<0.05).结论是饲料中添加0.2% DMPT、0.2% TMAO和0.2%甜菜碱对哲罗鲑具有促摄食和生长效果,有利于改善体成分和提高机体消化能力;饲料添加0.05% IMP对哲罗鲑促摄食和生长效果不明显,但有益于改善鱼体免疫状况.%The taimen with initial body weight of 9.39±0.26 g were used to conducted a 56 days growth ex-periment for studying the effects of four feeding attractants (dimethyl-β-propiothetin, trimethylamine oxide,betaine and sodium-5'-inosinate) on feed intake, growth performance, body composition, digestive enzyme ac-tivities and serum indices. Fishes were raised in water flow system. There were 5 diets, and each diet was ran-domly assigned to triplicate groups of 50 fishes. During the experiment, the water temperature fluctuated from 9.3 ℃ to 14.2℃ and the dissolved oxygen was above 8.0 mg·L-1. The results showed that the weight gain rate and specific growth rate of the taimen

  17. Effects of crossbreeding on slaughter traits and breast muscle chemical composition in chinese chickens

    Directory of Open Access Journals (Sweden)

    Ai-xia Huang

    2011-12-01

    Full Text Available We investigated the effects of crossbreeding on slaughter traits and the chemical composition of chicken breast muscle. Trials were conducted using 120 broilers from four lines: Xiao-Shan chicken (XS, Xian-Ju chicken (XJ, Xiao-Shan chicken♂♂ × Xian-Ju chicken♀♀ (Zhenan 1, ZNY1 and Xiao-Shan chicken♂♂ × (Guang-Xi Yellow chicken♂♂×Xian-Ju chicken♀♀ ♀♀ (Zhenan 2, ZNY2. The birds were slaughtered at 120 days of age and the slaughter traits were measured. Breast muscles were sampled to determine chemical composition. The slaughter traits of hybrid chickens were improved. Both hybrid strains had higher intramuscular fat (IMF and inosine-5'-monophosphate (inosinic acid, IMP. Concentrations of monounsaturated fatty acids (MUFA in breast muscles from the two hybrids were significantly higher than in the other two breeds (p < 0.05. The concentration of polyunsaturated fatty acids (PUFA in the breast muscles of the two hybrids was significantly lower than in the other two breeds (p < 0.05. ZNY2 had significantly lower (p < 0.05 concentrations of myristic acid (C14:0. The breast muscle of ZNY1 had significantly higher palmitic acid (C16:0 concentrations than XS, XJ, or ZNY2 (p < 0.05. The concentrations of oleic acid (C18:1 and eicosapentaenoic acid (C20:5n-3, EPA in breast muscle from the two hybrid lines were significantly higher than the other two breeds (p < 0.05. Breast muscles from XS and XJ chickens contained significantly higher docosahexenoic acid (C22:6n-3, DHA than the two hybrid lines (p < 0.05. The XS and XJ chickens had lower n-6/n-3 ratios than the two hybrids (p < 0.05. Breast muscles from ZNY1 and ZNY2 contained higher concentrations of essential amino acids (p < 0.05, total amino acids (p < 0.05, and some individual amino acids (p < 0.05. In conclusion, crossbreeding improved the slaughter traits of chickens and increased intramuscular fat and inosinic acid content in breast muscle. The fatty acid and amino acid

  18. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    Science.gov (United States)

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  19. Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

    Energy Technology Data Exchange (ETDEWEB)

    Ting, L; Shi, W; Lewandowicz, A; Singh, V; Mwakingwe, A; Birck, M R; Taylor Ringia, E A; Bench, G; Madrid, D C; Tyler, P C; Evans, G B; Furneaux, R H; Schramm, V L; Kim, K

    2004-05-19

    Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials.

  20. Treatment of 104 Cases of Chemotherapy-Induced Leukopenia by Injection of Drugs into Zusanli

    Institute of Scientific and Technical Information of China (English)

    尹先哲; 尹德印; 刘新群; 丁旭萌

    2001-01-01

    @@From April 1992 to April 1998, 104 cases of chemotherapy-induced leukopenia were treated by injection into Zusanli (ST 36) with a mixture consisting of dexamethasone, 654-2, ATP and inosine. The therapeutic results were satisfactory as reported in the following. Clinical Data In this series, all the 127 cases were definitely diagnosed by pathological examination. Of them, 93 were male and 34 female, ranging in age from 12 to 75 years. 38 cases were carcinoma of esophagus, 22 carcinoma of cardia of stomach, 21 cancer of lung, 11 hepatic carcinoma, 8 lymphoma, 8 mammary cancer, 7 carcinoma of colon, and 12 other kinds of the tumors. Leukocyte count was below 4.0×109/L in all the patients after being treated by chemotherapy.

  1. Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature.

    Science.gov (United States)

    Rice, Gillian I; Kasher, Paul R; Forte, Gabriella M A; Mannion, Niamh M; Greenwood, Sam M; Szynkiewicz, Marcin; Dickerson, Jonathan E; Bhaskar, Sanjeev S; Zampini, Massimiliano; Briggs, Tracy A; Jenkinson, Emma M; Bacino, Carlos A; Battini, Roberta; Bertini, Enrico; Brogan, Paul A; Brueton, Louise A; Carpanelli, Marialuisa; De Laet, Corinne; de Lonlay, Pascale; del Toro, Mireia; Desguerre, Isabelle; Fazzi, Elisa; Garcia-Cazorla, Angels; Heiberg, Arvid; Kawaguchi, Masakazu; Kumar, Ram; Lin, Jean-Pierre S-M; Lourenco, Charles M; Male, Alison M; Marques, Wilson; Mignot, Cyril; Olivieri, Ivana; Orcesi, Simona; Prabhakar, Prab; Rasmussen, Magnhild; Robinson, Robert A; Rozenberg, Flore; Schmidt, Johanna L; Steindl, Katharina; Tan, Tiong Y; van der Merwe, William G; Vanderver, Adeline; Vassallo, Grace; Wakeling, Emma L; Wassmer, Evangeline; Whittaker, Elizabeth; Livingston, John H; Lebon, Pierre; Suzuki, Tamio; McLaughlin, Paul J; Keegan, Liam P; O'Connell, Mary A; Lovell, Simon C; Crow, Yanick J

    2012-11-01

    Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

  2. Solid-phase Synthesis of Combinatorial 2,4-Disubstituted-1,3,5-Triazine via Amine Nucleophilic Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Sung Won [KIST Gangneung Institute, Gangneung (Korea, Republic of); Ham, Jungyeob [Gangneung-Wonju National University, Gangneung (Korea, Republic of); Chang, Young-Tae [National University of Singapore, Singapore (Singapore); Lee, Jae Wook [University of Science and Technology, Daejon (Korea, Republic of)

    2015-01-15

    In combinatorial chemistry, solid-phase synthesis is a popular approach formass production of small molecules. Compared to solution-phase synthesis, it is easy to prepare and purify a large number of heterocyclic small molecules via solid-phase chemistry; the overall reaction time is decreased as well. 1,3,5-Triazine is a nitrogen-containing heterocyclic aromatic scaffold that was shown to be a druggable scaffold in recent studies. These structures have been reported as anticancer, antimicrobial, and antiretroviral compounds, as CDKs and p38 MAP kinase inhibitors, as estrogen receptor modulators, and as inosine monophosphate dehydrogenase inhibitors. we designed and synthesized disubstituted triazine compounds as an analog of disubstituted pyrimidine compounds. These disubstituted triazine compounds possess a linear structure which may have biological activity similar to that of disubstituted pyrimidine. Here we report the solid-phase synthesis of disubstituted triazine compounds.

  3. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    Science.gov (United States)

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT.

  4. Increased level of soluble adenosine deaminase in bone marrow of visceral leishmaniasis patients: an inverse relation with parasite load.

    Science.gov (United States)

    Rai, Ambak K; Kumar, Prabin; Saini, Sheetal; Thakur, Chandreshwar P; Seth, Tulika; Mitra, Dipendra K

    2016-09-01

    Adenosine deaminase (ADA) which degrades adenosine to inosine, is known to be pro-inflammatory molecule in many diseases. Adenosine suppresses the functioning of the immune system and thus promotes dissemination of the parasite. In our previous finding, the level of soluble ADA in serum of visceral leishmaniasis (VL) was found to be increased as compared to healthy controls. However, it cannot be fairly interpreted unless their level is demonstrated at the disease site, where the parasite resides. We designed this study to correlate the level of soluble ADA (sADA) with parasitic load at the disease site i.e. bone marrow (BM). We found increased levels of sADA in BM as compared to the unaffected BM. Furthermore, a significant inverse correlation is observed between the parasite load and level of sADA at the disease site. PMID:27447233

  5. Dicty_cDB: Contig-U10771-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available dida dubliniensis inosine-5'-monophosphate deh... 62 2e-13 4 ( EA377314 ) Sequence 26137 from patent...ts) S1: 12 (24.3 bits) S2: 15 (30.2 bits) dna update 2008.11.21 Homology vs DNA Query= Contig-U10771-1 (Co...kkkkkkkkkkkkkkkkkkkk own update 2004. 6.10 Homology vs CSM-cDNA Query= Contig-U10771-1 (Contig-U10771-1Q) /CSM_Contig/Co...used: 200553593363865 X1: 11 (21.8 bits) S2: 23 (46.1 bits) protein update 2009. 6.28 Homolo...Y810896 |pid:none) Schistosoma japonicum SJCHGC05057 ... 246 2e-63 AK118098_1( AK118098 |pid:none) Arabidopsis thalian

  6. Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity

    DEFF Research Database (Denmark)

    Hansen, Jeanette; Conley, Lene; Hedegaard, Jakob;

    2012-01-01

    Acute physical activity elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. In the present time-course study, pigs were submitted to an acute bout of treadmill running until near exhaustion to determine the impact...... detected an upregulation of genes that are associated with muscle cell proliferation and differentiation, including MUSTN1, ASB5 and CSRP3, possibly reflecting activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression...... of adenosine-to-inosine edited mRNAs in the ribonucleoprotein bodies called paraspeckles. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery...

  7. Pharmacokinetic study of inosiplex tablets in healthy Chinese volunteers by hyphenated HPLC and tandem MS techniques

    Institute of Scientific and Technical Information of China (English)

    Mo Chen; Yuan Zhang; Xiao-Ting Que; Ya Ding; Lin Yang; Ai-Dong Wen; Tai-Jun Hang

    2013-01-01

    Inosiplex is a compound formulation composed of inosine and p-acetaminobenzoic acid (PABA) salt of N,N-dimethylamino-2-propanol (DIP). This study was to investigate the clinical plasma pharmacokinetic properties of DIP and PABA after single and multiple oral doses of inosiplex tablets in healthy Chinese volunteers. The established LC/MS/MS method for plasma DIP determination had a linear range of 0.02-10 mg/mL, and the HPLC method for plasma PABA determination had a linear range of 0.05-40 mg/mL. Linear pharmacokinetic characteristics were found with single oral doses of 0.5, 1.0 and 2.0 g. No obvious accumulation effects were observed for DIP and PABA.

  8. Genetic polymorphisms of the AMPD1 gene and their correlations with IMP contents in Fast Partridge and Lingshan chickens.

    Science.gov (United States)

    Hu, Jin; Yu, Ping; Ding, Xiaoling; Xu, Minglong; Guo, Baoping; Xu, Yinxue

    2015-12-15

    The object of this study was to evaluate associations between the adenosine monophosphate deaminase 1 (AMPD1) gene polymorphisms and inosine monophosphate acid (IMP) contents of chicken to provide a molecular marker for breeding. Three single nucleotide polymorphisms (SNPs), g.4064G/A, g.5573A/G and g.6805G/A were detected in exons IV, VI, and VIII of the AMPD1 gene in Fast Partridge and Lingshan chickens, respectively. All were purine conversion and caused no alteration in amino acid sequence. Statistical analysis revealed that Lingshan chicken with the homozygous genotype AA at position 4064 and 6805 had a significantly greater IMP content than those with the GG genotype (Pchicken with the genotype GG at position 6805 had a significantly greater IMP content compared with those with the AA genotype (Pchickens. The results in our study suggest that SNP 6805A/G can be used as a possible candidate marker of IMP content of chicken.

  9. High performance liquid chromatography analysis of 9-(2',3'-dideoxy-2'beta-fluoro-D-threo-penta furanosyl) adenine and its metabolite in human plasma using solid-phase extraction on a polyfluorinated reversed stationary phase.

    Science.gov (United States)

    Aboul-Enein, H Y; Abu-Zaid, S

    2001-06-01

    A quick and sensitive reversed-phase HPLC method has been developed for the analysis of 2'-beta -fluoro-2',3'-dideoxy adenosine (F-ddA), the acid-stable anti-AIDS drug, and its metabolite 2'-fluoro-2',3'-dideoxy inosine (F-ddI) in human plasma using polyfluorinated stationary phase column (Fluo fix, 15 cm, 4.0 mm i.d., 5 microm particle size). The mobile phase consisted of ammonium phosphate buffer solution (10 mM) adjusted with phosphoric acid 85% to pH 6.8:dimethyl formamide (97:3, v/v). F-ddA and F-ddI were monitored by UV-visible detector at 258 and 247 nm, respectively. The recoveries of F-ddA and F-ddI from plasma using a C(18) solid-phase extraction cartridge were 99.2% and 99.7%, respectively. PMID:11438969

  10. Genomic evidence for complementary purine metabolism in the pea aphid, Acyrthosiphon pisum, and its symbiotic bacterium Buchnera aphidicola.

    Science.gov (United States)

    Ramsey, J S; MacDonald, S J; Jander, G; Nakabachi, A; Thomas, G H; Douglas, A E

    2010-03-01

    The purine salvage pathway recycles purines to nucleotides, promoting efficient utilization of purine nucleotides. Exceptionally among animals with completely sequenced genomes, the pea aphid lacks key purine recycling genes that code for purine nucleoside phosphorylase and adenosine deaminase, indicating that the aphid can neither metabolize nucleosides to the corresponding purines, nor adenosine to inosine. Purine metabolism genes in the symbiotic bacterium Buchnera complement aphid genes, and Buchnera can meet its nucleotide requirement from aphid-derived guanosine. Buchnera demand for nucleosides may have relaxed the selection for purine recycling in the aphid, leading to the loss of key aphid purine salvage genes. Further, the coupled purine metabolism of aphid and Buchnera could contribute to the dependence of the pea aphid on this symbiosis.

  11. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    Energy Technology Data Exchange (ETDEWEB)

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  12. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  13. ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor.

    Science.gov (United States)

    Doria, Margherita; Tomaselli, Sara; Neri, Francesca; Ciafrè, Silvia Anna; Farace, Maria Giulia; Michienzi, Alessandro; Gallo, Angela

    2011-05-01

    The adenosine deaminases acting on RNA (ADAR) enzymes catalyse conversion of adenosine to inosine in dsRNA. A positive effect of ADAR1 on human immunodeficiency virus type 1 (HIV-1) replication has recently been reported. Here, we show that another ADAR enzyme, ADAR2, positively affects the replication process of HIV-1. We found that, analogously to ADAR1, ADAR2 enhances the release of progeny virions by an editing-dependent mechanism. However, differently from the ADAR1 enzyme, ADAR2 does not increase the infectious potential of the virus. Importantly, downregulation of ADAR2 in Jurkat cells significantly impairs viral replication. Therefore, ADAR2 shares some but not all proviral functions of ADAR1. These results suggest a novel role of ADAR2 as a viral regulator. PMID:21289159

  14. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    Science.gov (United States)

    Musser, Jeffrey M B; Heatley, J Jill; Koinis, Anastasia V; Suchodolski, Paulette F; Guo, Jianhua; Escandon, Paulina; Tizard, Ian R

    2015-01-01

    Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture. PMID:26222794

  15. 'Rod and ring' formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway.

    Science.gov (United States)

    Calise, S John; Purich, Daniel L; Nguyen, Thuy; Saleem, Dania A; Krueger, Claire; Yin, Joyce D; Chan, Edward K L

    2016-08-01

    'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway. PMID:27343244

  16. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Hazelton, Keith Z. [Yeshiva Univ., New York, NY (United States); Ho, Meng-Chaio [Yeshiva Univ., New York, NY (United States); Cassera, Maria B. [Yeshiva Univ., New York, NY (United States); Clinch, Keith [Industrial Research Ltd., Lower Hutt (New Zealand); Crump, Douglas R. [Industrial Research Ltd., Lower Hutt (New Zealand); Rosario Jr., Irving [Yeshiva Univ., New York, NY (United States); Merino, Emilio F. [Yeshiva Univ., New York, NY (United States); Almo, Steve C. [Yeshiva Univ., New York, NY (United States); Tyler, Peter C. [Industrial Research Ltd., Lower Hutt (New Zealand); Schramm, Vern L. [Yeshiva Univ., New York, NY (United States)

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  17. Mycophenolate mofetil for drug-induced vanishing bile duct syndrome

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Amoxicillin/clavulanate is associated with liver injury,mostly of a cholestatic pattern. While outcomes are usually benign, progression to cirrhosis and death has been reported. The role of immunosuppressive therapy for patients with a protracted course is unclear. We report the case of an elderly patient who developed prolonged cholestasis secondary to amoxicillin/clavulanate. Vanishing bile duct syndrome was confirmed by sequential liver biopsies. The patient responded to prednisone treatment,but could not be weaned off corticosteroids, even when azathioprine was added. Complete withdrawal of both prednisone and azathioprine was possible by using mycophenolate mofetil, an inosine monophosphate dehydrogenase inhibitor. Sustained remission has been maintained for more than 3 years with low-dose mycophenolate mofetil.

  18. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  19. Solid-phase Synthesis of Combinatorial 2,4-Disubstituted-1,3,5-Triazine via Amine Nucleophilic Reaction

    International Nuclear Information System (INIS)

    In combinatorial chemistry, solid-phase synthesis is a popular approach formass production of small molecules. Compared to solution-phase synthesis, it is easy to prepare and purify a large number of heterocyclic small molecules via solid-phase chemistry; the overall reaction time is decreased as well. 1,3,5-Triazine is a nitrogen-containing heterocyclic aromatic scaffold that was shown to be a druggable scaffold in recent studies. These structures have been reported as anticancer, antimicrobial, and antiretroviral compounds, as CDKs and p38 MAP kinase inhibitors, as estrogen receptor modulators, and as inosine monophosphate dehydrogenase inhibitors. we designed and synthesized disubstituted triazine compounds as an analog of disubstituted pyrimidine compounds. These disubstituted triazine compounds possess a linear structure which may have biological activity similar to that of disubstituted pyrimidine. Here we report the solid-phase synthesis of disubstituted triazine compounds

  20. RNA editing in Drosophila melanogaster: new targets and functionalconsequences

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

    2006-09-05

    Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR including components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

  1. Personalized Whole-Cell Kinetic Models of Metabolism for Discovery in Genomics and Pharmacodynamics

    DEFF Research Database (Denmark)

    Bordbar, Aarash; McCloskey, Douglas; Zielinski, Daniel C;

    2015-01-01

    challenge. Here, we constructed multi-omic, data-driven, personalized whole-cell kinetic models of erythrocyte metabolism for 24 healthy individuals based on fasting-state plasma and erythrocyte metabolomics and whole-genome genotyping. We show that personalized kinetic rate constants, rather than......-induced anemia) and how genetic variation (inosine triphosphatase deficiency) may protect against this side effect. This study demonstrates the feasibility of personalized kinetic models, and we anticipate their use will accelerate discoveries in characterizing individual metabolic variation.......Understanding individual variation is fundamental to personalized medicine. Yet interpreting complex phenotype data, such as multi-compartment metabolomic profiles, in the context of genotype data for an individual is complicated by interactions within and between cells and remains an unresolved...

  2. Discrimination of Umami Tastants Using Floating Electrode-Based Bioelectronic Tongue Mimicking Insect Taste Systems.

    Science.gov (United States)

    Lee, Minju; Jung, Je Won; Kim, Daesan; Ahn, Young-Joon; Hong, Seunghun; Kwon, Hyung Wook

    2015-12-22

    We report a floating electrode-based bioelectronic tongue mimicking insect taste systems for the detection and discrimination of umami substances. Here, carbon nanotube field-effect transistors with floating electrodes were hybridized with nanovesicles containing honeybee umami taste receptor, gustatory receptor 10 of Apis mellifera (AmGr10). This strategy enables us to discriminate between l-monosodium glutamate (MSG), best-known umami tastant, and non-umami substances with a high sensitivity and selectivity. It could also be utilized for the detection of MSG in liquid food such as chicken stock. Moreover, we demonstrated the synergism between MSG and disodium 5'-inosinate (IMP) for the umami taste using this platform. This floating electrode-based bioelectronic tongue mimicking insect taste systems can be a powerful platform for various applications such as food screening, and it also can provide valuable insights on insect taste systems. PMID:26563753

  3. Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

    Science.gov (United States)

    Kawamura, K.; Ferris, J. P.

    1999-01-01

    The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

  4. Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

    Directory of Open Access Journals (Sweden)

    Andrew Charles Penn

    2013-04-01

    Full Text Available The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal responsiveness during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signalling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus ‘hardwiring’ modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine resulting in a change in the nucleotide to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases and RNA secondary structure forming capacity. In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. We will describe here the recoding function of key RNA editing targets in the mammalian central nervous system (CNS and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarising new findings from high-throughput studies.

  5. To edit or not to edit: regulation of ADAR editing specificity and efficiency.

    Science.gov (United States)

    Deffit, Sarah N; Hundley, Heather A

    2016-01-01

    Hundreds to millions of adenosine (A)-to-inosine (I) modifications are present in eukaryotic transcriptomes and play an essential role in the creation of proteomic and phenotypic diversity. As adenosine and inosine have different base-pairing properties, the functional consequences of these modifications or 'edits' include altering coding potential, splicing, and miRNA-mediated gene silencing of transcripts. However, rather than serving as a static control of gene expression, A-to-I editing provides a means to dynamically rewire the genetic code during development and in a cell-type specific manner. Interestingly, during normal development, in specific cells, and in both neuropathological diseases and cancers, the extent of RNA editing does not directly correlate with levels of the substrate mRNA or the adenosine deaminase that act on RNA (ADAR) editing enzymes, implying that cellular factors are required for spatiotemporal regulation of A-to-I editing. The factors that affect the specificity and extent of ADAR activity have been thoroughly dissected in vitro. Yet, we still lack a complete understanding of how specific ADAR family members can selectively deaminate certain adenosines while others cannot. Additionally, in the cellular environment, ADAR specificity and editing efficiency is likely to be influenced by cellular factors, which is currently an area of intense investigation. Data from many groups have suggested two main mechanisms for controlling A-to-I editing in the cell: (1) regulating ADAR accessibility to target RNAs and (2) protein-protein interactions that directly alter ADAR enzymatic activity. Recent studies suggest cis- and trans-acting RNA elements, heterodimerization and RNA-binding proteins play important roles in regulating RNA editing levels in vivo. WIREs RNA 2016, 7:113-127. doi: 10.1002/wrna.1319. PMID:26612708

  6. Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.

    Science.gov (United States)

    Pfefferkorn, E R; Bzik, D J; Honsinger, C P

    2001-12-01

    In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine

  7. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699

  8. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699

  9. Concentration of Umami Compounds in Pork Meat and Cooking Juice with Different Cooking Times and Temperatures.

    Science.gov (United States)

    Rotola-Pukkila, Minna K; Pihlajaviita, Seija T; Kaimainen, Mika T; Hopia, Anu I

    2015-12-01

    This study examined the concentrations of umami compounds in pork loins cooked at 3 different temperatures and 3 different lengths of cooking times. The pork loins were cooked with the sous vide technique. The free amino acids (FAAs), glutamic acid and aspartic acid; the 5'-nucleotides, inosine-5'-monophosphate (IMP) and adenosine-5'-monophosphate (AMP); and corresponding nucleoside inosine of the cooked meat and its released juice were determined by high-performance liquid chromatography. Under the experimental conditions used, the cooking temperature played a more important role than the cooking time in the concentration of the analyzed compounds. The amino acid concentrations in the meat did not remain constant under these experimental conditions. The most notable effect observed was that of the cooking temperature and the higher amino acid concentrations in the released juice of meat cooked at 80 °C compared with 60 and 70 °C. This is most likely due to the heat induced hydrolysis of proteins and peptides releasing water soluble FAAs from the meat into the cooking juice. In this experiment, the cooking time and temperature had no influence on the IMP concentrations observed. However, the AMP concentrations increased with the increasing temperature and time. This suggests that the choice of time and temperature in sous vide cooking affects the nucleotide concentration of pork meat. The Sous vide technique proved to be a good technique to preserve the cooking juice and the results presented here show that cooking juice is rich in umami compounds, which can be used to provide a savory or brothy taste.

  10. Concentration of Umami Compounds in Pork Meat and Cooking Juice with Different Cooking Times and Temperatures.

    Science.gov (United States)

    Rotola-Pukkila, Minna K; Pihlajaviita, Seija T; Kaimainen, Mika T; Hopia, Anu I

    2015-12-01

    This study examined the concentrations of umami compounds in pork loins cooked at 3 different temperatures and 3 different lengths of cooking times. The pork loins were cooked with the sous vide technique. The free amino acids (FAAs), glutamic acid and aspartic acid; the 5'-nucleotides, inosine-5'-monophosphate (IMP) and adenosine-5'-monophosphate (AMP); and corresponding nucleoside inosine of the cooked meat and its released juice were determined by high-performance liquid chromatography. Under the experimental conditions used, the cooking temperature played a more important role than the cooking time in the concentration of the analyzed compounds. The amino acid concentrations in the meat did not remain constant under these experimental conditions. The most notable effect observed was that of the cooking temperature and the higher amino acid concentrations in the released juice of meat cooked at 80 °C compared with 60 and 70 °C. This is most likely due to the heat induced hydrolysis of proteins and peptides releasing water soluble FAAs from the meat into the cooking juice. In this experiment, the cooking time and temperature had no influence on the IMP concentrations observed. However, the AMP concentrations increased with the increasing temperature and time. This suggests that the choice of time and temperature in sous vide cooking affects the nucleotide concentration of pork meat. The Sous vide technique proved to be a good technique to preserve the cooking juice and the results presented here show that cooking juice is rich in umami compounds, which can be used to provide a savory or brothy taste. PMID:26524113

  11. Expression and functional activity of nucleoside transporters in human choroid plexus

    Directory of Open Access Journals (Sweden)

    Grujicic Danica

    2010-01-01

    Full Text Available Abstract Background Human equilibrative nucleoside transporters (hENTs 1-3 and human concentrative nucleoside transporters (hCNTs 1-3 in the human choroid plexus (hCP play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. Methods Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR; for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E and the quantification cycle (Cq were calculated. The uptake of [3H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. Results RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E-Cq value being only about 40 fold less that the E-Cq value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [3H]inosine by the CP samples was linear and consisted of an Na+-dependent component, which was probably mediated by hCNT3, and Na+-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl-6-thioinosine (NBMPR, when used at a concentration of 0.5 μM, a finding that

  12. All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

    International Nuclear Information System (INIS)

    We have studied the all or none cell response of Ca2+-dependent K+ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5 mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity = 1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca2+ of the individual K+ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mumol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mumol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6-8 times the measured rate of uptake of external 45Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca2+-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5 mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells

  13. A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

    Science.gov (United States)

    Wice, B M; Gordon, J I

    1992-01-01

    The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their

  14. 手术室常用抗生素与输液溶液的稳定性研究%The study of compatibility between antibiotics and infusion solutions

    Institute of Scientific and Technical Information of China (English)

    王倩; 王宇

    2012-01-01

    目的:观察临床常用15种抗生素分别与5种常用输液溶液的配伍稳定性.方法:模拟临床用药浓度选择15种抗生素分别与维生素C、肌苷、ATP、阿西洛韦和甲硝唑依次进行配伍,观察配伍液在室温条件下(25℃)0~8h的外观(颜色改变、气泡、可见沉淀产生)及pH值的变化.结果:①硫酸链霉素、硫酸妥布霉素和磷霉素钠与5种输液溶液均不能配伍;②乳糖酸红霉素不能与甲硝唑和阿西洛韦配伍应用;③硫酸庆大霉素不能与维生素C、肌酐和ATP配伍使用.结论:硫酸链霉素、硫酸妥布霉素和磷霉素钠不能常规进行药物配伍,需单独使用.合理配伍用药是治疗过程的重要环节.%Objective To investigate the compatibility between 15 kinds of antibiotics and 5 drugs, including vitamin C, inosine, ATP, axiluowei and metronidazole. Methods 15 kinds of antibiotics and 5 drugs were mixtured at room temperature, respectively and the physico-chemical properties of the mixture, including appearance of solution, precipitate, content, even changes of pH were studied. Results ①streptomycin sulfate, tobramycin and fosfomycin sodium are incompatible with 5 drugs and need to be used alone in clinically. ②Erythromycin lactobionate couldn't combine with metronidazole or Axiluowei. ③There is imcompatibility for gentamycin sulfate with vitamin C, inosine and ATP. Conclusions Treptomycin sulfate, tobramycin and fosfomycin sodium need to be used alone. Reasonable compatibility plays an important role in the process of therapy.

  15. Cyclosporine and methotrexate-related pharmacogenomic predictors of acute graft-versus-host disease.

    Science.gov (United States)

    Laverdière, Isabelle; Guillemette, Chantal; Tamouza, Ryad; Loiseau, Pascale; Peffault de Latour, Regis; Robin, Marie; Couture, Félix; Filion, Alain; Lalancette, Marc; Tourancheau, Alan; Charron, Dominique; Socié, Gérard; Lévesque, Éric

    2015-02-01

    Effective immunosuppression is mandatory to prevent graft-versus-host disease and to achieve a successful clinical outcome of hematopoietic stem cell transplantation. Here we tested whether germline single nucleotide polymorphisms in 20 candidate genes related to methotrexate and cyclosporine metabolism and activity influence the incidence of graft-versus-host disease in patients who undergo stem cell transplantation for hematologic disorders. Recipient genetic status of the adenosine triphosphate-binding cassette sub-family C1 and adenosine triphosphate-binding cassette sub-family C2 transporters, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ inosine monophosphate cyclohydrolase within the methotrexate pathway, and nuclear factor of activated T cells (cytoplasmic 1) loci exhibit a remarkable influence on severe acute graft-versus-host disease prevalence. Indeed, an increased risk of acute graft-versus-host disease was observed in association with single nucleotide polymorphisms located in 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (hazard ratio=3.04; P=0.002), nuclear factor of activated T cells (cytoplasmic 1) (hazard ratio=2.69; P=0.004), adenosine triphosphate-binding cassette sub-family C2 (hazard ratio=3.53; P=0.0018) and adenosine triphosphate-binding cassette sub-family C1 (hazard ratio=3.67; P=0.0005). While donor single nucleotide polymorphisms of dihydrofolate reductase and solute carrier family 19 (member 1) genes are associated with a reduced risk of acute graft-versus-host disease (hazard ratio=0.32-0.41; P=0.0009-0.008), those of nuclear factor of activated T cells (cytoplasmic 2) are found to increase such risk (hazard ratio=3.85; P=0.0004). None of the tested single nucleotide polymorphisms was associated with the occurrence of chronic graft-versus-host disease. In conclusion, by targeting drug-related biologically relevant genes, this work emphasizes the potential role of

  16. Purine biosynthesis in archaea: variations on a theme

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    Brown Anne M

    2011-12-01

    Full Text Available Abstract Background The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains. Results We searched the Integrated Microbial Genome system (IMG for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function. Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified. Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected or Enzyme Commission (E. C. numbers (57 proteins, 7.7%. There were also 57 proteins (7.7% assigned overly generic names and 78 proteins (10.6% without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified. Conclusions The patchy distribution of purine biosynthetic genes in archaea is

  17. Effects of Danshen Injection on the Malignant Obstructive Jaundice in the SD Rat Model

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To observe the effects of Danshen on the growth of hepatocellular carcinoma in the SD rats, a model of malignant obstructive jaundice was established by inoculation of transplanted tumor into the hepatic portal with the walker-256 hepatocarcine line, which resulted in the obstruction by the infiltration and metastasis of hepatocellular carcinoma. SD rats were divided into 4 groups: the rats were treated by 0.9 % NS (n=24, control group), inosine+vitamin C (n=40, InV group), Danshen (n=40, DS group) and 5-FU (n=40, 5-FU group), respectively. The liver function, morphological changes and the expressions of PCNA, VEGF and ICAM-1 in carcinoma foci, peri-carcinoma tissues,adjacent lobe (left-internal lobe) and lung tissues were observed after the treatment with the 4 agents.Our results showed that the protective effect of Danshen on liver function was significantly better than that of NS and 5-FU (P<0.01). No significant difference in protective effect was observed between DS group and InV group (P>0.05). Danshen also provided protective effect on the morphological damage of liver caused by obstructive jaundice. The rates of carcinoma-inhibition and metastasis inhibition were significantly higher than those of NS and inosine+vitamin C (P<0.01). No significant difference in this regard existed between DS group and 5-FU group (P>0.05). The expressions of PCNA,VEGF and ICAM-1 PCNA, VEGF and ICAM-1 in carcinoma foci, peri-carcinoma tissues, adjacent lobe (left-internal lobe) and lung tissues were lower than those in control group and InV group, with the differences being significant (P<0.01). No significant differences were found between DS group and 5-FU group in the expression levels of PCNA and VEGF (P>0.05) but ICAM-1 (P<0.05). It is concluded that Danshen injection not only has protective effects on liver injury caused by obstructive jaundice, but can inhibit the proliferation and growth of hepatocarcinoma,interfere with the vascularization of tumors

  18. The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

    International Nuclear Information System (INIS)

    In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment

  19. A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption.

    Science.gov (United States)

    Nielsen, Kirstine L; Telving, Rasmus; Andreasen, Mette F; Hasselstrøm, Jørgen B; Johannsen, Mogens

    2016-02-01

    The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology. PMID:26705142

  20. Overexpression of adenosine deaminase acting on RNA 1 in chordoma tissues is associated with chordoma pathogenesis by reducing miR-125a and miR-10a expression

    Science.gov (United States)

    KUANG, LEI; LV, GUOHUA; WANG, BING; LI, LEI; DAI, YULIANG; LI, YAWEI

    2015-01-01

    Chordoma is a rare, slow-growing primary malignant neoplasm of the axial skeleton, which arises from the remnants of the notochord. Emerging evidence suggests that microRNAs (miRs) are dysregulated in chordoma tissues and crucially involved in chordoma pathogenesis. In the present study, the expression of 11 candidate miRs were analyzed in chordoma tissues and miR-10a and miR-125a were found to be significantly downregulated compared with controls. Notably, the expression of the primary transcripts, pri-miR-125a and pri-miR-10a was unaltered, suggesting that disturbed microRNA expression may be induced by altered pri-miRNA processing. Previous studies have indicated that disturbed adenosine deaminase acting on RNA (ADAR) expression is able to alter mRNA and miRNA adenosine to inosine (A-to-I) levels associated with cancer pathogenesis. Therefore, the expression of ADAR1 and ADAR2 was analyzed in chordoma tissues. It was found that ADAR1 was significantly overexpressed, which was accompanied by enhanced pre-miR-10a and pri-miR-125a A-to-I editing. These findings suggest that ADAR2 overexpression causes enhanced pre-miR-10a and pri-miR-125a A-to-I editing, which alters mature miR-10a and miR-125a expression and may contribute to chordoma pathogenesis. PMID:25673044

  1. Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

    Science.gov (United States)

    MACBETH, MARK R.; LINGAM, ARUNTH T.; BASS, BRENDA L.

    2004-01-01

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs. PMID:15383678

  2. RNASequel: accurate and repeat tolerant realignment of RNA-seq reads.

    Science.gov (United States)

    Wilson, Gavin W; Stein, Lincoln D

    2015-10-15

    RNA-seq is a key technology for understanding the biology of the cell because of its ability to profile transcriptional and post-transcriptional regulation at single nucleotide resolutions. Compared to DNA sequencing alignment algorithms, RNA-seq alignment algorithms have a diminished ability to accurately detect and map base pair substitutions, gaps, discordant pairs and repetitive regions. These shortcomings adversely affect experiments that require a high degree of accuracy, notably the ability to detect RNA editing. We have developed RNASequel, a software package that runs as a post-processing step in conjunction with an RNA-seq aligner and systematically corrects common alignment artifacts. Its key innovations are a two-pass splice junction alignment system that includes de novo splice junctions and the use of an empirically determined estimate of the fragment size distribution when resolving read pairs. We demonstrate that RNASequel produces improved alignments when used in conjunction with STAR or Tophat2 using two simulated datasets. We then show that RNASequel improves the identification of adenosine to inosine RNA editing sites on biological datasets. This software will be useful in applications requiring the accurate identification of variants in RNA sequencing data, the discovery of RNA editing sites and the analysis of alternative splicing.

  3. Consistent levels of A-to-I RNA editing across individuals in coding sequences and non-conserved Alu repeats

    Directory of Open Access Journals (Sweden)

    Osenberg Sivan

    2010-10-01

    Full Text Available Abstract Background Adenosine to inosine (A-to-I RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. Results Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. Conclusions Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms.

  4. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    Science.gov (United States)

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  5. Umami compounds enhance the intensity of retronasal sensation of aromas from model chicken soups.

    Science.gov (United States)

    Nishimura, Toshihide; Goto, Shingo; Miura, Kyo; Takakura, Yukiko; Egusa, Ai S; Wakabayashi, Hidehiko

    2016-04-01

    We examined the influence of taste compounds on retronasal aroma sensation using a model chicken soup. The aroma intensity of a reconstituted flavour solution from which glutamic acid (Glu), inosine 5'-monophosphate (IMP), or phosphate was omitted was significantly lower (p<0.05) than that of the model soup. The aroma intensity of 0.4% NaCl solution containing the aroma chicken model (ACM) with added Glu and IMP was significantly higher (p<0.05) than that of 0.4% NaCl solution containing only ACM. The quantitative analyses showed that adding monosodium glutamate (MSG) to aqueous aroma solution containing only ACM enhanced the intensity of retronasal aroma sensation by 2.5-folds with increasing MSG concentration from 0% to 0.3%. Sensation intensity using an umami solution with added MSG and IMP was significantly higher than that with only MSG when the MSG concentration was 0.05%, 0.075%, or 0.1%. However, it plateaued when MSG concentration was beyond 0.3%. PMID:26593530

  6. Pickles, pectin, and penicillin.

    Science.gov (United States)

    Demain, Arnold L

    2004-01-01

    My professional life has been devoted to the study of microbial products and their biosynthesis, regulation, and overproduction. These have included primary metabolites (glutamic acid, tryptophan, inosinic acid, guanylic acid, vitamin B(12), riboflavin, pantothenic acid, ethanol, and lactic acid) and secondary metabolites (penicillin, cephalosporins, streptomycin, fosfomycin, gramicidin S, rapamycin, indolmycin, microcin B17, fumagillin, mycotoxins, Monascus pigments, and tetramethylpyrazine). Other areas included microbial nutrition, strain improvement, bioconversions of statins and beta-lactams, sporulation and germination, plasmid stability, gel microdroplets, and the production of double-stranded RNA, the polymer xanthan, and enzymes (polygalacturonase, protease, cellulase). Most of the studies were carried out with me by devoted and hardworking industrial scientists for 15 years at Merck & Co. and by similarly characterized students, postdoctorals, and visiting scientists during my 32 years at the Massachusetts Institute of Technology. I owe much of my success to my mentors from academia and industry. My recent research activities with undergraduate students at the Charles A. Dana Research Institute for Scientists Emeriti (R.I.S.E.) at Drew University have been very rewarding and are allowing me to continue my career.

  7. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

    Science.gov (United States)

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; Santos, Odelta dos; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-11-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

  8. Molecular cloning, sequence analysis and expression of genome segment 7 (S7) of Antheraea mylitta cypovirus (AmCPV) that encodes a viral structural protein.

    Science.gov (United States)

    Chavali, Venkata Ramana Murthy; Ghosh, Ananta K

    2007-10-01

    The Genome segment 7 (S7) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus (AmCPV) was converted to cDNA, cloned and sequenced. The nucleotide sequence showed that segment 7 consisted of 1789 nucleotides with an ORF of 530 amino acids and could encode a protein of approximately 61 kDa, termed P61. The 5' terminal sequence, AGTAAT and the 3' terminal sequence, AGAGC of the plus strand was found to be the same as genome segment 10 of AmCPV encoding polyhedrin. No sequence similarity was found by searching nucleic acid and protein sequence databases using BLAST. The secondary structure prediction showed the presence of 17 alpha-helices, 18 extended beta-sheets along the entire length of P61. The ORF of segment 7 was expressed in E. coli as His-tagged fusion protein, purified through Ni-NTA chromatography, and polyclonal antibody was raised in rabbit indicating that P61 is immunogenic. Immunoblot analysis using this antibody on viral infected cells as well as purified polyhedra showed that P61 is a viral structural protein. Motif scan search showed some similarity of P61 with Inosine monophosphate dehydrogenase (IMPDH) cystathionine-beta-synthase (CBS) domain at the C-terminus and it was hypothesized that by binding to single stranded viral RNA through its CBS domain P61 may help in virus replication or transcription.

  9. Distribution of ADAT-Dependent Codons in the Human Transcriptome

    Directory of Open Access Journals (Sweden)

    Àlbert Rafels-Ybern

    2015-07-01

    Full Text Available Nucleotide modifications in the anticodons of transfer RNAs (tRNA play a central role in translation efficiency, fidelity, and regulation of translation, but, for most of these modifications, the details of their function remain unknown. The heterodimeric adenosine deaminases acting on tRNAs (ADAT2-ADAT3, or ADAT are enzymes present in eukaryotes that convert adenine (A to inosine (I in the first anticodon base (position 34 by hydrolytic deamination. To explore the influence of ADAT activity on mammalian translation, we have characterized the human transcriptome and proteome in terms of frequency and distribution of ADAT-related codons. Eight different tRNAs can be modified by ADAT and, once modified, these tRNAs will recognize NNC, NNU and NNA codons, but not NNG codons. We find that transcripts coding for proteins highly enriched in these eight amino acids (ADAT-aa are specifically enriched in NNC, NNU and NNA codons. We also show that the proteins most enriched in ADAT-aa are composed preferentially of threonine, alanine, proline, and serine (TAPS. We propose that the enrichment in ADAT-codons in these proteins is due to the similarities in the codons that correspond to TAPS.

  10. Anti-rods/rings: a human model of drug-induced autoantibody generation

    Directory of Open Access Journals (Sweden)

    S. John eCalise

    2015-02-01

    Full Text Available In recent years, autoantibodies targeting subcellular structures described as the rods and rings pattern in HEp-2 ANA have been presented as a unique case of autoantibody generation. These rod and ring structures (RR are at least partially composed of inosine monophosphate dehydrogenase type 2 (IMPDH2, and their formation can be induced in vitro by several small-molecule inhibitors, including some IMPDH2 inhibitors. Autoantibodies targeting these relatively unknown structures have been almost exclusively observed in hepatitis C (HCV patients who have undergone treatment with pegylated interferon-α/ribavirin (IFN/RBV combination therapy. To date, anti-RR antibodies have not been found in treatment-naïve HCV patients or in patients from any other disease groups, with few reported exceptions. Here, we describe recent advances in characterizing the RR structure and the strong association between anti-RR antibody response and HCV patients treated with IFN/RBV, detailing why anti-RR can be considered a human model of drug-induced autoantibody generation.

  11. Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism

    Directory of Open Access Journals (Sweden)

    Jessica R. Gooding

    2015-10-01

    Full Text Available Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS from islet β cells, heralds the onset of type 2 diabetes (T2D. To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP and an increase in adenylosuccinate (S-AMP, suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS. Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1. S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D.

  12. Experimental proof for the regulation of Salmonella typhimurium purB by purR

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Salmonella typhimurium purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of inosine 5′-monophos- phate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence of purB was obtained by the genetic map and sequence homologous analysis. The conserved pur operator in purB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 - 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that the purB is under the control of purR. We also answered why previous study had conflicting report concerning the regulation of purB by purR by identifying the junction site of purB to lacZ in a purB-MudJ (lacZ, Kanr) fusion strain. This result strongly sup-ports that the purB is the second gene in the ycfC-purB op-eron.

  13. ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.

    Science.gov (United States)

    Zipeto, Maria Anna; Court, Angela C; Sadarangani, Anil; Delos Santos, Nathaniel P; Balaian, Larisa; Chun, Hye-Jung; Pineda, Gabriel; Morris, Sheldon R; Mason, Cayla N; Geron, Ifat; Barrett, Christian; Goff, Daniel J; Wall, Russell; Pellecchia, Maurizio; Minden, Mark; Frazer, Kelly A; Marra, Marco A; Crews, Leslie A; Jiang, Qingfei; Jamieson, Catriona H M

    2016-08-01

    Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling. PMID:27292188

  14. Expressions of multiple umami taste receptors in oral and gastrointestinal tissues, and umami taste synergism in chickens.

    Science.gov (United States)

    Yoshida, Yuta; Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

    2015-10-23

    Umami taste is one of the five basic taste qualities, along with sweet, bitter, sour, and salty, and is elicited by some l-amino acids and their salts, including monopotassium l-glutamate (MPG). The unique characteristic of umami taste is that it is synergistically enhanced by 5'-ribonucleotides such as inosine 5'-monophosphate (IMP). Unlike the other four basic taste qualities, the presence of umami taste sense in avian species is not fully understood. In this study, we demonstrated the expression of multiple umami taste receptor candidates in oral and gastrointestinal tract tissues in chickens using RT-PCR analysis. We first showed the metabotropic glutamate receptors (mGluRs) expressed in these tissues. Furthermore, we examined the preference for umami taste in chickens, focusing on the synergistic effect of umami taste as determined by the two-feed choice test. We concluded that chickens preferred feed containing both added MPG and added IMP over feeds containing either added MPG or added IMP alone and over the control feed. These results suggest that the umami taste sense and synergism are conserved in chickens. PMID:26361143

  15. Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.

    Science.gov (United States)

    Parry, R J; Hoyt, J C

    1997-02-01

    Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase. PMID:9023226

  16. Mycophenolic acid formulations in adult renal transplantation – update on efficacy and tolerability

    Directory of Open Access Journals (Sweden)

    Déla Golshayan

    2009-04-01

    Full Text Available Déla Golshayan1,2, M Pascual2, Bruno Vogt11Service of Nephrology and Hypertension, 2Transplantation Centre and Transplantation Immunopathology Laboratory, Department of Medicine, Centre Hospitalier Universitaire Vaudois (CHUV, Lausanne University, 1011 Lausanne, SwitzerlandAbstract: The description more than 30 years ago of the role of de novo purine synthesis in T and B lymphocytes clonal proliferation opened the possibility for selective immunosuppression by targeting specific enzymatic pathways. Mycophenolic acid (MPA blocks the key enzyme inosine monophosphate dehydrogenase and the production of guanosine nucleotides required for DNA synthesis. Two MPA formulations are currently used in clinical transplantation as part of the maintenance immunosuppressive regimen. Mycophenolate mofetil (MMF was the first MPA agent to be approved for the prevention of acute rejection following renal transplantation, in combination with cyclosporine and steroids. Enteric-coated mycophenolate sodium (EC-MPS is an alternative MPA formulation available in clinical transplantation. In this review, we will discuss the clinical trials that have evaluated the efficacy and safety of MPA in adult kidney transplantation for the prevention of acute rejection and their use in new combination regimens aiming at minimizing calcineurin inhibitor toxicity and chronic allograft nephropathy. We will also discuss MPA pharmacokinetics and the rationale for therapeutic drug monitoring in optimizing the balance between efficacy and safety in individual patients.Keywords: kidney transplantation, immunosuppression, mycophenolic acid, mycophenolate mofetil, enteric-coated mycophenolate sodium, acute rejection, chronic allograft nephropathy

  17. Metabolic profiles and free radical scavenging activity of Cordyceps bassiana fruiting bodies according to developmental stage.

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    Sun-Hee Hyun

    Full Text Available The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana.

  18. Pickles, pectin, and penicillin.

    Science.gov (United States)

    Demain, Arnold L

    2004-01-01

    My professional life has been devoted to the study of microbial products and their biosynthesis, regulation, and overproduction. These have included primary metabolites (glutamic acid, tryptophan, inosinic acid, guanylic acid, vitamin B(12), riboflavin, pantothenic acid, ethanol, and lactic acid) and secondary metabolites (penicillin, cephalosporins, streptomycin, fosfomycin, gramicidin S, rapamycin, indolmycin, microcin B17, fumagillin, mycotoxins, Monascus pigments, and tetramethylpyrazine). Other areas included microbial nutrition, strain improvement, bioconversions of statins and beta-lactams, sporulation and germination, plasmid stability, gel microdroplets, and the production of double-stranded RNA, the polymer xanthan, and enzymes (polygalacturonase, protease, cellulase). Most of the studies were carried out with me by devoted and hardworking industrial scientists for 15 years at Merck & Co. and by similarly characterized students, postdoctorals, and visiting scientists during my 32 years at the Massachusetts Institute of Technology. I owe much of my success to my mentors from academia and industry. My recent research activities with undergraduate students at the Charles A. Dana Research Institute for Scientists Emeriti (R.I.S.E.) at Drew University have been very rewarding and are allowing me to continue my career. PMID:15487928

  19. Beneficial effects of intermittent hypobaric hypoxia on the body

    Institute of Scientific and Technical Information of China (English)

    Yi ZHANG; Zhao-nian ZHOU

    2012-01-01

    Myocardial ischemia and reperfusion (I/R) is a common problem in clinic and there is no satisfactory method for prevention or treatment of I/R injury so far.Chronic intermittent hypobaric hypoxia (CIHH),similar to the concept of ischemia preconditioning(IPC)or altitude hypoxia adaptation (AHA),has been recognized to confer a protective effect on heart against I/R injury with a longer protective effect than IPC and a less adverse effect than AHA.It has been proved that CIHH increases myocardial tolerance to ischemia or hypoxia,reserving cardiac function and preventing arrhythmia during I/R.Multiple mechanisms or pathway underlying the cardiac protection of ClHH have been proposed,such as induction of heatshock protein,enhancement of myocardial antioxidation capacity,increase of coronary flow and myocardial capillary angiogenesis,activation of adenosine triphosphate (ATP)-sensitive potassium channels,inhibition of mitochondrial permeability transition pores,and activation of protein kinase C (PKC) and induced nitric oxide synthase (iNOS).In addition,CIHH has been found having many beneficial effects on the body,such as promotion of health,increase of oxygen utilization,and prevention or treatment for some diseases.The beneficial effects of ClHH and potential mechanisms are reviewed mainly based on the researches performed by our group.

  20. Perturbing A-to-I RNA editing using genetics and homologous recombination.

    Science.gov (United States)

    Staber, Cynthia J; Gell, Selena; Jepson, James E C; Reenan, Robert A

    2011-01-01

    Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those "most successful" phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deaminases acting on RNA) are highly conserved and are present in every higher metazoan genome sequenced to date. The fruit fly, Drosophila melanogaster, represents an ideal model organism for studying A-to-I editing, both in terms of fundamental biochemistry and in relation to determining adaptive downstream effects on physiology and behavior. The Drosophila genome contains a single structural gene for ADAR (dAdar), yet the fruit fly transcriptome has the widest range of conserved and validated ADAR targets in coding mRNAs of any known organism. In addition, many of the genes targeted by dADAR have been genetically identified as playing a role in nervous system function, providing a rich source of material to investigate the biological relevance of this intriguing process. Here, we discuss how recent advances in the use of ends-out homologous recombination (HR) in Drosophila make possible both the precise control of the editing status for defined adenosine residues and the engineering of flies with globally altered RNA editing of the fly transcriptome. These new approaches promise to significantly improve our understanding of how mRNA modification contributes to insect physiology and ethology.

  1. A novel multi-biofunctional protein from brown rice hydrolysed by endo/endo-exoproteases.

    Science.gov (United States)

    Selamassakul, Orrapun; Laohakunjit, Natta; Kerdchoechuen, Orapin; Ratanakhanokchai, Khanok

    2016-06-15

    Brown rice, which is a less allergenic food grain and contains essential amino acids, was hydrolysed by bromelain and PROTEASE FP51® to improve its functionalities and taste for food applications. The hydrolysate prepared by bromelain (eb-RPH) had high protein solubility, surface hydrophobicity, low molecular weight peptides, hydrophobic amino acids (leucine, valine and glycine) and flavor amino acids (glutamic acid and aspartic acid). The eb-RPH exhibited higher 1,1-diphenyl-2-picrylhydrazyl (DPPH˙) and 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic (ABTS˙(+)) radical-scavenging activities of 76.62% and 52.96%, respectively, and possessed a better foaming capacity (221.76%) and emulsifying capacity (32.34%) than the hydrolysate prepared by PROTEASE FP51® (ep-RPH) did. The eb-RPH gave the desired taste, which is attributed to volatile flavor compounds (benzaldehyde, benzeneacetaldehyde and 2-acetyl-1-pyrroline) and non-volatile flavor compounds, such as monosodium glutamate, 5'-guanosine monophosphate and 5'-inosine monophosphate (0.07, 0.03 and 0.05 mg mL(-1), respectively). Brown rice peptides generated by bromelain were novel bioactive peptides with multifunctional properties. PMID:27186602

  2. A Novel Computational Strategy to Identify A-to-I RNA Editing Sites by RNA-Seq Data: De Novo Detection in Human Spinal Cord Tissue

    Science.gov (United States)

    Picardi, Ernesto; Gallo, Angela; Galeano, Federica; Tomaselli, Sara; Pesole, Graziano

    2012-01-01

    RNA editing is a post-transcriptional process occurring in a wide range of organisms. In human brain, the A-to-I RNA editing, in which individual adenosine (A) bases in pre-mRNA are modified to yield inosine (I), is the most frequent event. Modulating gene expression, RNA editing is essential for cellular homeostasis. Indeed, its deregulation has been linked to several neurological and neurodegenerative diseases. To date, many RNA editing sites have been identified by next generation sequencing technologies employing massive transcriptome sequencing together with whole genome or exome sequencing. While genome and transcriptome reads are not always available for single individuals, RNA-Seq data are widespread through public databases and represent a relevant source of yet unexplored RNA editing sites. In this context, we propose a simple computational strategy to identify genomic positions enriched in novel hypothetical RNA editing events by means of a new two-steps mapping procedure requiring only RNA-Seq data and no a priori knowledge of RNA editing characteristics and genomic reads. We assessed the suitability of our procedure by confirming A-to-I candidates using conventional Sanger sequencing and performing RNA-Seq as well as whole exome sequencing of human spinal cord tissue from a single individual. PMID:22957051

  3. Harmful effects of the azathioprine metabolite 6-mercaptopurine in vascular cells: induction of mineralization.

    Directory of Open Access Journals (Sweden)

    Jasmin Prüfer

    Full Text Available Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells in vitro. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in in vitro and ex vivo models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.

  4. Multiple univariate data analysis reveals the inulin effects on the high-fat-diet induced metabolic alterations in rat myocardium and testicles in the preobesity state.

    Science.gov (United States)

    Duan, Yixuan; An, Yanpeng; Li, Ning; Liu, Bifeng; Wang, Yulan; Tang, Huiru

    2013-07-01

    Obesity is a worldwide epidemic and a well-known risk factor for many diseases affecting billions of people's health and well-being. However, little information is available for metabolic changes associated with the effects of obesity development and interventions on cardiovascular and reproduction systems. Here, we systematically analyzed the effects of high-fat diet (HFD) and inulin intake on the metabolite compositions of myocardium and testicle using NMR spectroscopy. We developed a useful high-throughput method based on multiple univariate data analysis (MUDA) to visualize and efficiently extract information on metabolites significantly affected by an intervention. We found that HFD caused widespread metabolic changes in both rat myocardium and testicles involving fatty acid β-oxidation together with the metabolisms of choline, amino acids, purines and pyrimidines even before HFD caused significant body-weight increases. Inulin intake ameliorated some of the HFD-induced metabolic changes in both myocardium (3-HB, lactate and guanosine) and testicle tissues (3-HB, inosine and betaine). A remarkable elevation of scyllo-inositol was also observable with inulin intake in both tissues. These findings offered essential information for the inulin effects on the HFD-induced metabolic changes and demonstrated this MUDA method as a powerful alternative to traditionally used multivariate data analysis for metabonomics.

  5. Smaller muscle ATP reduction in women than in men by repeated bouts of sprint exercise.

    Science.gov (United States)

    Esbjörnsson-Liljedahl, Mona; Bodin, Kristina; Jansson, Eva

    2002-09-01

    It was hypothesized that the reduction of high-energy phosphates in muscle after repeated sprints is smaller in women than in men. Fifteen healthy and physically active women and men with an average age of 25 yr (range of 19-42 yr) performed three 30-s cycle sprints (Wingate test) with 20 min of rest between sprints. Repeated blood and muscle samples were obtained. Freeze-dried pooled muscle fibers of types I and II were analyzed for high-energy phosphates and their breakdown products and for glycogen. Accumulation of plasma ATP breakdown products, plasma catecholamines, and blood lactate, as well as glycogen reduction in type I fibers, was all lower in women than in men during sprint exercise. Repeated sprints induced smaller reduction of ATP and smaller accumulation of IMP and inosine in women than in men in type II muscle fibers, with no gender differences in changes of ATP and its breakdown products during the bouts of exercise themselves. This indicates that the smaller ATP reduction in women than in men during repeated sprints was created during recovery periods between the sprint exercises and that women possess a faster recovery of ATP via reamination of IMP during these recovery periods.

  6. The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Skardelly, Marco, E-mail: Marco.Skardelly@med.uni-tuebingen.de [Department of Neurosurgery, University Hospital, Leipzig (Germany); Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig (Germany); Glien, Anja; Groba, Claudia; Schlichting, Nadine [Department of Neurosurgery, University Hospital, Leipzig (Germany); Kamprad, Manja [Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig (Germany); Meixensberger, Juergen [Department of Neurosurgery, University Hospital, Leipzig (Germany); Milosevic, Javorina [Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig (Germany)

    2013-12-10

    In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.

  7. A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

    Science.gov (United States)

    Ota, Hiroko; Sakasegawa, Shin-Ichi; Yasuda, Yuko; Imamura, Shigeyuki; Tamura, Tomohiro

    2008-12-01

    The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

  8. Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains.

    Science.gov (United States)

    Di Francesco, P; Tavazzi, B; Gaziano, R; Lazzarino, G; Casalinuovo, I A; Di Pierro, D; Garaci, E

    1998-01-01

    This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

  9. Large-scale analysis of structural, sequence and thermodynamic characteristics of A-to-I RNA editing sites in human Alu repeats

    Directory of Open Access Journals (Sweden)

    Eisenberg Eli

    2010-07-01

    Full Text Available Abstract Background Alu repeats in the human transcriptome undergo massive adenosine to inosine RNA editing. This process is selective, as editing efficiency varies greatly among different adenosines. Several studies have identified weak sequence motifs characterizing the editing sites, but these alone do not account for the large diversity observed. Results Here we build a dataset of 29,971 editing sites and use it to characterize editing preferences. We focus on structural aspects, studying the double-stranded RNA structure of the Alu repeats, and show the editing frequency of a given site to depend strongly on the micro-structure it resides in. Surprisingly, we find that interior loops, and especially the nucleotides at their edges, are more likely to be edited than helices. In addition, the sequence motifs characterizing editing sites vary with the micro-structure. Finally, we show that thermodynamic stability of the site is important for its editing. Conclusions Analysis of a large dataset of editing events reveals more information on sequence and structural motifs characterizing the A-to-I editing process

  10. In Silico Design of Human IMPDH Inhibitors Using Pharmacophore Mapping and Molecular Docking Approaches

    Directory of Open Access Journals (Sweden)

    Rui-Juan Li

    2015-01-01

    Full Text Available Inosine 5′-monophosphate dehydrogenase (IMPDH is one of the crucial enzymes in the de novo biosynthesis of guanosine nucleotides. It has served as an attractive target in immunosuppressive, anticancer, antiviral, and antiparasitic therapeutic strategies. In this study, pharmacophore mapping and molecular docking approaches were employed to discover novel Homo sapiens IMPDH (hIMPDH inhibitors. The Güner-Henry (GH scoring method was used to evaluate the quality of generated pharmacophore hypotheses. One of the generated pharmacophore hypotheses was found to possess a GH score of 0.67. Ten potential compounds were selected from the ZINC database using a pharmacophore mapping approach and docked into the IMPDH active site. We find two hits (i.e., ZINC02090792 and ZINC00048033 that match well the optimal pharmacophore features used in this investigation, and it is found that they form interactions with key residues of IMPDH. We propose that these two hits are lead compounds for the development of novel hIMPDH inhibitors.

  11. Binding mode of inhibitors and Cryptosporidium parvum IMP dehydrogenase: A combined ligand- and receptor-based study.

    Science.gov (United States)

    Li, R-J; Wang, Y-L; Wang, Q-H; Huang, W-X; Wang, J; Cheng, M-S

    2015-01-01

    A combined ligand- and target-based approach was used to analyse the interaction models of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) with selective inhibitors. First, a ligand-based pharmacophore model was generated from 20 NAD(+) competitive CpIMPDH inhibitors with the HipHop module. The characteristic of the NAD(+) binding site of CpIMPDH was then described, and the binding modes of the representative inhibitors were studied by molecular docking. The combination of the pharmacophore model and the docking results allowed us to evaluate the pharmacophore features and structural information of the NAD(+) binding site of CpIMPDH. This research supports the proposal of an interaction model inside the NAD(+) binding site of CpIMPDH, consisting of four key interaction points: two hydrophobic-aromatic groups, a hydrophobic-aliphatic group and a hydrogen bond donor. This study also provides guidance for the design of more potent CpIMPDH inhibitors for the treatment of Cryptosporidium infections. PMID:25978645

  12. Chemical Constituents of Croton caudatus Geisel. var. tomentosus Hook.%毛叶巴豆化学成分的研究

    Institute of Scientific and Technical Information of China (English)

    邹国安; 王媛; 杨峻山; 邹忠梅

    2009-01-01

    目的 研究大戟科(Euphorbiaceae)植物毛叶巴豆(Croton caudatus Geisel.var.tomentosus Hook.)茎中的化学成分.方法 利用硅胶柱,凝胶柱色谱等常规分离技术分离纯化化合物,根据现代波谱学技术(MS,1H-NMR,13C-NMR)进行结构鉴定.结果 从毛叶巴豆茎的体积分数95%乙醇提取物中分离得到11个化合物,分别鉴定为:三十六烷酸乙酯(ethylhexatriacontanoate,1),棕榈酸(palmic acid,2),硬脂酸(stearic acid,3),浙贝素(zheberiesinol,4),香草醛(vanillin,5),香草酸(vanillic acid,6),丁香酸(syringic acid,7),二十八烷酸(octacosanoic acid,8),琥珀酸(succinic acid,9),肌苷(inosine,10),异橙黄酮(isosinensetin,11).结论 所有化合物均为首次从该植物中分离得到,其中化合物4,7,9,10,11为首次从该属植物中分得.

  13. A Review of the Potential Utility of Mycophenolate Mofetil as a Cancer Therapeutic

    Directory of Open Access Journals (Sweden)

    Nazanin Majd

    2014-01-01

    Full Text Available Tumor cells adapt to their high metabolic state by increasing energy production. To this end, current efforts in molecular cancer therapeutics have been focused on signaling pathways that modulate cellular metabolism. However, targeting such signaling pathways is challenging due to heterogeneity of tumors and recurrent oncogenic mutations. A critical need remains to develop antitumor drugs that target tumor specific pathways. Here, we discuss an energy metabolic pathway that is preferentially activated in several cancers as a potential target for molecular cancer therapy. In vitro studies have revealed that many cancer cells synthesize guanosine triphosphate (GTP, via the de novo purine nucleotide synthesis pathway by upregulating the rate limiting enzyme of this pathway, inosine monophosphate dehydrogenase (IMPDH. Non-proliferating cells use an alternative purine nucleotide synthesis pathway, the salvage pathway, to synthesize GTP. These observations pose IMPDH as a potential target to suppress tumor cell growth. The IMPDH inhibitor, mycophenolate mofetil (MMF, is an FDA-approved immunosuppressive drug. Accumulating evidence shows that, in addition to its immunosuppressive effects, MMF also has antitumor effects via IMPDH inhibition in vitro and in vivo. Here, we review the literature on IMPDH as related to tumorigenesis and the use of MMF as a potential antitumor drug.

  14. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    Science.gov (United States)

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT. PMID:26968365

  15. Kinetic studies and evaluation of potential compounds for the chemotherapy of Leishmaniasis using LdNH-MBP

    Energy Technology Data Exchange (ETDEWEB)

    Renno, M.N.; Figueroa-Villar, J.D. [Instituto Militar de Engenharia (IME), Rio de Janeiro, RJ (Brazil). Dept. de Quimica; Silva, N.B. da; Tinoco, L.W. [Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Nucleo de Pesquisas de Produtos Naturais; Borja-Cabrera, G.P.; Palatnik-de-Sousa, C.B.P. [Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Inst. de Microbiologia

    2008-07-01

    Full text: Protozoan parasites rely exclusively on purine salvage from the host for DNA and RNA synthesis and nucleoside hydrolases (N Hs) are the enzymes that catalyze the N-rib osyl hydrolysis of all commonly occurring purine and pi rimidine nucleosides, thus being excellent targets for the design of antiparasitic compounds. The general aim of our work with Leishmania donovani NH (LdNH) is to find new inhibitors for this enzyme as potential agents for the chemotherapy of visceral leishmaniasis. In this part of the work we expressed LdNH bound to maltose-binding protein (MBP) in E. coli using the pMAL-C2x vector. After purification by affinity chromatography the enzyme activity was monitored by UV (280 nm) and {sup 1}H NMR spectroscopy using inosine as substrate. All the assays were carried out at 25 deg C in phosphate buffer (pH 8.0) in water (UV) and D{sub 2}O (NMR). Our results show that LdNH-MBP behaves kinetically in the same way as it have been reported for free LdNH, thus confirming that LdNH-MBP maintains the appropriate folding and activity of the enzyme active site, thus being a good model to develop and evaluate new inhibitors of LdNH. As an example, the kinetics tests with AZT have shown that this compound is not an effective inhibitor of this enzyme.

  16. Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture.

    Science.gov (United States)

    Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

    2010-12-01

    Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

  17. Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

    Directory of Open Access Journals (Sweden)

    Irina S.-R. Waisertreiger

    2010-01-01

    Full Text Available Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP deoxynucleoside triphosphate and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.

  18. Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

    Science.gov (United States)

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Masaaki; Kimura, Kazuhiro; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2016-08-01

    Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells. PMID:27421851

  19. Glutamine deprivation initiates reversible assembly of mammalian rods and rings.

    Science.gov (United States)

    Calise, S John; Carcamo, Wendy C; Krueger, Claire; Yin, Joyce D; Purich, Daniel L; Chan, Edward K L

    2014-08-01

    Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (5 μm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

  20. A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

    Science.gov (United States)

    Médici, Rosario; Garaycoechea, Juan I; Valino, Ana L; Pereira, Claudio A; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

    2014-04-01

    Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

  1. A-to-I RNA editing: A new mechanism of genomic information modification

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A-to-I RNA editing, the important event of gene modification, which takes place at post-transcriptional level, was firstly reported in 1991. The molecular mechanism of A-to-I RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, thereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues, however, up to date their substrates are mainly found in the central nervous system. It has recently been noticed that imperfect editing of these RNA substrates play critical roles in corresponding diseases, indicating that A-to-I RNA editing may be quite important in physiological or pathophysiological processes. Finding more new substrates of ADARs, especially in peripheral tissues, and performing functional research on new genes will be helpful to elucidate the biological significance of A-to-I RNA editing.

  2. In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

    Science.gov (United States)

    Koči, J; Jeffery, B; Riviere, J E; Monteiro-Riviere, N A

    2015-03-01

    In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

  3. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Science.gov (United States)

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  4. Loss or major reduction of umami taste sensation in pinnipeds

    Science.gov (United States)

    Sato, Jun J.; Wolsan, Mieczyslaw

    2012-08-01

    Umami is one of basic tastes that humans and other vertebrates can perceive. This taste is elicited by L-amino acids and thus has a special role of detecting nutritious, protein-rich food. The T1R1 + T1R3 heterodimer acts as the principal umami receptor. The T1R1 protein is encoded by the Tas1r1 gene. We report multiple inactivating (pseudogenizing) mutations in exon 3 of this gene from four phocid and two otariid species (Pinnipedia). Jiang et al. (Proc Natl Acad Sci U S A 109:4956-4961, 2012) reported two inactivating mutations in exons 2 and 6 of this gene from another otariid species. These findings suggest lost or greatly reduced umami sensory capabilities in these species. The widespread occurrence of a nonfunctional Tas1r1 pseudogene in this clade of strictly carnivorous mammals is surprising. We hypothesize that factors underlying the pseudogenization of Tas1r1 in pinnipeds may be driven by the marine environment to which these carnivorans (Carnivora) have adapted and may include: the evolutionary change in diet from tetrapod prey to fish and cephalopods (because cephalopods and living fish contain little or no synergistic inosine 5'-monophosphate that greatly enhances umami taste), the feeding behavior of swallowing food whole without mastication (because the T1R1 + T1R3 receptor is distributed on the tongue and palate), and the saltiness of sea water (because a high concentration of sodium chloride masks umami taste).

  5. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    Science.gov (United States)

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  6. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    Science.gov (United States)

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  7. Current methods of the analysis of immunosuppressive agents in clinical materials: A review.

    Science.gov (United States)

    Mika, Adriana; Stepnowski, Piotr

    2016-08-01

    More than 100000 solid organ transplantations are performed every year worldwide. Calcineurin (cyclosporine A, tacrolimus), serine/threonine kinase (sirolimus, everolimus) and inosine monophosphate dehydrogenase inhibitor (mycophenolate mofetil), are the most common drugs used as immunosuppressive agents after solid organ transplantation. Immunosuppressive therapy, although necessary after transplantation, is associated with many adverse consequences, including the formation of secondary metabolites of drugs and the induction of their side effects. Calcineurin inhibitors are associated with nephrotoxicity, cardiotoxicity and neurotoxicity; moreover, they increase the risk of many diseases after transplantation. The review presents a study of the movement of drugs in the body, including the processes of absorption, distribution, localisation in tissues, biotransformation and excretion, and also their accompanying side effects. Therefore, there is a necessity to monitor immunosuppressants, especially because these drugs are characterised by narrow therapeutic ranges. Their incorrect concentrations in a patient's blood could result in transplant rejection or in the accumulation of toxic effects. Immunosuppressive pharmaceuticals are macrolide lactones, peptides, and high molecular weight molecules that can be metabolised to several metabolites. Therefore the two main analytical methods used for their determination are high performance liquid chromatography with various detection methods and immunoassay methods. Despite the rapid development of new analytical methods of analysing immunosuppressive agents, the application of the latest generation of detectors and increasing sensitivity of such methods, there is still a great demand for the development of highly selective, sensitive, specific, rapid and relatively simple methods of immunosuppressive drugs analysis. PMID:26874932

  8. Umami Increases Consumer Acceptability, and Perception of Sensory and Emotional Benefits without Compromising Health Benefit Perception.

    Science.gov (United States)

    Miyaki, Takashi; Retiveau-Krogmann, Annlyse; Byrnes, Erin; Takehana, Shunji

    2016-02-01

    This study was undertaken to understand how consumers in the United States perceive umami-rich products, specifically low sodium chicken noodle soup. Results suggest that the addition of monosodium l-glutamate (MSG) at a concentration of 0.1% to 0.5%, alone or in synergy with 5'-ribonucleotides of inosine monophosphate (IMP) at 0.1% not only increases consumer acceptance but also positively impacts other aspects of consumer perception. Regardless of concentration of MSG and IMP, samples enhanced in umami compounds were perceived as more savory, flavorful, and less bland while providing a more homemade, fresh, and healthy wholesome taste than a control sample. From a functional and emotional benefit standpoint, when consuming umami-rich samples, consumers reported feeling significantly higher general satisfaction (they felt more content, relaxed, satisfied, less disappointed, dissatisfied…) and heightened positive emotions (happy, excited, indulgent…) than under the control condition. The feeling of being healthy while consuming the dish was not compromised. Last, when asked how they would feel if serving the soup sample to their family or friends, consumers projected feeling more positively under the umami-rich conditions (more happy, competent, loving, less dissatisfied or disappointed) compared to the control condition. PMID:26720057

  9. Evaluation of Pacific white shrimp (Litopenaeus vannamei health during a superintensive aquaculture growout using NMR-based metabolomics.

    Directory of Open Access Journals (Sweden)

    Tracey B Schock

    Full Text Available Success of the shrimp aquaculture industry requires technological advances that increase production and environmental sustainability. Indoor, superintensive, aquaculture systems are being developed that permit year-round production of farmed shrimp at high densities. These systems are intended to overcome problems of disease susceptibility and of water quality issues from waste products, by operating as essentially closed systems that promote beneficial microbial communities (biofloc. The resulting biofloc can assimilate and detoxify wastes, may provide nutrition for the farmed organisms resulting in improved growth, and may aid in reducing disease initiated from external sources. Nuclear magnetic resonance (NMR-based metabolomic techniques were used to assess shrimp health during a full growout cycle from the nursery phase through harvest in a minimal-exchange, superintensive, biofloc system. Aberrant shrimp metabolomes were detected from a spike in total ammonia nitrogen in the nursery, from a reduced feeding period that was a consequence of surface scum build-up in the raceway, and from the stocking transition from the nursery to the growout raceway. The biochemical changes in the shrimp that were induced by the stressors were essential for survival and included nitrogen detoxification and energy conservation mechanisms. Inosine and trehalose may be general biomarkers of stress in Litopenaeus vannamei. This study demonstrates one aspect of the practicality of using NMR-based metabolomics to enhance the aquaculture industry by providing physiological insight into common environmental stresses that may limit growth or better explain reduced survival and production.

  10. A distant cis acting intronic element induces site-selective RNA editing.

    Science.gov (United States)

    Daniel, Chammiran; Venø, Morten T; Ekdahl, Ylva; Kjems, Jørgen; Öhman, Marie

    2012-10-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome. PMID:22848101

  11. [Free radicals and hepatic ischemia-reperfusion].

    Science.gov (United States)

    Szijártó, Attila

    2015-11-22

    The critical importance of the ischemic-reperfusive injury is well documented with regards to numerous organs and clinical conditions. Oxygen free radicals play a central role in the mediation of the injury, which dominantly influences the prevalence of postoperative complications, (long term) organ damage, and the potential manifestation of systemic reactions. The both anatomically and pathophysiologically unique ischemic-reperfusive injury of the liver, which is expressively vulnerable to free radicals, is of utmost importance in liver surgery. Several techniques (adaptive maneuvers, chemical agents) are known to ameliorate the reperfusive injury. Based on the prior research of the workgroup of the author, the aim of the current article is to overview the set of measures capable of attenuating ischemic-reperfusive injury (ischemic preconditioning, -perconditioning, administration of adenosine, -inosine, -levosimendan, and -poly-ADP-ribose-polymerase inhibitor), with special attention to the ischemic-reperfusive injury of the liver, as well as the special pathophysiological role of free radicals in mediating hepatic damage.

  12. Clastogenic Factors as Potential Biomarkers of Increased Superoxide Production

    Directory of Open Access Journals (Sweden)

    Ingrid Emerit

    2007-01-01

    Full Text Available The formation of clastogenic factors (CF and their damaging effects are mediated by superoxide, since superoxide dismutase is regularly protective. CF are produced via superoxide and stimulate the production of superoxide by monocytes and neutrophils. This results in a selfsustaining and longlasting process of clastogenesis, which may exceed the DNA repair system and ultimately lead to cancer (Emerit, 1994. An increased cancer risk is indeed observed in conditions accompanied by CF formation. These include irradiated persons, patients with chronic inflammatory diseases, HIV-infected persons and the chromosomal breakage syndromes ataxia telangiectasia, Bloom’s syndrome and Fanconi’s anemia. Biochemical analysis has identifi ed lipid peroxidation products, arachidonic acid metabolites, nucleotides of inosine and cytokines, in particular tumor necrosis factor alpha, as the clastogenic and also superoxide stimulating components of CF. Due to their chromosome damaging effects, these oxidants can be detected with classical cytogenetic techniques. Their synergistic action renders the CF-test particularly sensitive for the detection of a pro-oxidant state. Correlations were observed between CF and other biomarkers of oxidative stress such as decreases in total plasma thiols or increases in TBARS or chemiluminescence. Correlations between CF and disease activity, between CF and radiation exposure, suggest the study of CF for monitoring these conditions. CF may also be useful as biochemical markers and intermediate endpoints for the evaluation of promising antioxidant drugs. CF formation represents a link between chronic inflammation and carcinogenesis. Prophylactic use of superoxide scavengers as anticarcinogens is therefore suggested.

  13. High Concentrations of the Antibiotic Spiramycin in Wastewater Lead to High Abundance of Ammonia-Oxidizing Archaea in Nitrifying Populations.

    Science.gov (United States)

    Zhang, Yu; Tian, Zhe; Liu, Miaomiao; Shi, Zhou Jason; Hale, Lauren; Zhou, Jizhong; Yang, Min

    2015-08-01

    To evaluate the potential effects of antibiotics on ammonia-oxidizing microbes, multiple tools including quantitative PCR (qPCR), 454-pyrosequencing, and a high-throughput functional gene array (GeoChip) were used to reveal the distribution of ammonia-oxidizing archaea (AOA) and archaeal amoA (Arch-amoA) genes in three wastewater treatment systems receiving spiramycin or oxytetracycline production wastewaters. The qPCR results revealed that the copy number ratios of Arch-amoA to ammonia-oxidizing bacteria (AOB) amoA genes were the highest in the spiramycin full-scale (5.30) and pilot-scale systems (1.49 × 10(-1)), followed by the oxytetracycline system (4.90 × 10(-4)), with no Arch-amoA genes detected in the control systems treating sewage or inosine production wastewater. The pyrosequencing result showed that the relative abundance of AOA affiliated with Thaumarchaeota accounted for 78.5-99.6% of total archaea in the two spiramycin systems, which was in accordance with the qPCR results. Mantel test based on GeoChip data showed that Arch-amoA gene signal intensity correlated with the presence of spiramycin (P amoA functional gene structures by variance partitioning analysis. This study revealed the selection of AOA in the presence of high concentrations of spiramycin in activated sludge systems. PMID:26125322

  14. An enzymatic atavist revealed in dual pathways for water activation.

    Directory of Open Access Journals (Sweden)

    Donghong Min

    2008-08-01

    Full Text Available Inosine monophosphate dehydrogenase (IMPDH catalyzes an essential step in the biosynthesis of guanine nucleotides. This reaction involves two different chemical transformations, an NAD-linked redox reaction and a hydrolase reaction, that utilize mutually exclusive protein conformations with distinct catalytic residues. How did Nature construct such a complicated catalyst? Here we employ a "Wang-Landau" metadynamics algorithm in hybrid quantum mechanical/molecular mechanical (QM/MM simulations to investigate the mechanism of the hydrolase reaction. These simulations show that the lowest energy pathway utilizes Arg418 as the base that activates water, in remarkable agreement with previous experiments. Surprisingly, the simulations also reveal a second pathway for water activation involving a proton relay from Thr321 to Glu431. The energy barrier for the Thr321 pathway is similar to the barrier observed experimentally when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when Arg418 is protonated, which predicts that the substitution of Glu431 with Gln will shift the pH-rate profile to the right. This prediction is confirmed in subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway was present in the ancestral enzyme, but was lost when the eukaryotic lineage diverged. We propose that the primordial IMPDH utilized the Thr321 pathway exclusively, and that this mechanism became obsolete when the more sophisticated catalytic machinery of the Arg418 pathway was installed. Thus, our simulations provide an unanticipated window into the evolution of a complex enzyme.

  15. Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone.

    Science.gov (United States)

    Barnes, Alan J; Baker, David R; Hobby, Kirsten; Ashton, Simon; Michopoulos, Filippos; Spagou, Konstantina; Loftus, Neil J; Wilson, Ian D

    2014-01-01

    1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted (1)H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more "human-like" profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4.  (1)H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone. PMID:24350779

  16. Facile "one-pot" synthesis of poly(methacrylic acid)-based hybrid monolith via thiol-ene click reaction for hydrophilic interaction chromatography.

    Science.gov (United States)

    Lv, Xumei; Tan, Wangming; Chen, Ye; Chen, Yingzhuang; Ma, Ming; Chen, Bo; Yao, Shouzhuo

    2016-07-01

    A novel sol-gel "one-pot" approach in tandem with a radical-mediated thiol-ene reaction for the synthesis of a methacrylic acid-based hybrid monolith was developed. The polymerization monomers, tetramethoxysilane (TMOS) and 3-mercaptopropyl trimethoxysilane (MPTS), were hydrolyzed in high-concentration methacrylic acid solution that also served as a hydrophilic functional monomer. The resulting solution was then mixed with initiator (2, 2'-azobis (2-methylpropionamide) dihydrochloride) and porogen (urea, polyethylene glycol 20,000) in a capillary column and polymerized in water bath. The column had a uniform porous structure and a good permeability. The evaluation of the monolith was performed by separation of small molecules including nucleosides, phenols, amides, bases and Triton X-100. The calibration curves for uridine, inosine, adenosine and cytidine were determined. All the calibration curves exhibited good linear regressions (R(2)≥0.995) within the test ranges of 0.5-40μg/mL for four nucleosides. Additionaliy, atypical hydrophilic mechanism was proved by elution order from low to high according to polarity retention time increased with increases in the content of the organic solvent in the mobile phase. Further studies indicated that hydrogen bond and electrostatic interactions existed between the polar analytes and the stationary phase. This was the mechanism of retention. The excellent separation of the BSA digest showed good hydrophility of the column and indicated the potential in separation of complex biological samples. PMID:27264742

  17. p38α Activates Purine Metabolism to Initiate Hematopoietic Stem/Progenitor Cell Cycling in Response to Stress.

    Science.gov (United States)

    Karigane, Daiki; Kobayashi, Hiroshi; Morikawa, Takayuki; Ootomo, Yukako; Sakai, Mashito; Nagamatsu, Go; Kubota, Yoshiaki; Goda, Nobuhito; Matsumoto, Michihiro; Nishimura, Emi K; Soga, Tomoyoshi; Otsu, Kinya; Suematsu, Makoto; Okamoto, Shinichiro; Suda, Toshio; Takubo, Keiyo

    2016-08-01

    Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery. PMID:27345838

  18. Diversity of Endonuclease V: From DNA Repair to RNA Editing

    Directory of Open Access Journals (Sweden)

    Isao Kuraoka

    2015-09-01

    Full Text Available Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism.

  19. The umami taste: from discovery to clinical use.

    Science.gov (United States)

    Stańska, Katarzyna; Krzeski, Antonii

    2016-06-30

    In the diversity of the flavor world only five basic tastes have been described. The newest one, umami, has been identified about one hundred years ago by Kikunae Ikeda but widely accepted just in the second half of the twentieth century by international scientific world. There are three umami substances: monosodium glutamate (MSG), inosine-5'-monophosphate (IMP), guanylo-5'-monophosphate (GMP). A real breakthrough in umami history concerned the finding about independent receptors for umami: T1R1 and T1R3 (taste receptors type 1 member 1 and member 3). The palatable, delicious taste of umami and its mechanism determined a lot of research studies on this highlight. Umami substances elicit salivary secretion, enhance appetite and increase food palatability. They are desirable to improve the quality of diet. Moreover, the association between umami substances and the suppression of obesity has been found. Studies suggest that umami is engaged in metabolism but also increases satiety and reduces the post-ingestive recovery of hunger. PMID:27387211

  20. Impact of high hydrostatic pressure on non-volatile and volatile compounds of squid muscles.

    Science.gov (United States)

    Yue, Jin; Zhang, Yifeng; Jin, Yafang; Deng, Yun; Zhao, Yanyun

    2016-03-01

    The effects of high hydrostatic pressure processing (HHP at 200, 400 or 600MPa) on non-volatile and volatile compounds of squid muscles during 10-day storage at 4°C were investigated. HHP increased the concentrations of Cl(-) and volatile compounds, reduced the level of PO4(3-), but did not affect the contents of 5'-uridine monophosphate (UMP), 5'-guanosine monophosphate (GMP), 5'-inosine monophosphate (IMP), Na(+) and Ca(2+) in squids on Day 0. At 600MPa, squids had the highest levels of 5'-adenosine monophosphate, Cl(-) and lactic acid, but the lowest contents of CMP and volatile compounds on Day 10. Essential free amino acids and succinic acids were lower on Day 0 than on Day 10. HHP at 200MPa caused higher equivalent umami concentration (EUC) on Day 0, and the EUC decreased with increasing pressure on Day 10. Generally, HHP at 200MPa was beneficial for improving EUC and volatile compounds of squids. PMID:26471521

  1. Flavor Preferences Conditioned by Dietary Glutamate.

    Science.gov (United States)

    Ackroff, Karen; Sclafani, Anthony

    2016-07-01

    Our understanding of the molecular basis of umami taste and its appetitive qualities has been greatly aided by studies in laboratory rodents. This review describes methods for testing responses to the prototypical umami substance monosodium glutamate (MSG) in rodents. Two techniques, forced exposure to MSG and 2-bottle choice tests with ascending concentrations, were used to evaluate the responses to the taste of umami itself, and 2 other methods used oral or postoral MSG to modify the responses to other flavors. Intake and preference for MSG are enhanced in mice by experience with MSG and with other nutrients with positive postoral effects. In addition, flavor preferences are enhanced in mice and rats by gastric or intestinal MSG infusions via an associative learning process. Even mice with an impaired or absent ability to taste MSG can learn to prefer a flavor added to an MSG solution, supporting the notion that glutamate acts postorally. The more complex flavor of dashi seasoning, which includes umami substances (inosinate, glutamate), is attractive to rodents, but dashi does not condition flavor preferences. Details of the postoral glutamate detection process and the nature of the signal involved in learned preferences are still uncertain but probably involve gastric or intestinal sensors or both and vagal transmission. Some findings suggest that postoral glutamate effects may enhance food preferences in humans, but this requires further study. PMID:27422522

  2. Deamination of amino acids as a source for ammonia production in human skeletal muscle during prolonged exercise

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; van der Vusse, G J; Söderlund, K;

    1995-01-01

    1. The influence of pre-exercise muscle glycogen content on ammonia production, adenine nucleotide breakdown and amino acid metabolism was investigated during prolonged exercise in six subjects having one leg with a normal and one leg with a low muscle glycogen content. One-leg knee-extensor exer......1. The influence of pre-exercise muscle glycogen content on ammonia production, adenine nucleotide breakdown and amino acid metabolism was investigated during prolonged exercise in six subjects having one leg with a normal and one leg with a low muscle glycogen content. One-leg knee......-extensor exercise was performed for 90 min, at a workload of 60-65% of the maximal power output, first with one leg and then with the other. 2. During exercise ammonia was released in gradually increasing amounts and plateaued after 1 h exercise at a rate of approximately 80 mumol min-1. The total ammonia...... production was 9.1 +/- 0.4 and 9.5 +/- 1.4 mmol (kg dry muscle)-1 in the normal and low glycogen content leg, respectively. 3. Levels of muscle phosphocreatine (PC), total adenine nucleotides and inosine monophosphate (IMP) were similar at rest and after 90 min of exercise. 4. Only minor differences were...

  3. Review. Elimination of viruses in plants: twenty years of progress

    Directory of Open Access Journals (Sweden)

    A. Panattoni

    2013-02-01

    Full Text Available To shed light on trends about elimination of viruses from plants, a bibliographic research was conducted to identify thermotherapy, chemotherapy and tissue culture trials published from 1991 through 2010. Among woody plants, grapevine, apple and peach are the most frequent targets of sanitation protocols because their health status is strictly regulated. Even if thermotherapy represents the preferred method for the host, grapevine viruses can also be eliminated with chemotherapy and tissue culture; apple viruses respond to chemotherapy as well. Although a similar trend was reported among herbaceous plants, chemotherapy was the most frequently used technique in potato. With regard to virus, thermotherapy was successfully applied against viruses belonging to 13 families and an unassigned genus. Instead, chemotherapy and tissue culture techniques eradicated viruses belonging to fewer families (nine. An interpretation of thermotherapy effects considers the new metabolic “pathways” triggered by the natural antiviral response emitted by the infected plant, with particular reference to virus-induced gene silencing. With regard to chemotherapy, several groups of antiviral drugs belong to inosine monophosphate dehydrogenase inhibitors, S-adenosylhomocysteine hydrolase inhibitors, neuraminidase inhibitors. Tissue culture, usually adopted to regenerate plantlets in biotechnological breeding programs, represents the less used tool for eliminate viruses from plants.

  4. Study on Development and Application of Natural Flavor Enhancer%天然风味增强剂的开发及应用研究进展

    Institute of Scientific and Technical Information of China (English)

    周进杰; 冯涛; 庄海宁

    2013-01-01

    风味增强剂在人们的饮食中扮演着重要的角色,是人们生活质量的标志.传统的风味增强剂包括谷氨酸及其盐类、鸟苷酸及其盐类、肌苷酸及其盐类、核苷酸及其盐类、甘氨酸及其钠盐、麦芽酚、乙基麦芽酚和L-亮氨酸等.文章按来源把天然风味增强剂分为微生物源、动物源和植物源,并对其在食品工业中的应用进行综述.%Flavor enhancer plays an important role in people's daily diet,which is a symbol of people's quality of life.The traditional flavor enhancers include glutamic acid and its salt,guanosine acid and its salt,inosinic acid and its salt,nucleotide and its salt,glycine and its sodium salt,maltol,ethyl maltol and L-leucine,etc.According to the sources,natural flavor enhancer can be divided into microbial source,animal source and plant source,and its application in food industry is reviewed in this paper.

  5. Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells.

    Science.gov (United States)

    Ortega, M T; Jeffery, B; Riviere, J E; Monteiro-Riviere, N A

    2016-02-01

    We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow-derived mesenchymal stem cells (BMSC) were differentiated into enterocyte-like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC50 for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml(-1) for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml(-1) for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml(-1) for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml(-1) for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml(-1) for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml(-1) for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml(-1) for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml(-1) for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml(-1) for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml(-1) for ginger root extract; > 200 and 116.78 ± 7.35 mg ml(-1) for sorbose. Lemon grass oil was evaluated at 0.003-0.9 in BMSC and .03-0.9 mg ml(-1) in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta-oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells. PMID

  6. The Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection

    Science.gov (United States)

    Kong, Yimeng; Pan, Bohu; Chen, Longxian; Wang, Hongbing; Hao, Pei; Li, Xuan

    2016-01-01

    The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. Here we present a study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system. PMID:27467689

  7. Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.

    Science.gov (United States)

    DasSarma, Priya; Negi, Vidya Devi; Balakrishnan, Arjun; Karan, Ram; Barnes, Susan; Ekulona, Folasade; Chakravortty, Dipshikha; DasSarma, Shiladitya

    2014-07-31

    Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.

  8. Synthesis of Tetra-Acetyl Ribofuranose by Sulfuric Ion Supported on Titanium Dioxide and Zirconium Dioxide%SO2-4-TiO2-SO2-4-ZrO2催化合成四乙酰呋喃核糖的研究

    Institute of Scientific and Technical Information of China (English)

    赵景联; 王云海

    2001-01-01

    1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose was synthesized from inosine and acetic anhydride in the presence of sulfuric ion supported on titanium dioxide and zirconium dioxide. Sulfuric ion supported on titanium dioxide and zirconium dioxide were prepared by immersing titanium dioxide and zirconium dioxide in sulfuric acid, and then baked at a fixed temperature. The catalyst is a mixture of sulfuric ion on titanium and sulfuric ion on zirconium in a fixed weight-rate. The yield of 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose can reach up to 84.6% under certain reactive conditions.%对用复合固体超强酸SO2-4-TiO2-SO2-4-ZrO2催化肌苷制备1,2,3,5-O-四乙酰-β-D-呋喃核糖进行了研究.研究结果表明,将TiO2和自制的ZrO2分别用硫酸溶液浸泡,在一定温度下焙烧,制成SO2-4-ZrO2和SO2-4-TiO2,再将SO2-4-ZrO2和SO2-4-TiO2按一定质量比混合制成复合固体超强酸催化剂,用肌苷和乙酸酐为原料,在一定条件下进行催化酯化反应,可使1,2,3,5-O-四乙酰-β-D-呋喃核糖的收率达到84.6%.

  9. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    Science.gov (United States)

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  10. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    Science.gov (United States)

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-03-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons. PMID:26717837

  11. Evaluation of different methods of stunning/killing sea bass (Dicentrarchus labrax) by tissue stress/quality indicators.

    Science.gov (United States)

    Zampacavallo, Giulia; Parisi, Giuliana; Mecatti, Massimo; Lupi, Paola; Giorgi, Gianluca; Poli, Bianca Maria

    2015-05-01

    The aim of the study was to evaluate the effect on the final product quality of certain innovative stunning/killing methods for sea bass as substitutes for the most common methods used by European farmers. The changes in tissue stress/quality parameters were monitored from the first hours after death and during the shelf life of the fish. Two trials were conducted in July and November on n. 231 sea bass stunned/killed by ice-water slurry, by single gas or mixture of gases in ice-water and by single- or two-stage electrical stunning/killing methods. Behavioural responses, stun/death time, rigor index, muscular and ocular pH, lactic acid, ATP and catabolites at death and within the 24 h after death were determined. In the November trial, the sensorial evaluation, rigor index, IMP, inosine, hypoxanthine, and K1 values were also evaluated during refrigerated storage until spoilage. The stunning/killing in ice-water appeared to induce low effects on the analysed parameters and preserve a good product quality as indicated by the highest pH and ATP values at death, the delayed full rigor onset and the 1 day longer shelf life (14 days) in comparison with the single- or two-stage electrical stunning/killing. The gas mixture addition provided a 40 % shortening of the time to obtain stunning/killing and 14 days of shelf life. The actual level of quality loss with the different killing conditions and the actual impact of a significant shortage of rigor mortis onset and pH drop on the possible pre-rigor filleting remain to be studied in depth. PMID:25892757

  12. Effects of the Sheep Polyclonal Antibodies Against the Porcine Adipocyte Plasma Membrane Proteins on Porcine Carcass Composition and Meat Quality

    Institute of Scientific and Technical Information of China (English)

    GAO Shi-zheng; HU Hong-mei; LIU Ling-yun; ZHANG Xi; LIU Yong-gang; GE Chang-rong

    2007-01-01

    To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin (ASIg) or sheep nonimmune serum immunoglobulin (NSIg). At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lung weight, dressing percentage,and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate,cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH,meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.

  13. Regional differences in the electrically stimulated release of endogenous and radioactive adenosine and purine derivatives from rat brain slices.

    Science.gov (United States)

    Pedata, F; Pazzagli, M; Tilli, S; Pepeu, G

    1990-10-01

    The release of both radioactive and endogenous purines was investigated in rat brain cortical, hippocampal and striatal slices at rest and following stimulation with electrical fields. Purines were labelled by incubating the slices with 3H-adenine. The purine efflux at rest and that evoked by electrical stimulation (10 Hz. 5 min) was analyzed by HPLC with ultraviolet absorbance detection. Both radioactive and endogenous purines in the effluent consisted mainly of hypoxanthine, xanthine, inosine and adenosine. No qualitative differences in the composition of the released purines were found in the three areas investigated. Electrical stimulation evoked a net increase in both radioactive and endogenous purine release. However the increase in 3H-adenosine following electrical stimulation was twice as large as that of endogenous adenosine. The electrically evoked release of both radioactive and endogenous purines was greatest in hippocampal slices and progressively smaller in cortical and striatal slices. In the three areas the addition of 0.5 microM tetrodotoxin to the superfusing Krebs solution brought about a similar (83-100%) reduction in evoked 3H-purine and endogenous purine release. Superfusion of the slices with calcium-free Krebs solution containing 0.5 mM EGTA reduced evoked release of 3H-purines by 58-60% and that of endogenous purine components by 54-89%. The results demonstrate similar characteristics for both radioactive and endogenous purine release but indicate that the most recently synthetized adenosine is the most readily available for release. The features of the electrically evoked purine release support a neuronal origin of adenosine and derivatives and are consistent with the hypothesis of discrete regional differences in adenosine neuromodulation. PMID:2255336

  14. Effect of anti-leukopenic drugs on the recovery of immunocompetent cells

    International Nuclear Information System (INIS)

    This study was performed to examine whether antileukopenic drugs can also promote the functional recovery of lymphocytes after radiation treatment. This was tested by investigating the ability of the drugs to restore the antibody forming capacity in 300R irradiated mice. Anti-leukopenic drugs were studied cobalt chlorophyllin sodium salt, adenine, glutathione, inosine, cepharanthine (CE), glycyrrhizin (GL) and purified vaccine lymph (PVL). CE, GL and PVL were shown to be effective in restoring the antibody forming capacity of the irradiated mice, as judged by an increased number of antibody forming cells (PFC) per spleen. Eight mg/kg and 20 mg/kg doses of CE increased the number of PFC in the cultures of irradiated spleen cells. Four mg/kg to 240 mg/kg doses of GL had virtually no effect on the numbers of PFC in the cultures. Specific doses of CE have opposite effects on the different types of cells in the immune response. CE-treated peritoneal exudate cells (PEC) from irradiated mice cultured with normal T and B cells generated significantly more PFC than any other combinations. These findings indicate that CE enhances the immune response by promoting the proliferation and/or differentiation of T and B precursor cells with some soluble substances from CE-treated PEC (macrophages), and CE somewhat suppresses the function of mature T and B cells by acting on these cells directly. In conclusion, among the 7 anti-leukopenic drugs, Cepharanthin was most effective in promoting the functional recovery of the lymphocytes assessed by the humoral immune response. Glycyrrhizin was also effective, but only when it was administered to the mice on a continuous basis. (J.P.N.)

  15. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

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    Shreoshi Pal Choudhuri

    Full Text Available Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  16. Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjusated polymers

    Institute of Scientific and Technical Information of China (English)

    HE Fang; YU MingHui; WANG Shu

    2009-01-01

    A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deami-nase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine (biotin-aptamer),a signaling probe single-stranded DNA-tagged fiuorescein at terminus (ssDNA-FI) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized,and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA (biotin-dsDNA-FI).The ssONA-FI is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.

  17. Changes in Biogenic Amines and ATP-Related Compounds and Their Relation to Other Quality Changes in Common Carp (Cyprinus carpio var. Jian) Stored at 20 and 0°C.

    Science.gov (United States)

    Zhang, Yuemei; Qin, Na; Luo, Yongkang; Shen, Huixing

    2015-09-01

    Biogenic amines, ATP-related compounds, sensory attributes, total volatile basic nitrogen, microbial flora (total viable bacteria, Pseudomonas, Aeromonas, and H2S-producing bacteria), and free amino acids were determined in common carp (Cyprinus carpio var. Jian) stored at 20 and 0°C. Pseudomonas and H2S-producing bacteria became the dominant bacteria in carp stored at 20 and 0°C, whereas Aeromonas rapidly increased only in carp stored at 0°C. Inosine monophosphate, which is responsible for flavor and freshness, increased to a maximum of 2.37 l mol/g after 12 h at 20°C and to 4.72 l mol/g after 3 days at 0°C. Putrescine and cadaverine were the dominant amines in carp and their concentrations were significantly correlated (P < 0.05) with total volatile basic nitrogen and sensory scores in all samples during the storage. Significant correlations also were observed between histamine and total volatile basic nitrogen and sensory scores only in samples stored at 20°C. Arginine decreased while putrescine increased in all samples. A significant decrease (P < 0.05) in histidine was observed after 24 h of storage, which coincided with an increase in histamine after 36 h in samples stored at 20°C. Hypoxanthine concentrations were significantly correlated with the microbial species (P < 0.01) and sensory scores (P < 0.05) and seems to be a reliable marker for quality of carp fillets stored at 20 and 0°C.

  18. Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

    Science.gov (United States)

    Choudhry, H; Albukhari, A; Morotti, M; Haider, S; Moralli, D; Smythies, J; Schödel, J; Green, C M; Camps, C; Buffa, F; Ratcliffe, P; Ragoussis, J; Harris, A L; Mole, D R

    2015-08-20

    Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

  19. Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

    Science.gov (United States)

    Oishi, Yo; Huang, Zhi-Li; Fredholm, Bertil B; Urade, Yoshihiro; Hayaishi, Osamu

    2008-12-16

    Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

  20. Altered A-to-I RNA Editing in Human Embryogenesis

    Science.gov (United States)

    Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  1. Altered A-to-I RNA editing in human embryogenesis.

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    Ronit Shtrichman

    Full Text Available Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2 and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC, as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs, which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.

  2. Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing.

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    Sivan Osenberg

    Full Text Available Adenosine to Inosine (A-to-I RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs. We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

  3. Alu Sequences in Undifferentiated Human Embryonic Stem Cells Display High Levels of A-to-I RNA Editing

    Science.gov (United States)

    Osenberg, Sivan; Paz Yaacov, Nurit; Safran, Michal; Moshkovitz, Sharon; Shtrichman, Ronit; Sherf, Ofra; Jacob-Hirsch, Jasmine; Keshet, Gilmor; Amariglio, Ninette; Itskovitz-Eldor, Joseph; Rechavi, Gideon

    2010-01-01

    Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions. PMID:20574523

  4. Apoptosis induced by nucleosides in the human hepatoma HepG2

    Institute of Scientific and Technical Information of China (English)

    Suh-Ching Yang; Che-Lin Chiu; Chi-Chang Huang; Jiun-Rong Chen

    2005-01-01

    AIM: To investigate the apoptotic effects of nucleosides on the human hepatoma HepG2.METHODS: The nucleosides included inosine (I), cytidine(C), uridine (U), thymidine (T), adenosine (A), and guanosine (G). Cells were incubated by the mediums with or without nucleosides at 37 ℃ in a 50 mL/L CO2 humidified atmosphere.RESULTS: It was found that the cell viabilities were significantly decreased, when cells were treated with 30 mmol/L I, 30 mmol/L C, 30 mmol/L U, 30 mmol/L T,0.5 mmol/L A, and 0.5 mmol/L G after 12 h incubation (P<0.05). About the apoptotic phenomenon, the cell percentages of sub-G1 cells were significantly increased in the mediums containing nucleosides such as C, U, T,A, and G (P<0.05). Furthermore, the caspase-3 activity was increased, when the cells were incubated with T(P<0.05). The protein expressions of p53 and p21 showed no difference in each group. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the contents of soluble Fas ligand contents were increased in HepG2 cells following I, U, T, and A treatment (P<0.05).But, TNF-α and cytochrome c were undetectable.CONCLUSION: Thymidine may induce the apoptosis in HepG2, but the effective dosages and reactive time must be investigated in the future study. However, the apoptosis-inducing abilities of other nucleosides were still unclear in this study.

  5. Implication of metabolomic profiles to wide thermoneutral zone in Mongolian gerbils (Meriones unguiculatus).

    Science.gov (United States)

    Shi, Yaolong; Wang, Dehua

    2016-07-01

    Mongolian gerbils (Meriones unguiculatus) have evolved a wide thermoneutral zone (26.5-38.9 °C) and high upper critical temperature, and appear to have a high tolerance for heat exposure. Here, we use a metabolomic approach to measure global metabolite profiles for gerbils between lower (27 °C) and upper critical temperatures (38 °C) to investigate the role of metabolomic characterization in maintaining basal metabolic rates within a wide thermoneutral zone. We found that in serum and liver, 14 and 19 metabolites were significantly altered, respectively. In the aerobic respiration-related tricarboxylic cycle (TCA), 5 intermediates (isocitric acid, cis-aconitic acid, α-ketoglutaric acid, fumaric acid and malic acid) were increased in serum in 38 °C animals; however, no such increase was found in the liver. A stable level of hepatic TCA cycle intermediates may be related to the steady state of aerobic respiration at 38 °C. Metabolomic results also revealed that acute heat exposure caused increased oxidative stress and low molecular weight antioxidants in Mongolian gerbils. Increased methionine and 2-hydroxybutyrate suggest an accelerated synthesis of glutathione. Increased urate and its precursors, inosine and hypoxanthine, were detected at 38 °C. Glucuronate, threonate and oxalate involved in ascorbate synthesis and degradation were increased in serum at 38 °C. In conclusion, although dramatic metabolomic variation was found, a stable hepatic TCA cycle may contribute to maintaining a constant basal metabolic rate within a wide thermoneutral zone in Mongolian gerbils. PMID:26749160

  6. Simultaneous quantification and splenocyte-proliferating activities of nucleosides and bases in Cervi cornu Pantotrichum

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    Ying Zong

    2014-01-01

    Full Text Available Background: Cervi Cornu Pantotrichum has been a well known traditional Chinese medicine, which is young horn of Cervus Nippon Temminck (Hualurong: HLR. At present, the methods used for the quality control of Cervi Cornu Pantotrichum show low specificity. Objective: To describe a holistic method based on chemical characteristics and splenocyte-proliferating activities to evaluate the quality of HLR. Materials and Methods: The nucleosides and bases from HLR were identified by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS, and six of them were chosen to be used for simultaneous HPLC quantification according to the results of proliferation of mouse splenocytes in vitro. Results: In this study, eight nucleosides and bases have been identified. In addition, uracil, hypoxanthine, uridine, inosine, guanosine, and adenosine were chosen to be used for simultaneous HPLC quantification. Simultaneous quantification of these six substances was performed on ten groups of HLR under the condition of a TIANHE Kromasil C 18 column (5 μm, 4.6 mm × 250 mm i.d. and a gradient elution of water and acetonitrile. Of the ten groups, HLR displayed the highest total nucleoside contents (TNC, sum of adenosine and uracil, 0.412 mg/g with the strongest splenocyte-proliferating activities. Conclusion: These results suggest that TNC (such as particularly highly contained adenosine and uracil in HLR has a certain correlation with the activity of splenocyte-proliferating, and it may be used as a quality control for HLR. This comprehensive method could be applied to other traditional Chinese medicines to ameliorate their quality control.

  7. Global metabolomic analysis of human saliva and plasma from healthy and diabetic subjects, with and without periodontal disease.

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    Virginia M Barnes

    Full Text Available Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine, increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate and ω-6 fatty acid (linoleate and arachidonate signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic

  8. Screening and characterization of purine nucleoside degrading lactic acid bacteria isolated from Chinese sauerkraut and evaluation of the serum uric acid lowering effect in hyperuricemic rats.

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    Ming Li

    Full Text Available Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99% to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P<0.05. The preventive treatment of DM9218-A caused a greater reduction in serum uric acid concentration in hyperuricemic rats than the later treatment (P<0.05. Our results suggest that DM9218-A may be a promising candidate as an adjunctive treatment in patients with hyperuricemia during the onset period of disease. DM9218-A also has potential

  9. The characteristics exosporium antigens from different vaccine strains of bacillus antracis

    International Nuclear Information System (INIS)

    To develop of both test-systems for rapid detection and identification of B. anthracis spores and a new subunit vaccine the antigens on the spore surface should be characterized. Exosporium consists of two layers-basal and peripheral and has been form by protein, amino- and neutral polysaccharides, lipids and ash. Number of anthrax exosporium proteins was described and identified: glycoprotein BclA, BclB, alanine racemase, inosine hydrolase, glycosyl hydrolase, superoxid dismutase, ExsF, ExsY, ExsK,CotB,CotY and SoaA. So far no glycosylated proteins other then highly immunogenic glycoproteins BclA, BclB were detected in the B. anthracis spore extract although several exosporium-specific glycoprotein have been described in other members of the B.cereus family- B. thuringiensis and B. cereus. Although EA1 protein originally described as main component of S-layer from vegetative cells he can regular observed in different exosporium preparations and additionally some anti- EA1 monoclonal antibodies able to recognize spore surface. We have revealed that EA1 isolated from spore of Russians strain STI-1contain carbohydrate which determine immunogenicity of this antigen. Because some time ago we have found that exosporium protein's pattern variable among B. anthracis strains we investigated exosporium from spore of different strains of B. anthracis including STI-1, Ames, Stern and others. We have comparative characterized antigens by using Western Blotting, Two-Dimensional electrophoresis and Mass Spec analysis. The results of analysis will be presented and discussed.(author)

  10. Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes.

    Science.gov (United States)

    Kocbuch, Katarzyna; Sakowicz-Burkiewicz, Monika; Grden, Marzena; Szutowicz, Andrzej; Pawelczyk, Tadeusz

    2009-01-01

    In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10(-8) M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10(-11) M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10(-8) M insulin comparing to cells grown in 10(-11) M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.

  11. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    Science.gov (United States)

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  12. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

    Science.gov (United States)

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  13. Engineering targeted chromosomal amplifications in human breast epithelial cells.

    Science.gov (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh

    2015-07-01

    Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

  14. Bronx River bed sediments phosphorus pool and phosphorus compound identification

    Science.gov (United States)

    Wang, J.; Pant, H. K.

    2008-12-01

    Phosphorus (P) transport in the Bronx River degraded water quality, decreased oxygen levels, and resulted in bioaccumulation in sediment potentially resulting in eutrophication, algal blooms and oxygen depletion under certain temperature and pH conditions. The anthropogenic P sources are storm water runoff, raw sewage discharge, fertilizer application in lawn, golf course and New York Botanical Garden; manure from the Bronx zoo; combined sewoverflows (CSO's) from parkway and Hunts Point sewage plant; pollutants from East River. This research was conducted in the urban river system in New York City area, in order to control P source, figure out P transport temporal and spatial variations and the impact on water quality; aimed to regulate P application, sharing data with Bronx River Alliance, EPA, DEP and DEC. The sediment characteristics influence the distribution and bioavailbility of P in the Bronx River. The P sequential extraction gave the quantitative analysis of the P pool, quantifying the inorganic and organic P from the sediments. There were different P pool patterns at the 15 sites, and the substantial amount of inorganic P pool indicated that a large amount P is bioavailable. The 31P- NMR (Nuclear Magnetic Resonance Spectroscopy) technology had been used to identify P species in the 15 sites of the Bronx River, which gave a qualitative analysis on phosphorus transport in the river. The P compounds in the Bronx River bed sediments are mostly glycerophophate (GlyP), nucleoside monophosphates (NMP), polynucleotides (PolyN), and few sites showed the small amount of glucose-6-phosphate (G6P), glycerophosphoethanoamine (GPEA), phosphoenopyruvates (PEP), and inosine monophosphate (IMP). The land use spatial and temporal variations influence local water P levels, P distributions, and P compositions.

  15. Possible Processes for Origin of First Chemoheterotrophic Microorganisms with Modeling of Physiological Processes of Bacterium Bacillus subtilis as a Model System in 2H2O

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    Ignat Ignatov

    2015-09-01

    Full Text Available We studied possible processes for origin of first chemoheterotrophic microorganisms with modeling of physiological processes of a Gram-positive chemoheterotrophic bacterium Bacillus subtilis, producer of purine ribonucleoside inosine as a model system in heavy water. The physiological influence of deuterium on the chemoheterotrophic bacterium B. subtilis was studied on a heavy water (HW medium with a maximal concentration of 2H2O (89–90 atom% 2H. Also various suitable samples of hot mineral water and sea water derived from different sources of Bulgaria were investigated using IR- and DNES-spectroscopy. It was shown that hot alkaline mineral water with temperature from +65 0C to +95 0C and pH value from 9 to 11 is more suitable for the origination of first organic forms than other analyzed water samples. There were discussed the reactions of condensation and dehydration occurring in alkaline aqueous solutions at t = +65–95 0C and рН = 9–10, resulting in synthesis from separate molecules the larger organic molecules as short polipeptides and pyrines, as well as the possible mechanisms of the deuterium accumulation in form of H2HO in hot water. The metabolism of the bacterium B. subtilis and the resistance to deuterium was also analyzed on an evolutionary level taking into account the hydrological conditions of primodial hydrosphere and the presence of H2HO, as well as the qualitative and quantitative composition of the cellular protein, amino acids and carbohydrates on media with maximum deuterium content. It was demonstrated on the example of chemoheterotrophic bacteria that first microorganisms might have been originated in hot mineral water with Ca2+ (0.5-1.0 g/l at t = + 65-95 0C and pH = 9–11, that is more suitable for maintenance and origin of life than other analyzed water samples.

  16. Global regulation of nucleotide biosynthetic genes by c-Myc.

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    Yen-Chun Liu

    Full Text Available BACKGROUND: The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2 on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

  17. Extracellular-purine metabolism in blood vessels (part I). Extracellular-purine level in blood of patients with abdominal aortic aneurysm.

    Science.gov (United States)

    Lecka, Joanna; Molski, Stanislaw; Komoszynski, Michal

    2010-09-01

    Adenosine and adenosine derivatives are the main regulators of purinoceptors (P1 and P2) mediated hemostasis and blood pressure. Since impaired hemostasis and high blood pressure lead to atherosclerosis and to the development of aneurysm, in this study we tested and compared the concentration of extracellular purines (e-purines) in the blood in of patients having abdominal aortic aneurysm with that from healthy volunteers. Whereas adenine nucleosides and nucleotides level in human blood plasma was analysed using reverse phase high performance liquid chromatography (HPLC), cholesterol concentration was estimated by an enzymatic assay. We did not find any correlation between e-purines concentration and the age of healthy volunteers. Furthermore, the sum level of e-purines (ATP, ADP, AMP, adenosine, and inosine) in the control group did not exceed 70 microM, while it was nearly two-fold higher in the blood of patients having abdominal aortic aneurysm, (123 microM). In a special case of people with Leriche Syndrome, a disease characterized by deep atherosclerotic changes, the e-purines level had further increased. Additionally, we also report typical atherosclerotic changes in the aorta using histological assays as well as total cholesterol rise. The significant rise in cholesterol concentration in the blood of the patients with abdominal aortas aneurysm, compared with the control groups, was not unique since 23% of the healthy people also exceeded the normal level of cholesterol. Therefore, our results strongly indicate that the estimation of e-purines concentration in the blood may serve as another indicator of atherosclerosis and warrant further consideration as a futuristic diagnostic tool.

  18. Preventive effect of α-lipoic acid on prepulse inhibition deficits in a juvenile two-hit model of schizophrenia.

    Science.gov (United States)

    Deslauriers, J; Racine, W; Sarret, P; Grignon, S

    2014-07-11

    Some pathophysiological models of schizophrenia posit that prenatal inflammation sensitizes the developing brain to second insults in early life and enhances brain vulnerability, thereby increasing the risk of developing the disorder during adulthood. We previously developed a two-hit animal model, based on the well-established prenatal immune challenge with poly-inosinic/cytidylic acid (polyI:C), followed by juvenile restraint stress (RS). We observed an additive disruption of prepulse inhibition (PPI) of acoustic startle in juvenile mice submitted to both insults. Previous studies have also reported that oxidative stress is associated with pathophysiological mechanisms of psychiatric disorders, including schizophrenia. We report here that PPI disruption in our two-hit animal model of schizophrenia is associated with an increase in oxidative stress. These findings led us to assess whether α-lipoic acid, an antioxidant, can prevent both increase in oxidative status and PPI deficits in our juvenile in vivo model of schizophrenia. In the offspring submitted to prenatal injection of polyI:C and to RS, treatment with α-lipoic acid prevented the development of PPI deficits 24h after the last period of RS. α-Lipoic acid also improved PPI performance in control mice. The reversal effect of α-lipoic acid pretreatment on these behavioral alterations was further accompanied by a normalization of the associated oxidative status and dopaminergic and GABAergic abnormalities in the prefrontal cortex. Based on our double insult paradigm, these results support the hypothesis that oxidative stress plays an important role in the development of PPI deficits, a well-known behavior associated with schizophrenia. These findings form the basis of future studies aiming to unravel mechanistic insights of the putative role of antioxidants in the treatment of schizophrenia, especially during the prodromal stage.

  19. ITPA Gene Polymorphisms Significantly Affect Hemoglobin Decline and Treatment Outcomes in Patients Coinfected With HIV and HCV

    Science.gov (United States)

    Osinusi, Anu; Naggie, Susanna; Poonia, Seerat; Trippler, Martin; Hu, Zonghui; Funk, Emily; Schlaak, Joerg; Fishbein, Dawn; Masur, Henry; Polis, Michael; Kottilil, Shyam

    2012-01-01

    Published studies have described a strong association with a single-nucleotide polymorphism (SNP) in the inosine triphosphate pyrophosphatase (ITPA) gene and ribavirin (RBV)-induced hemolytic anemia in HCV-infected patients receiving pegylated interferon (pegIFN) and RBV. This study sought to evaluate the effect of these polymorphisms on anemia, hemoglobin reduction, HCV kinetics, and treatment outcomes. Sixty-three patients coinfected with HIV and HCV and 58 patients infected with HCV only were treated with pegIFN/RBV were genotyped using the ABI Taq-Man allelic discrimination kit for the 2 ITPA SNP variants rs1127354 and rs7270101. A composite variable of ITPA deficiency using both SNPs was created as previously reported. Statistical analysis was performed using Mann-Whitney test or Chi square/Fishers exact test for categorical data and mixed model analysis for multiple variables. Thirty-five patients (30%) were predicted to have reduced ITPA activity. ITPA deficiency was found to be protective against the development of hemoglobin reduction >3 g/dl over the course of treatment. The rates of hemoglobin reduction >3 g/dl decreased in correlation with the severity of ITPA deficiency. ITPA deficiency was associated with slower hemoglobin decline early in treatment (week 4, P = 0.020) and rapid virologic response (RVR) at week 4 (P = 0.017) in patients coinfected with HIV and HCV. ITPA polymorphisms are associated with hemoglobin decline and in patients coinfected with HIV and HCV it is also associated with early virologic outcomes. Determination of ITPA polymorphisms may allow prediction of RBV-induced anemia and earlier initiation of supportive care to ensure optimal therapeutic outcomes. PMID:22585729

  20. Taste coding of complex naturalistic taste stimuli and traditional taste stimuli in the parabrachial pons of the awake, freely licking rat.

    Science.gov (United States)

    Sammons, Joshua D; Weiss, Michael S; Victor, Jonathan D; Di Lorenzo, Patricia M

    2016-07-01

    Several studies have shown that taste-responsive cells in the brainstem taste nuclei of rodents respond to sensory qualities other than gustation. Such data suggest that cells in the classical gustatory brainstem may be better tuned to respond to stimuli that engage multiple sensory modalities than to stimuli that are purely gustatory. Here, we test this idea by recording the electrophysiological responses to complex, naturalistic stimuli in single neurons in the parabrachial pons (PbN, the second neural relay in the central gustatory pathway) in awake, freely licking rats. Following electrode implantation and recovery, we presented both prototypical and naturalistic taste stimuli and recorded the responses in the PbN. Prototypical taste stimuli (NaCl, sucrose, citric acid, and caffeine) and naturalistic stimuli (clam juice, grape juice, lemon juice, and coffee) were matched for taste quality and intensity (concentration). Umami (monosodium glutamate + inosine monophosphate) and fat (diluted heavy cream) were also tested. PbN neurons responded to naturalistic stimuli as much or more than to prototypical taste stimuli. Furthermore, they convey more information about naturalistic stimuli than about prototypical ones. Moreover, multidimensional scaling analyses showed that across unit responses to naturalistic stimuli were more widely separated than responses to prototypical taste stimuli. Interestingly, cream evoked a robust and widespread response in PbN cells. Collectively, these data suggest that natural foods are more potent stimulators of PbN cells than purely gustatory stimuli. Probing PbN cells with pure taste stimuli may underestimate the response repertoire of these cells.

  1. Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

    Science.gov (United States)

    Inoue, Masashi; Glendinning, John I; Theodorides, Maria L; Harkness, Sarah; Li, Xia; Bosak, Natalia; Beauchamp, Gary K; Bachmanov, Alexander A

    2007-12-19

    The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system. PMID:17911381

  2. Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study.

    Science.gov (United States)

    Surowiec, Izabella; Karimpour, Masoumeh; Gouveia-Figueira, Sandra; Wu, Junfang; Unosson, Jon; Bosson, Jenny A; Blomberg, Anders; Pourazar, Jamshid; Sandström, Thomas; Behndig, Annelie F; Trygg, Johan; Nording, Malin L

    2016-07-01

    Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas

  3. Study on meat quality of famous pig breeds in Zhejiang%浙江地方名猪肉品质的研究

    Institute of Scientific and Technical Information of China (English)

    汪雨龙; 张晓军; 杨锋

    2015-01-01

    试验旨在通过对影响猪肉品质的指标测定,包括外观特性、氨基酸组成、矿物质含量、呈味物质和质构分析5个方面,系统评价了嘉兴黑猪、两头乌两个浙江地方猪种与外三元猪和普通猪的肉质差异. 结果表明:嘉兴黑猪、两头乌肉色和大理石纹级别较外三元和普通猪高,同时嘉兴黑猪还富含铁和锌元素,肌苷酸含量丰富,嫩度好. 四个猪种氨基酸总量,各成分之间含量,呈味物质差异大.%Meat quality differences between Zhejiang local pig breeds Jiaxing black pig,two-end black pig and hybrid and common pig were evaluated systematically through determining the indexes that effected pork quality. The indexes included appearance characteristics,amino acid composition,mineral content,flavoring materials and texture analysis. The results showed that the level of meat color and mar-bling of Zhejiang black pig and two-end black pig were higher than the hybrid and common pig. Mean-while Jiaxing black pig was rich in both iron and zinc. And it was of rich content inosine monophosphate and good tenderness. There were big differences in total amino acid,the content of each ingredient and flavoring materials among the four kinds of pig breeds.

  4. The Regulation of Exosporium-Related Genes in Bacillus thuringiensis

    Science.gov (United States)

    Peng, Qi; Kao, Guiwei; Qu, Ning; Zhang, Jie; Li, Jie; Song, Fuping

    2016-01-01

    Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus group. Spores of B. cereus group species are encircled by exosporium, which is composed of an external hair-like nap and a paracrystalline basal layer. Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we studied the regulation of several exosporium-related genes in Bt. A SigK consensus sequence is present upstream of genes encoding hair-like nap proteins (bclA and bclB), basal layer proteins (bxpA, bxpB, cotB, and exsY ), and inosine hydrolase (iunH). Mutation of sigK decreased the transcriptional activities of all these genes, indicating that the transcription of these genes is controlled by SigK. Furthermore, mutation of gerE decreased the transcriptional activities of bclB, bxpB, cotB, and iunH but increased the expression of bxpA, and GerE binds to the promoters of bclB, bxpB, cotB, bxpA, and iunH. These results suggest that GerE directly regulates the transcription of these genes, increasing the expression of bclB, bxpB, cotB, and iunH and decreasing that of bxpA. These findings provide insight into the exosporium assembly process at the transcriptional level. PMID:26805020

  5. Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

    Directory of Open Access Journals (Sweden)

    Tsuneo Kakuda

    Full Text Available Mitochondrial DNA (mtDNA serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs, which relies on a novel amplified product-length polymorphisms (APLP method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp, this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

  6. Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

    Science.gov (United States)

    Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

    2016-01-01

    Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

  7. Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

    Directory of Open Access Journals (Sweden)

    Nieves Ayllón

    Full Text Available Tudor staphylococcal nuclease (Tudor-SN and Argonaute (Ago are conserved components of the basic RNA interference (RNAi machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

  8. Simultaneous quantification and splenocyte-proliferating activities of nucleosides and bases in Cervi cornu Pantotrichum

    Science.gov (United States)

    Zong, Ying; Wang, Yu; Li, Hang; Li, Na; Zhang, Hui; Sun, Jiaming; Niu, Xiaohui; Gao, Xiaochen

    2014-01-01

    Background: Cervi Cornu Pantotrichum has been a well known traditional Chinese medicine, which is young horn of Cervus Nippon Temminck (Hualurong: HLR). At present, the methods used for the quality control of Cervi Cornu Pantotrichum show low specificity. Objective: To describe a holistic method based on chemical characteristics and splenocyte-proliferating activities to evaluate the quality of HLR. Materials and Methods: The nucleosides and bases from HLR were identified by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), and six of them were chosen to be used for simultaneous HPLC quantification according to the results of proliferation of mouse splenocytes in vitro. Results: In this study, eight nucleosides and bases have been identified. In addition, uracil, hypoxanthine, uridine, inosine, guanosine, and adenosine were chosen to be used for simultaneous HPLC quantification. Simultaneous quantification of these six substances was performed on ten groups of HLR under the condition of a TIANHE Kromasil C18 column (5 μm, 4.6 mm × 250 mm i.d.) and a gradient elution of water and acetonitrile. Of the ten groups, HLR displayed the highest total nucleoside contents (TNC, sum of adenosine and uracil, 0.412 mg/g) with the strongest splenocyte-proliferating activities. Conclusion: These results suggest that TNC (such as particularly highly contained adenosine and uracil) in HLR has a certain correlation with the activity of splenocyte-proliferating, and it may be used as a quality control for HLR. This comprehensive method could be applied to other traditional Chinese medicines to ameliorate their quality control. PMID:25422536

  9. Application of Membrane Bioreactor in Advanced Treatment of Pharmaceutical Wastewater%膜-生物反应器在制药废水深度处理中的应用

    Institute of Scientific and Technical Information of China (English)

    李振红; 徐洪斌; 王磊; 刘琛

    2011-01-01

    由于国家颁布了新的发酵类制药工业水污染物排放标准,COD排放标准由300mg/L降至120 mg/L,以肌苷生产为主的制药厂原有污水处理工艺不能满足排放要求.在原污水处理工艺后采用浸没一体式MBR工艺对制药废水进行深度处理中试试验,考察处理效果.试验结果表明,浸没一体式MBR工艺在DO质量浓度分别为2,4,6 mg/L时,出水COD去除率分别为63%,75%,80%,出水NH3-N的去除率分别为88.5%,93.6%和94%.%The new discharge standards of water pollutants for pharmaceutical industry in fermentation products category has been issued and the discharge standard of COD is reduced from 300 mg/L to 120 mg/L. The original wastewater treatment process for the pharmaceutical factory mainly in inosine production can not meet the discharge requirements. The pilot test of following the original sewage treating technology for the advanced treatment of pharmaceutical wastewater was carried out. The test results showed that with the integrated and submerged membrane bioreactor process, when DO was kept at 2,4 and 6 mg/L, the effluent COD removal rates were 63%, 75% and 80% and ammonia nitrogen removal rate were 88.5%, 93.6% and 94% respectively.

  10. Safety assessment of potential food ingredients in canine hepatocytes.

    Science.gov (United States)

    Zhang, Leshuai W; Koci, Juraj; Jeffery, Brett; Riviere, Jim E; Monteiro-Riviere, Nancy A

    2015-04-01

    This research aimed to develop in vitro methods to assess hazard of canine food ingredients. Canine hepatocytes were harvested and cell viability of clove-leaf oil (CLO), eugenol (EUG), lemongrass oil (LGO), guanosine monophosphate (GMP), inosine monophosphate (IMP), sorbose, ginger-root extract (GRE), cinnamon-bark oil (CBO), cinnamaldehyde (CINA), thymol oil (TO), thymol (THYM), and citric acid were assessed with positive controls: acetaminophen (APAP), aflatoxin B1 and xylitol. Molecular Toxicology PathwayFinder array (MTPF) analyzed toxicity mechanisms for LGO. LC50 for APAP was similar among human (3.45), rat (2.35), dog (4.26 mg/ml). Aflatoxin B1 had an LC50 of 4.43 (human), 5.78 (rat) and 6.05 (dog) µg/ml; xylitol did not decrease viability. LC50 of CLO (0.185 ± 0.075(SD)), EUG (0.165 ± 0.112), LGO (0.220 ± 0.012), GRE (1.54 ± 0.31) mg/ml; GMP (166.03 ± 41.83), GMP + IMP (208.67 ± 15.27) mM; CBO (0.08 ± 0.03), CINA (0.11 ± 0.01), TO (0.21 ± 0.03), THYM (0.05 ± 0.01), citric acid (1.58 ± 0.08) mg/ml, while sorbose was non-toxic. LGO induced upregulation of 16 and down-regulation of 24 genes, which CYP and heat shock most affected. These results suggest that in vitro assays such as this may be useful for hazard assessment of food ingredients for altered hepatic function. PMID:25660481

  11. Use of nucleotides in weanling rats with diarrhea induced by a lactose overload: effect on the evolution of diarrhea and weight and on the histopathology of intestine, liver and spleen

    Directory of Open Access Journals (Sweden)

    Norton R.

    2001-01-01

    Full Text Available Until recently, dietary sources of nucleotides were thought not to be essential for good nutrition. Certain states with higher metabolic demands may require larger amounts that cannot be provided by endogenous production. The objective of the present study was to determine the action of nucleotides on the recovery from lactose-induced diarrhea in weaned rats. Thirty-six weanling Fisher rats were divided into two groups. Group 1 received a standard diet and group 2 received a diet containing lactose in place of starch. On the 10th day, six animals per group were sacrificed for histopathological evaluation. The remaining animals were divided into two other subgroups, each with 6 animals, receiving a control diet, a control diet with nucleotides (0.05% adenosine monophosphate, 0.05% guanosine monophosphate, 0.05% cytidine monophosphate, 0.05% uridine monophosphate and 0.05% inosine monophosphate, a diet with lactose, and a diet with lactose and nucleotides. On the 32nd day of the experiment all animals were sacrificed. Animals with diarrhea weighed less than animals without diarrhea. The introduction of nucleotides did not lead to weight gain. Mean diet consumption was lower in the group that continued to ingest lactose, with the group receiving lactose plus nucleotides showing a lower mean consumption. Animals receiving lactose had inflammatory reaction and deposits of periodic acid-Schiff-positive material in intestinal, hepatic and splenic tissues. The introduction of nucleotides led to an improvement of the intestinal inflammatory reaction. In lactose-induced diarrhea, when the stimulus is maintained - lactose overload - the nucleotides have a limited action on the weight gain and on recovery of intestinal morphology, although they have a protective effect on hepatic injury and improve the inflammatory response.

  12. Transformation and action of extracellular NAD+ in perfused rat and mouse livers

    Institute of Scientific and Technical Information of China (English)

    Ana Carla BROETTO-BLAZON; Fabricio BRACHT; Livia BRACHT; Ana Maria KELMER-BRACHT; Adelar BRACHT

    2009-01-01

    Aim: Transformation and possible metabolic effects of extracellular NAD+ were investigated in the livers of mice (Mus mus-culus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains). Methods: The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD+ was monitored using high-performance liquid chromatography. Results: In the mouse liver, the single-pass metabolism of 100 μmol/L NAD+ was almost complete; ADP-ribose and nicoti-namide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main trans-formation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also iden-tified. On a weight basis, the transformation of NAD+ was more efficient in the mouse liver. In the rat liver, 100 μmol/L NAD+ transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibi-tion was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 μmol/L NAD+. Conclusion: It can be concluded that the functions of extracellular NAD+ are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.

  13. Taste coding of complex naturalistic taste stimuli and traditional taste stimuli in the parabrachial pons of the awake, freely licking rat.

    Science.gov (United States)

    Sammons, Joshua D; Weiss, Michael S; Victor, Jonathan D; Di Lorenzo, Patricia M

    2016-07-01

    Several studies have shown that taste-responsive cells in the brainstem taste nuclei of rodents respond to sensory qualities other than gustation. Such data suggest that cells in the classical gustatory brainstem may be better tuned to respond to stimuli that engage multiple sensory modalities than to stimuli that are purely gustatory. Here, we test this idea by recording the electrophysiological responses to complex, naturalistic stimuli in single neurons in the parabrachial pons (PbN, the second neural relay in the central gustatory pathway) in awake, freely licking rats. Following electrode implantation and recovery, we presented both prototypical and naturalistic taste stimuli and recorded the responses in the PbN. Prototypical taste stimuli (NaCl, sucrose, citric acid, and caffeine) and naturalistic stimuli (clam juice, grape juice, lemon juice, and coffee) were matched for taste quality and intensity (concentration). Umami (monosodium glutamate + inosine monophosphate) and fat (diluted heavy cream) were also tested. PbN neurons responded to naturalistic stimuli as much or more than to prototypical taste stimuli. Furthermore, they convey more information about naturalistic stimuli than about prototypical ones. Moreover, multidimensional scaling analyses showed that across unit responses to naturalistic stimuli were more widely separated than responses to prototypical taste stimuli. Interestingly, cream evoked a robust and widespread response in PbN cells. Collectively, these data suggest that natural foods are more potent stimulators of PbN cells than purely gustatory stimuli. Probing PbN cells with pure taste stimuli may underestimate the response repertoire of these cells. PMID:27121585

  14. A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

    Directory of Open Access Journals (Sweden)

    Mortensen Uffe H

    2011-09-01

    Full Text Available Abstract Background Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA, an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH, which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF. Results In accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class. Conclusions We identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.

  15. The effects of intragastric infusion of umami solutions on amygdalar and lateral hypothalamic neurons in rats.

    Science.gov (United States)

    Davaasuren, Munkhzul; Matsumoto, Jumpei; Chinzorig, Choijiljav; Nakamura, Tomoya; Takamura, Yusaku; Patrono, Enrico; Kondoh, Takashi; Ono, Taketoshi; Nishijo, Hisao

    2015-10-01

    Previous behavioral studies have suggested that l-glutamate, an umami substance, is detected in the gut, and that this information regarding glutamate is conveyed from the gut to the amygdala and the lateral hypothalamus (LH) through the vagus nerve to establish glutamate preference. In this study, we investigated the roles of the amygdala and LH in the information processing of gut glutamate. We recorded the activity of amygdalar and LH neurons during the intragastric administration of five test solutions (monosodium l-glutamate [MSG, 60 mmol/L]; inosine monophosphate [IMP, 60 mmol/L]; a mixture of MSG and IMP; NaCl [60 mmol/L]; or physiological saline) in intact and subdiaphragmatic vagotomized awake rats. In intact rats, 349 and 189 neurons were recorded from the amygdala and LH, respectively, while in vagotomized rats, 104 and 90 neurons were recorded from the amygdala and LH, respectively. In intact rats, similar percentages of neurons (30-60%) in the amygdala and LH responded to the intragastric infusion of the solutions. Vagotomy significantly altered responses to the MSG and NaCl solutions. In particular, vagotomy suppressed the inhibitory responses to the NaCl solution. Furthermore, vagotomy increased the response similarity between the MSG and NaCl solutions, suggesting that vagotomy impaired the coding of the postingestive consequences of the MSG solution in the amygdala and LH, which are unique for glutamate. The present results provide the first neurophysiological evidence that amygdalar and LH neurons process glutamate signals from the gut. PMID:26438732

  16. 奶汤鱼肚风味形成与烹制%Flavor Formation and Cooking of Fish Maw with Milk Soup

    Institute of Scientific and Technical Information of China (English)

    李保定

    2014-01-01

    Fish maw with milk soup is a traditional dish in Henan Province.Use empirical method to analyze the reason for the formation of flavors.Studies have shown that amino acids and other nutri-ents in fish maw,ham,bamboo shoots,mushroom are the basis for the formation of flavors of cui-sine.Inosine 5'-monophosphate (5'-IMP and 5'-guanosine monophosphate 5'-GMP)in mushroom and umami substances of amino acids in fish maw make the flavors of cuisine multiplied.The amino acids, nucleotides,guanylic acid and succinic acid in milk soup are the main source of umami substances.%奶汤鱼肚是河南传统名菜。运用实证法分析了菜肴风味形成的原因。研究表明:鱼肚、火腿、冬笋、香菇所含的氨基酸及其他营养素是菜肴风味形成的基础,其中香菇中5'-磷酸肌甘酸(5'-I MP 和5'-磷酸鸟苷酸5'-G MP )与鱼肚中的氨基酸类鲜味物质协同,使菜肴风味倍增。奶汤当中的氨基酸、核苷酸、鸟甘酸和琥珀酸是呈现鲜味物质的主要来源。

  17. Effects of 3 Feeding Modes on the Volatile and Nonvolatile Compounds in the Edible Tissues of Female Chinese Mitten Crab (Eriocheir sinensis).

    Science.gov (United States)

    Zhuang, Kejin; Wu, Na; Wang, Xichang; Wu, Xugan; Wang, Shuai; Long, Xiaowen; Wei, Xuan

    2016-04-01

    To reveal the impact of different feeding modes on the flavor quality of female Chinese mitten crab (Eriocheir sinensis) this study was conducted to compare the sensory evaluation scores, flavor compounds in meat and hepatopancreas of female E. sinensis fed with 3 feeding modes, that is, natural diets (NDs), traditional diets (TDs), and formulated diets (FDs). The result showed that crabs fed with ND had significantly lower sensory scores than the other 2 feeding modes in both edible tissues. The odor and taste profiles were evaluated by Electronic nose (E-nose) and tongue (E-tongue) techniques, respectively; results of perchloric acid showed each edible tissue had significant differences among the 3 modes. Contents of volatile compounds were measured by Headspace-solid phase micro extraction combined with gas chromatography-mass spectrometer. A total of 35 and 44 volatile compounds were identified in meat and hepatopancreas, respectively. ND mode of meat had the highest relative odor activity value (ROAV) summation among the 3 diet modes. TD mode of hepatopancreas had significantly higher ROAV summations. Based on the analysis of free amino acids and 5'-nucleotides, nonvolatile compounds were evaluated by equivalent umami concentration (EUC) and taste active values (TAVs) methods. For both meat and hepatopancreas, TD had the highest contents of umami amino acid, as for the 5'-nucleotide, FD had the highest 5'-inosin monophosphate concentrations. Overall, the EUC and TAVs of TD were higher than that of FD, whereas ND mode had the lowest values in the 2 edible tissues. In conclusion, TD mode had the best performance in terms of sensory evaluation, ROAVs of aroma-active compounds, and nonvolatile active compounds. PMID:26919186

  18. Lenticular energy metabolism during exogenous calcium deprivation and during recovery: effects of dextran-40.

    Science.gov (United States)

    Glonek, T; Kopp, S J; Greiner, J V; Sanders, D R

    1985-02-01

    Phosphatic metabolites of the intact rabbit lens were quantitated as a function of time by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy during in vitro incubations at 37 degrees C in calcium-sufficient and calcium-deficient modified Earle's buffer with and without the osmotic agent, Dextran-40. Intralenticular pH was determined from the resonance shift position of inorganic orthophosphate (Pi). Incubation of lenses in calcium-deficient buffer resulted in a pronounced, time-dependent decrease in lenticular adenosine triphosphate (ATP) levels. The half-life of ATP within the lens was 11 hr under these experimental conditions. A concomitant, essentially stoichiometric increase in adenosine diphosphate and Pi levels was observed also. The other phosphatic metabolites were unaffected by exogenous calcium deprivation except for adenosine and inosine monophosphate which accumulated with time. Dextran-40 (6%), which has been shown to prevent lens swelling under these same experimental conditions, did not influence the metabolic responses of the lens to external calcium deprivation and did not facilitate subsequent restoration of lens phosphatic metabolites following restoration of a physiologic calcium concentration to the supporting medium. The Dextran-40 did, however, promote the retention of intralenticular pH environment during the experimental period. These findings suggest that the previously reported Dextran-40-dependent recovery of intralenticular sodium and potassium concentrations to control levels following 10 hr of incubation in calcium-deficient media cannot be attributed to a direct energy-sparing action of Dextran-40 on lenticular energy metabolism. Instead, the mechanistic basis for the action of Dextran-40 would appear to be related to its colloid osmotic properties and its ability to prevent lenticular swelling, which otherwise occurs in the absence of Dextran under these experimental conditions. PMID:2579839

  19. Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

    Science.gov (United States)

    Ochi, Kozo; Nishizawa, Tomoyasu; Inaoka, Takashi; Yamada, Akiyo; Hashimoto, Kohsuke; Hosaka, Takeshi; Okamoto, Susumu; Ozeki, Yoshihiro

    2012-08-01

    The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

  20. Metabonmontics study on the urine of natural aging rats administrated with Erzhi Pills%二至丸对自然衰老小鼠尿液代谢产物的影响

    Institute of Scientific and Technical Information of China (English)

    赵益; 苏文; 张启云; 李冰涛; 徐国良; 刘红宁

    2011-01-01

    目的:研究长期给予滋阴药二至丸对小鼠内源性代谢产物的影响.方法:二至丸组自小鼠6月龄起按1.8g/kg给药,至18月龄时结束时收集尿液,经过液相色谱分离,三重四极杆质谱检测尿液中内源性代谢物信息,采用主成分分析法(Principal component analysis,PCA)降维,正交信号校正偏最小二乘法判别分析法(Orthogonal signal correction-partial least square discriminate analysis,OSC-PLS-DA),分析二至丸小鼠尿样的代谢组学特征及生物标记物.结果:二至丸组与空白对照组相比,小鼠尿液代谢物共有36种发生改变,其中与延缓衰老的药理作用有:Malonic acid(m/z105)、3-O-a-L-Fucopyranosyl-D-glucose(m/z327.1)、Cytosine(m/z243)、17a-Ethynylestradiol(m/z296.1)、inosinic acid(m/z348.2)、N-Acetylaspartylglutamate(m/z305.1)、(R)-3-Hydroxydodecanoic acid (m/z245)、Oleic acid(m/z283.1)、cholesterol esters(m/z701.5).结论:二至丸长期给药对自然衰老小鼠的内源性物质有影响,可能能够从提高免疫力、调节神经功能、调整内分泌、促进物质代谢等方面延缓衰老,这为二至丸抗衰老的开发和利用提高基础.

  1. Impact of ribavirin dosage in chronic hepatitis C patients treated with simeprevir, pegylated interferon plus ribavirin combination therapy.

    Science.gov (United States)

    Tahata, Yuki; Hiramatsu, Naoki; Oze, Tsugiko; Urabe, Ayako; Morishita, Naoki; Yamada, Ryoko; Yakushijin, Takayuki; Hosui, Atsushi; Oshita, Masahide; Kaneko, Akira; Hagiwara, Hideki; Mita, Eiji; Ito, Toshifumi; Yamada, Yukinori; Inada, Masami; Katayama, Kazuhiro; Tamura, Shinji; Imai, Yasuharu; Hikita, Hayato; Sakamori, Ryotaro; Yoshida, Yuichi; Tatsumi, Tomohide; Hayashi, Norio; Takehara, Tetsuo

    2016-10-01

    The factors associated with sustained virologic response (SVR) in chronic hepatitis C (CH-C) genotype 1 patients treated with simeprevir (SMV), pegylated interferon (Peg-IFN) plus ribavirin (RBV) triple therapy have not been fully investigated. Two hundred and twenty-nine treatment-naïve CH-C patients treated with SMV triple therapy were enrolled in this study. The overall SVR rate was 87% in per-protocol analysis. In multivariate analysis, the interleukin (IL) 28B genotype (rs8099917, TT vs. non-TT, odds ratio [OR]: 0.044, P = 0.001) and RBV dose (< 10/10-12/ ≥ 12 mg/kg/day, OR: 4.513, P = 0.041) were significant factors associated with SVR. In patients with the IL28B non-TT genotype, RBV dose affected SVR dose-dependently in stratified analysis of RBV dose (P = 0.015); it was 44% (8/18) for patients administered <10 mg/kg/day of RBV, 78% (14/18) for those administered 10-12 mg/kg/day of RBV, and 100% (3/3) for those administered ≥12 mg/kg/day of RBV, whereas in patients with the IL28B TT genotype, a significant correlation between SVR and RBV dose was not observed (P = 0.229). Regarding RBV dose reduction of less than 10 mg/kg/day, the inosine triphosphate pyrophosphatase (ITPA) genotype (rs1127354, CC vs. non-CC, OR: 0.239, P = 0.003) and age (by 1 y.o., OR: 1.084, P = 0.002) were significant independent factors. RBV dosage affected SVR dose-dependently in patients with the IL28B non-TT genotype treated with SMV triple therapy. Special attention to anemia progression and RBV dosage should be paid to aged patients with the ITPA CC genotype. J. Med. Virol. 88:1776-1784, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991414

  2. When does ALS start? ADAR2-GluA2 hypothesis for the etiology of sporadic ALS

    Directory of Open Access Journals (Sweden)

    Takuto eHideyama

    2011-11-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is the most common adult-onset motor neuron disease. More than 90% of ALS cases are sporadic, and the majority of sporadic ALS patients do not carry mutations in genes causative of familial ALS; therefore, investigation specifically targeting sporadic ALS is needed to discover the pathogenesis. The motor neurons of sporadic ALS patients express unedited GluA2 mRNA at the Q/R site in a disease-specific and motor neuron-selective manner. GluA2 is a subunit of the AMPA receptor, and it has a regulatory role in the Ca2+-permeability of the AMPA receptor after the genomic Q codon is replaced with the R codon in mRNA by adenosine-inosine conversion, which is mediated by adenosine deaminase acting on RNA 2 (ADAR2. Therefore, ADAR2 activity may not be sufficient to edit all GluA2 mRNA expressed in the motor neurons of ALS patients. To investigate whether deficient ADAR2 activity plays pathogenic roles in sporadic ALS, we generated genetically modified mice (AR2 in which the ADAR2 gene was conditionally knocked out in the motor neurons. AR2 mice showed an ALS-like phenotype with the death of ADAR2-lacking motor neurons. Notably, the motor neurons deficient in ADAR2 survived when they expressed only edited GluA2 in AR2/GluR-BR/R (AR2res mice, in which the endogenous GluA2 alleles were replaced by the GluR-BR allele that encoded edited GluA2. In heterozygous AR2 mice with only one ADAR2 allele, approximately 20% of the spinal motor neurons expressed unedited GluA2 and underwent degeneration, indicating that half-normal ADAR2 activity is not sufficient to edit all GluA2 expressed in motor neurons. It is likely therefore that the expression of unedited GluA2 causes the death of motor neurons in sporadic ALS. We hypothesize that a progressive downregulation of ADAR2 activity plays a critical role in the pathogenesis of sporadic ALS and that the pathological process commences when motor neurons express unedited GluA2.

  3. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

    Directory of Open Access Journals (Sweden)

    Deforce Dieter

    2006-08-01

    Full Text Available Abstract Background Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence gene family can be performed using quantitative PCR (qPCR. In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans biofilms, using the geNorm Visual Basic Application (VBA for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. Results The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable as follows: ACT1 (β-actin/PMA1 (adenosine triphosphatase, RIP (ubiquinol cytochrome-c reductase complex component, RPP2B (cytosolic ribosomal acidic protein P2B, LSC2 (succinyl-CoA synthetase β-subunit fragment, IMH3 (inosine-5'-monophosphate dehydrogenase fragment, CPA1 (carbamoyl-phosphate synthethase small subunit and GAPDH (glyceraldehyde-3-phosphate dehydrogenase. Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control, for 24 hours. Conclusion In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2 for normalization, when analysing differences in gene expression levels between C. albicans biofilm

  4. Dipeptidyl peptidase IV inhibition potentiates amino acid- and bile acid-induced bicarbonate secretion in rat duodenum.

    Science.gov (United States)

    Inoue, Takuya; Wang, Joon-Ho; Higashiyama, Masaaki; Rudenkyy, Sergiy; Higuchi, Kazuhide; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2012-10-01

    Intestinal endocrine cells release gut hormones, including glucagon-like peptides (GLPs), in response to luminal nutrients. Luminal L-glutamate (L-Glu) and 5'-inosine monophosphate (IMP) synergistically increases duodenal HCO3- secretion via GLP-2 release. Since L cells express the bile acid receptor TGR5 and dipeptidyl peptidase (DPP) IV rapidly degrades GLPs, we hypothesized that luminal amino acids or bile acids stimulate duodenal HCO3- secretion via GLP-2 release, which is enhanced by DPPIV inhibition. We measured HCO3- secretion with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or intravenous (iv) injection (3 μmol/kg) of the DPPIV inhibitor NVP728. The loop was also perfused with a selective TGR5 agonist betulinic acid (BTA, 10 μM) or the non-bile acid type TGR5 agonist 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide (CCDC; 10 μM). DPPIV activity visualized by use of the fluorogenic substrate was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP728 (0.1 mM). An iv injection of NVP728 enhanced L-Glu/IMP-induced HCO3- secretion, whereas luminal perfusion of NVP728 had no effect. BTA or CCDC had little effect on HCO3- secretion, whereas NVP728 iv markedly enhanced BTA- or CCDC-induced HCO3- secretion, the effects inhibited by a GLP-2 receptor antagonist. Coperfusion of the TGR5 agonist enhanced L-Glu/IMP-induced HCO3- secretion with the enhanced GLP-2 release, suggesting that TGR5 activation amplifies nutrient sensing signals. DPPIV inhibition potentiated luminal L-Glu/IMP-induced and TGR5 agonist-induced HCO3- secretion via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal HCO3- secretion.

  5. Clinical utility of pharmacogenomics in the management of hepatitis C

    Directory of Open Access Journals (Sweden)

    Trinks J

    2014-10-01

    elaborating safer and more effective therapeutic strategies specifically designed for each patient. In conclusion, the individualization of treatment regimens for patients with hepatitis C, that may lead to a universal cure in future years, is becoming a reality due to recent developments in biomarker and genomic medicine. In light of these advances, we review the scientific evidence and clinical implications of recent findings related to host genetic factors in the management of HCV infection. Keywords: hepatitis C virus, pharmacogenomics, interleukin 28B, inosine triphosphatase

  6. Topographic organizations of taste-responsive neurons in the parabrachial nucleus of C57BL/6J mice: An electrophysiological mapping study.

    Science.gov (United States)

    Tokita, K; Boughter, J D

    2016-03-01

    The activities of 178 taste-responsive neurons were recorded extracellularly from the parabrachial nucleus (PbN) in the anesthetized C57BL/6J mouse. Taste stimuli included those representative of five basic taste qualities, sweet, salty, sour, bitter and umami. Umami synergism was represented by all sucrose-best and sweet-sensitive sodium chloride-best neurons. Mediolaterally the PbN was divided into medial, brachium conjunctivum (BC) and lateral subdivisions while rostrocaudally the PbN was divided into rostral and caudal subdivisions for mapping and reconstruction of recording sites. Neurons in the medial and BC subdivisions had a significantly greater magnitude of response to sucrose and to the mixture of monopotassium glutamate and inosine monophosphate than those found in the lateral subdivision. In contrast, neurons in the lateral subdivision possessed a more robust response to quinine hydrochloride. Rostrocaudally no difference was found in the mean magnitude of response. Analysis on the distribution pattern of neuron types classified by their best stimulus revealed that the proportion of neuron types in the medial vs. lateral and BC vs. lateral subdivisions was significantly different, with a greater amount of sucrose-best neurons found medially and within the BC, and a greater amount of sodium chloride-, citric acid- and quinine hydrochloride-best neurons found laterally. There was no significant difference in the neuron-type distribution between rostral and caudal PbN. We also assessed breadth of tuning in these neurons by calculating entropy (H) and noise-to-signal (N/S) ratio. The mean N/S ratio of all neurons (0.43) was significantly lower than that of H value (0.64). Neurons in the caudal PbN had a significantly higher H value than in the rostral PbN. In contrast, mean N/S ratios were not different both mediolaterally and rostrocaudally. These results suggest that although there is overlap in taste quality representation in the mouse PbN, taste

  7. Screening of serum uric acid-lowering Lactic acid bacteria and its effect on hyperuricemia in rat models%降血尿酸乳酸菌筛选及其对高尿酸血症模型大鼠作用研究

    Institute of Scientific and Technical Information of China (English)

    杨殿斌; 袁杰利

    2013-01-01

    目的 提出利用具特殊活性乳酸菌来预防、治疗高尿酸血症方法,筛选具有较高降血尿酸活性的乳酸菌.方法 基于RP-HPLC方法,通过检测培养液培养前后肌苷、鸟苷含量变化,筛选出具有最快核苷分解速率的乳酸菌株作为候选菌株.并将其施用于高尿酸血症模型大鼠,观察对模型大鼠血尿酸含量影响.结果 获得具有最佳核苷分解速率的菌株DM9218.动物实验结果表明DM9218干预组的血尿酸浓度(219.25±21.98) μmol/L明显低于模型组(311.75±27.07) μmol/L(P<0.05).结论 菌株DM9218具有最快的核苷分解速率,对高尿酸血症大鼠具有明显的降血尿酸作用.菌株DM9218为植物乳杆菌(Lactobacillus plantarum).%Objective To propose a method for the prevention and treatment of hyperuricemia by Lactic acid bacteria (LAB) with specific activity, and screen LAB strains with high serum uric acid-iowering activity. Methods Based on RP-HPLC method, the changes of inosine and guanosine contents in the culture medium before and after cultivation were determined; The LAB strains which presented the fastest rates of decomposition of nucleosides were selected as the candidate strains, and administered to the model rats. The effect of the strains on the serum uric acid level of the rats were observed. Results The strain DM9218 with an optimal decomposition rate of nucleo-side was obtained. Animal experimental results showed that the serum uric acid level of DM9218 intervention group [ (219.25 ± 21.98) μmol/L] was obviously lower than that of the model group [(311.75 ± 27.07) μmol/L] (P<0.05). Conclusion DM9218 has the fastest decomposition rate of nucleosides and obvious effect of lowering serum uric acid in hyperuricemia rats. DM9218 is identified as Lactobacillus plantarum.

  8. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  9. Small RNA and A-to-I Editing in Autism Spectrum Disorders

    Science.gov (United States)

    Eran, Alal

    One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I receding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated A-to-I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism

  10. 云南金钱槭茎化学成分%Chemical Constituents from the Stems of Dipteronia dyeriana Henry

    Institute of Scientific and Technical Information of China (English)

    郭蓉; 王跃虎; 石亚娜; 李兴玉; 李文昌; 龙春林

    2012-01-01

    对云南金钱槭枝条部分的化学成分进行了研究,从其乙醇提取物中共分离鉴定了16个化合物,通过波谱学方法鉴定为:erythro-4,7,9-三羟基-3,3’-二甲氧基-8-O-4'-新木脂素-9’-O-β-D-吡喃葡萄糖苷(1),Hyuganoside Ⅲa (2),Hyuganoside Ⅲb (3),erythro-Buddlenol B (4),erythro-7’,8’-Didehydrobuddlenol B(5),(±)-丁香脂素(6),臭矢菜素A(7),柑橘苷A(8),(4R)-p-薄荷-1-烯-7,8-二醇7-O-β-D-吡喃葡萄糖苷(9),2-甲氧基-3-(3-吲哚基)丙酸(10),肌苷(11),Tachioside (12),Isotachioside (13),3-O-(β-D-吡喃葡萄糖基)-1-(3,5-二甲氧基-4-羟基苯基)-1-丙酮(14),反式异松柏苷(15),4-[(E)-3-乙氧基-1-丙烯基]-2-甲氧基苯酚(16).其中化合物5为新的倍半木脂素,其余化合物均首次从该属植物中分离得到.%Sixteen compounds were isolated from the EtOH extracts of the branches of Dipteronia dyeriana. Based on spectroscopic methods, they were identified as erythro-4, 7,9-trihydroxy -3,3 '-dimethoxy-8-O-4'-neolignan -9'-O-β-D-glucopyranoside (1) ,hyuganoside IIIa (2) ,hyuganoside IIIb (3) , erythro-buddlenol B (4) ,erythro-7' ,8'-didehydro-buddlenol B (5) ,( ± ) -syringaresinol (6) , cleomiscosin A (7) ,citroside A (8) , (4R)-p-menth-l-ene-7,8-diol 7-0-β-D-glucopyranoside (9) ,2-methoxy-3-(3-indolyl) propanoic acid (10),inosine ( 11) , tachioside ( 12 ) , isotachioside (13) ,3-O-(β-D-glucopyranosyl)-l-(3,5-dimethoxy-4-hydroxyphenyl) -1-propanone (14) ,trans-isoconiferin (15) and 4-[(E)-3-ethoxyprop-l-enyl]-2-methoxyphenol (16). Compound 5 was a new sesquilignan, while other compounds were obtained from the genus for the first time.

  11. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

    Science.gov (United States)

    Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

    2014-09-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

  12. 特异茶树资源生物碱测定及相关基因表达分析%Determination of Alkaloid and Analysis of Gene Expression in Peculiar Tea Plant

    Institute of Scientific and Technical Information of China (English)

    夏丽飞; 陈林波; 梁名志; 邓威威; 田易萍; 刘本英; 刘军

    2013-01-01

    The component of caffeine and theobromine of five tea plants was determined by high performance liquid chromatography.The results showed that the theobromine content was low,but caffeine content was high in the same tea plant.The theobromine was accumulated in the tea resource of low caffeine content.The expression level of sAMS and TIDH were nearly affected by the change of caffeine content,according to the differences of expression level of S-adenosylmethionine Synthetase gene(sAMS),Inosine 5'-monophosphate Dehydroge-nase (TIDH) and Caffeine Synthase gene (TCSI) analyzed by RT-PCR in different tea species and the date of caffeine content.TCSI could be expressed whatever in the high caffeine content material or in the low one,but its expression level was higher in the high caffeine content material.The reason could be that the Caffeine Synthase of vitality was low in the low caffeine content tea plant,or other regulation mechanisms could lead to the blockage of biosynthesis of caffeine and the accumulation of theobromine.%利用高效液相色谱对5份茶树资源中的咖啡碱、可可碱组分进行测定,发现咖啡碱含量高的茶树资源可可碱含量低,而咖啡碱含量低茶树资源的可可碱被积累.利用RT-PCR分析了S-腺苷甲硫氨酸合成酶基因(sAMS)、次黄嘌呤苷-5-单酸脱氢酶基因(TIDH)、咖啡碱合成酶基因(TCSI)在咖啡碱含量不同的茶树品种间的表达水平差异,并结合咖啡碱含量数据发现,sAMS和TIDH的表达水平对咖啡碱含量变化影响不明显.TCSI在高咖啡碱和低咖啡碱材料中均有表达,且TCSI在高咖啡碱材料中的表达比在低咖啡碱材料中高.推测可能是低咖啡碱茶树资源中咖啡碱合成酶活性低或者存在其他的调控机制,导致咖啡碱的生物合成受阻,使得可可碱被积累.

  13. Mycophenolate pharmacokinetics and pharmacodynamics in belatacept treated renal allograft recipients – a pilot study

    Directory of Open Access Journals (Sweden)

    Stenstrøm Jean

    2009-07-01

    Full Text Available Abstract Background Mycophenolic acid (MPA is widely used as part of immunosuppressive regimens following allograft transplantation. The large pharmacokinetic (PK and pharmacodynamic (PD variability and narrow therapeutic range of MPA provide a potential for therapeutic drug monitoring. The objective of this pilot study was to investigate the MPA PK and PD relation in combination with belatacept (2nd generation CTLA4-Ig or cyclosporine (CsA. Methods Seven renal allograft recipients were randomized to either belatacept (n = 4 or cyclosporine (n = 3 based immunosuppression. Samples for MPA PK and PD evaluations were collected predose and at 1, 2 and 13 weeks posttransplant. Plasma concentrations of MPA were determined by HPLC-UV. Activity of inosine monophosphate dehydrogenase (IMPDH and the expressions of two IMPDH isoforms were measured in CD4+ cells by HPLC-UV and real-time reverse-transcription PCR, respectively. Subsets of T cells were characterized by flow cytometry. Results The MPA exposure tended to be higher among belatacept patients than in CsA patients at week 1 (P = 0.057. Further, MPA concentrations (AUC0–9 h and C0 increased with time in both groups and were higher at week 13 than at week 2 (P = 0.031, n = 6. In contrast to the postdose reductions of IMPDH activity observed early posttransplant, IMPDH activity within both treatment groups was elevated throughout the dosing interval at week 13. Transient postdose increments were also observed for IMPDH1 expression, starting at week 1. Higher MPA exposure was associated with larger elevations of IMPDH1 (r = 0.81, P = 0.023, n = 7 for MPA and IMPDH1 AUC0–9 h at week 1. The maximum IMPDH1 expression was 52 (13–177% higher at week 13 compared to week 1 (P = 0.031, n = 6. One patient showed lower MPA exposure with time and did neither display elevations of IMPDH activity nor IMPDH1 expression. No difference was observed in T cell subsets between treatment groups. Conclusion The

  14. Alterações bioquímicas post-mortem de matrinxã Brycon cephalus (Günther, 1869 procedente da piscicultura, mantido em gelo Post-mortem biochemical alterations in aquacultured matrinxã fish Brycon cephalus (Günther, 1869 when stored on ice

    Directory of Open Access Journals (Sweden)

    Gilvan Machado Batista

    2004-12-01

    be inosine (HxR forming, in experimental conditions. The K value pointed out that matrinxã specimens remained "quite fresh" up to 16 days storage time on ice corroborating the sensory, tasting evaluation.

  15. Charge transfer from 2-aminopurine radical cation and radical anion to nucleobases: A pulse radiolysis study

    Energy Technology Data Exchange (ETDEWEB)

    Manoj, P. [School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686 560, Kerala (India); Mohan, H. [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Mittal, J.P. [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Manoj, V.M. [School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686 560, Kerala (India); Aravindakumar, C.T. [School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686 560, Kerala (India)], E-mail: CT-Aravindakumar@rocketmail.com

    2007-01-08

    Pulse radiolysis study has been carried out to investigate the properties of the radical cation of 2-aminopurine (2AP) and the probable charge transfer from the radical cation and radical anion of 2AP to natural nucleobases in aqueous medium. The radical cation of 2AP was produced by the reaction of sulfate radical anion (SO{sub 4}{sup dot-}). The time resolved absorption spectra obtained by the reaction of SO{sub 4}{sup dot-} with 2AP at neutral pH have two distinct maxima at 380 and 470nm and is assigned to the formation of a neutral radical of the form 2AP-N{sup 2}(-H){sup dot} (k{sub 2}=4.7x10{sup 9}dm{sup 3}mol{sup -1}s{sup -1} at pH 7). This neutral radical is formed from the deprotonation reaction of a very short-lived radical cation of 2AP. The transient absorption spectra recorded at pH 10.2 have two distinct maxima at 400 and 480nm and is assigned to the formation of a nitrogen centered radical (2AP-N(9){sup dot}). As the hole transport from 2AP to guanine is a highly probable process, the reaction of SO{sub 4}{sup dot-} is carried out in the presence of guanosine, adenosine and inosine. The spectrum obtained in the presence of guanosine was significantly different from that in the absence and it showed prominent absorption maxima at 380 and 470nm, and a weak broad maximum centered around 625nm which match well with the reported spectrum of a neutral guanine radical (G(-H){sup dot}). The electron transfer reaction from the radical anion of 2AP to thymine (T), cytidine (Cyd) and uridine (Urd) was also investigated at neutral pH. Among the three pyrimidines, only the transient spectrum in the presence of T gave a significant difference from the spectral features of the electron adduct of 2AP, which showed a prominent absorption maximum at 340nm and this spectrum is similar to the electron adduct spectrum of T. The preferential reduction of thymine by 2AP{sup dot-} and the oxidation of guanosine by 2AP{sup dot+} clearly follow the oxidation

  16. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    Science.gov (United States)

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste. PMID:26701297

  17. Dipeptidyl peptidase IV inhibition potentiates amino acid- and bile acid-induced bicarbonate secretion in rat duodenum.

    Science.gov (United States)

    Inoue, Takuya; Wang, Joon-Ho; Higashiyama, Masaaki; Rudenkyy, Sergiy; Higuchi, Kazuhide; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2012-10-01

    Intestinal endocrine cells release gut hormones, including glucagon-like peptides (GLPs), in response to luminal nutrients. Luminal L-glutamate (L-Glu) and 5'-inosine monophosphate (IMP) synergistically increases duodenal HCO3- secretion via GLP-2 release. Since L cells express the bile acid receptor TGR5 and dipeptidyl peptidase (DPP) IV rapidly degrades GLPs, we hypothesized that luminal amino acids or bile acids stimulate duodenal HCO3- secretion via GLP-2 release, which is enhanced by DPPIV inhibition. We measured HCO3- secretion with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or intravenous (iv) injection (3 μmol/kg) of the DPPIV inhibitor NVP728. The loop was also perfused with a selective TGR5 agonist betulinic acid (BTA, 10 μM) or the non-bile acid type TGR5 agonist 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide (CCDC; 10 μM). DPPIV activity visualized by use of the fluorogenic substrate was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP728 (0.1 mM). An iv injection of NVP728 enhanced L-Glu/IMP-induced HCO3- secretion, whereas luminal perfusion of NVP728 had no effect. BTA or CCDC had little effect on HCO3- secretion, whereas NVP728 iv markedly enhanced BTA- or CCDC-induced HCO3- secretion, the effects inhibited by a GLP-2 receptor antagonist. Coperfusion of the TGR5 agonist enhanced L-Glu/IMP-induced HCO3- secretion with the enhanced GLP-2 release, suggesting that TGR5 activation amplifies nutrient sensing signals. DPPIV inhibition potentiated luminal L-Glu/IMP-induced and TGR5 agonist-induced HCO3- secretion via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal HCO3- secretion. PMID:22821947

  18. 补肾养血法治疗狼疮肾炎环磷酰胺冲击疗法后引起的白细胞减少症临床研究%Clinical Study on the Reinforcing Kidney and Nourishing Blood Method in Treating Leukopenia Caused by Cyclophosphamide Pulse Therapy for Lupus Nephritis

    Institute of Scientific and Technical Information of China (English)

    程淑红; 宋纯东

    2011-01-01

    Objective:To observe the clinical effects of reinforcing kidney and nourishing blood method in treating leukopenia caused by cyclophosphamide pulse therapy for lupus nephritis. Methods: 30 cases of lupus nephritis were randomly divided into treatment group with 16 cases and control group with 14 cases. The treatment group was treated with kidney reinforcing and blood nourishing herbs and the control group was treated with Leucogen,Vitamin B4 tablets and inosine tablets. Both groups were treated for two weeks as a course of treatment. Results:The total effective rate of the treatment group was 93. 7% and that of the control group was 71% ,so the curative effect of the treatment group was superior to that of the control group( P < 0. 01). Conclusion; Kidney reinforcing and blood nourishing herbs have excellent therapeutic effect in treating leukopenia caused by cyclophosphamide pulse therapy for lupus nephritis.%目的:观察补肾养血法治疗狼疮肾炎环磷酰胺冲击疗法后引起的白细胞减少症的临床疗效.方法:将30例患者随机分为治疗组16例与对照组14例,治疗组给予补肾养血中药治疗,对照组口服刺可君、维生素B4片、肌苷片,两组均以14 d为1疗程.结果:治疗组有效率93.7%,对照组有效率71.0%,治疗组疗效明显优于对照组(P<0.01).结论:补肾养血中药治疗狼疮肾炎环磷酰胺冲击疗法后引起的白细胞减少症有较好的临床疗效.

  19. Mizoribine versus mycophenolate mofetil or intravenous cyclophosphamide for induction treatment of active lupus nephritis

    Institute of Scientific and Technical Information of China (English)

    Feng Xuebing; Gu Fei; Chen Weiwei; Liu Yan; Wei Hua; Liu Lin; Yin Songlou

    2014-01-01

    Background Lupus nephritis (LN) is one of the most serious manifestations of systemic lupus erythematosus.Although there have been substantial improvements in LN treatment over the last decade,the outcome remains unoptimistic in a considerable percentage of patients.The aim of this study was to evaluate the efficacy and safety of mizoribine (MZR),a novel selective inhibitor of inosine monophosphate dehydrogenase,as induction treatment for active LN in comparison with mycophenolate mofetil (MMF) and intravenous cyclophosphamide (CYC).Methods Ninety patients with active LN were observed.Thirty patients were given MZR orally at the dose of 300 mg every other day.Thirty patients took MMF at 2 g per day in two divided doses.Thirty patients received CYC intravenously 0.5 g every 2 weeks.Therapeutic effects and adverse events (AEs) were evaluated at the end of 24-week treatment.Oneway analysis of variance (ANOVA) followed by Dunn's test was applied to compare the difference among the groups.For comparing categorical data between two groups,X2 test was employed.Results Early responses at week 12 were achieved by 73.3%,90.0%,and 96.7% in MZR,MMF,and CYC groups,respectively.There was no significant difference in the complete remission rates (22.7%,24.0%,and 25.0%,respectively) or overall response rates (68.2%,72.0%,and 75.0%,respectively) among the three groups at week 24.The most prominent drop-down of Systemic Lupus Erythematosus Disease Activity Index scores was observed in MMF or CYC group,and the decline of health assessment questionnaire scores in MZR or MMF group was more prominent than that in the CYC group at week 12.Serum complement 3 (C3) or C4 levels were elevated in all groups after the treatments.CYC was more effective in inhibiting anti-double-stranded DNA antibody,while MZR was more effective in inhibiting antinuclear antibody.The incidences of AEs in patients treated with CYC were significantly higher than those in patients treated with MZR

  20. Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

    Science.gov (United States)

    Liu, Xiancheng; Huang, Keyi; Hou, Qunhao; Sun, Zhicheng; Wang, Binhua; Lin, Gang; Li, Dongming; Liu, Yong; Xu, Xiaowen; Hu, Chengyu

    2016-10-01

    As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional

  1. Renal Parenchyma Lithotomy by Hypothermic Renal Vascular Block%低温下原位肾动脉阻断治疗复杂性肾结石

    Institute of Scientific and Technical Information of China (English)

    乙从亮; 杨登伦; 任春凯; 朱巍; 余秋健; 陈令秋

    2014-01-01

    目的:探讨开放性手术低温条件下采用肾动脉阻断治疗复杂肾结石患者的方法以及临床疗效。方法:对32例复杂性肾结石患者采用静脉注射肌苷2.0 g,原位低温下阻断肾动脉后行肾实质切开取石术。术前经CT、KUB、IVP等检查诊断鹿角形结石或(和)多发结石。平均年龄48(19~65)岁。左肾24例,右肾8例。病史6个月~20年。结石最大直径2.5~7.0 cm,平均为3.5 cm。,术后随访。结果:本组均采用原位低温肾动脉阻断肾实质切开取石术。血流阻断时间15~40分钟,平均25分钟,术中出血量80~300 ml,平均180ml,肾实质切口大小3~5 cm,平均3.5cm,手术操作时间90~150分钟,平均110分钟。术中无大出血及血管损伤。术后并发大出血1例,再次手术缝合止血顺利出院,3例术后肉眼血尿2~3个月,经保守治疗后治愈,无并发症。结论:小切口原位低温下阻断肾动脉配合静脉注射肌苷行肾实质切开取石手术具有方法容易掌握、出血少、创伤小、肾功能损害小、手术安全等特点,是治疗复杂性肾结石安全、有效的方法之一,易于在无条件行微创手术的医院开展。%Objective:Explore the open surgery by renal artery block under the condition of low temperature treatment of complex method and clinical efficacy in patients with kidney stones .Methods:32 cases of patients with renal stones complexity by intravenous in-jection of inosine 2.0 g, block the renal artery in situ low temperature renal parenchyma incision after nephrolithotomy .Preoperative CT, the KUB, IVP, such as inspection diagnosis antlers or (and) multiple stones.The average age is 48 (19-65).The left kidney was 24 cases, 8 cases of right kidney .History was 6 months to 20 years.The stone diameter is from 2.5 to 7.0 cm, with an average of 3.5 cm. Results:All use of in situ cryogenic renal artery block the renal parenchyma

  2. Primary study on characteristics of human breast milk nucleosides in some areas of Shanghai%上海部分地区母乳核苷特征的初步研究

    Institute of Scientific and Technical Information of China (English)

    步军; 孙建华; 吴圣楣

    2015-01-01

    HPLC to yield TPAN value. Results The medium values were 100mol/L for cytidine, 47mol/L for uridine, 15mol/L for guanosine, and 21mol/L for adenosine, and they were found in all of the samples. Less than half of milk samples (32/71) contained inosine with low concentration, and the accumulation of the above four nucleotides were considered as TPAN with medium value of 193mol/L. There were differences in the value of uridine and guanosine between colostrum and mature milk (Z value was -2. 64 and -2. 13, respectively, both P<0. 05), and the TPAN value of colostrum was higher than mature milk (Z= -2. 09,P<0. 05). Conclusion The average TPAN level and the composition of nucleosides of breast milk from lactating women in Shanghai are similar to these in population in European, American and other Asian measured by the same methods. Cytidine is the predominant nucleoside in human milk, and inosine is found only in some samples.

  3. 丹曲林对大鼠缺血-再灌注心肌嘌呤代谢的影响%Effect of dantrolene on purine metabolism in the rats with myocardial ischemia-reperfusion

    Institute of Scientific and Technical Information of China (English)

    赵晓丽; 王丽艳; 郭彦青; 曹颖; 于公元; 李海东

    2011-01-01

    Objective To investigate the effect of dantrolene on purine metabolism in rats with myocardial ischemia - reperfusion. Methods Twenty - four male rats were randomly divided into three groups: control group, ischemia - reperfusion group and dantrolene - treated group. The hearts were perfused in a Langendorff model system. Hearts were perfused with Krebs for 30 min as a preischemic control. For ischemia - reperfusion measurement, hearts were subjected to further 30 min period of myocardial ischemia and 60 min of reperfusion. The dantrolene - treated group was subjected to perfusion in the presence of the 5 μmol/L dantrolene before ischemia and reperfusion. Tetrazolium chloride ( TTC)staining, lactate dehydrogenase ( LDH) release were used to evaluate the tissue injury. The levels of adenosine, inosine, hypoxanthine, xanthine, uric acid, and the activity of four enzymes in purine metabolism: 5' - nucleotidase ( 5' - NT ) , adenosine deaminase ( ADA ) , purine nucleoside phosphorylase ( PNP) , and xanthine oxidase ( XO) were measured by high - performance liquid chromatography ( HPLC) . Results Pretreaed with 5 μmol/L dantrolene, the myocardial infarction size was decreased by 12.6% , LDH release was decreased by 39.7% . The levels of purine metabolites in dantrolene - treated group were significantly decreased as compared with ischemia - reperfusion group ( P<0.05) . The content of adenosine, inosine, hypoxanthine, xanthine and uric acid was decreased to 69.1%, 77.9% , 80.5% , 74.6% and 87.3% of ischemia - reperfusion myocardium, respectively.The activity of 5' - NT, ADA, and XO in dantrolene - treated group was significantly increased ( P<0.01 ), and the activity of PNP was significantly reduced as compared with ischemia - reperfusion group (P<0.05 ) . Conclusion Pretreatment with dantrolene preserves the intracellular enzyme activity,significantly improves energy metabolism and produces a cardioprotective effect against ischemia -reperfusion

  4. 玉山黑猪肉营养特性分析%Nutritional Characteristics of Meat from Yushan Black Pig

    Institute of Scientific and Technical Information of China (English)

    李瑞丽; 胡丽芳; 唐书升; 陆文英; 李小光; 彭贵福; 罗林广

    2013-01-01

    研究在优质肉生产中科学利用玉山黑猪这一优良地方猪种资源.对9头玉山黑猪的背最长肌中肌内脂肪、氨基酸、肌苷酸以及脂肪酸和背脂中的脂肪酸,从营养学角度进行分析评价.结果表明:玉山黑猪背最长肌中17种氨基酸总含量为19.16%,其中鲜味氨基酸和必需氨基酸占总氨基酸的比例分别为38.96%和44.78%,具有较浓的鲜味和较高的蛋白质“生物效价”,而且氨基酸评分结果表明玉山黑猪的必需氨基酸比例适宜,有利于人体吸收;玉山黑猪肉中肌内脂肪、肌苷酸含量分别为6.62%、2.99%,是其肉质鲜嫩多汁的物质基础;此外,玉山黑猪背最长肌和背脂的不饱和脂肪酸的比例都较高,其背最长肌亚油酸和亚麻酸的含量分别为12.73%和0.86%,表明玉山黑猪脂肪酸的营养价值比较高.%The aim of this study was nutritional assessment of intramuscular fat (IMF),amino acid,inosinic acid (IMP),fatty acid profile ofLongissimus dorsi muscles and fatty acid profile of back fat from Yushan black pigs (n =9).The results showed that,17 kinds of amino acids were together 19.16% in Longissimus dorsi muscles with delicious amino acids and essential amino acids accounting for 38.96% and 44.78%,respectively.Moreover,strong umami taste and high protein bioavailability were found.Amino acid scoring showed good balance of essential amino acids and good absorbability in the human body.The contents of IMF and IMP were 6.62% and 2.99%,respectively,which contibuted to desired tenderness and juiciness of pork from Yushan black pigs.Furthremore,both Longissimus dorsi muscles and back fat from Yushan black pigs indicated high precentages of unsaturated fatty acids; linoleic acid and linolenic acid were 12.73% and 0.86% in Longissimus dorsi muscles,respectively,suggesting high nutirtional value.

  5. Screening for the Pathogenic Mutations in a Autosomal Dominant Retinitis Pigmentosa Pedigree%一个常染色体显性遗传视网膜色素变性家系突变基因筛查

    Institute of Scientific and Technical Information of China (English)

    王飞; 周新; 宋贵波; 丁锋; 王春芳

    2010-01-01

    目的 对一个四代常染色体显性遗传视网膜色素变性家系进行突变基因的筛查.方法 选取了常见的11个视网膜色素变性(retinitis pigmentosa,RP)相关致病基因:视紫红质(rhodopsin,RHO,RP4) 基因、视网膜变性慢蛋白(peripherin 2/retinal degeneration slow PRPH2/RDS,RP7) 基因、视网膜色素变性1 (retinitis pigmentosa 1,RP1) 基因、前体mRNA处理因子31(pre-mRNA processing factor 31,PRPF31,RP11) 基因、次黄苷单磷酸脱氢酶1型(inosine monophosphate dehydrogenase 1,IMPDH1,RP10) 基因、视杆外节蛋白1 (rod outer segment protein 1,ROM1) 基因、神经视网膜亮氨酸拉链(neural retina leucine zipper,NRL,RP27) 基因、Pim1激酶相关蛋白1(pim1-kinase associated protein 1,PAP1,RP9) 基因、视锥-视杆同源盒(cone-rod homeobox-containing,CRX)基因、前体mRNA处理因子3(pre-mRNA processing factor 3,PRPF3,RP18) 基因、前体mRNA处理因子8(pre-mRNA processing factor 8,PRPF8,RP13) 基因.通过聚合酶链式反应扩增这11个RP相关致病基因的外显子和邻近拼接位点,扩增产物进行正、反双向测序.结果 ①对常见的11个RP相关致病基因的64个片段的研究显示,除了基因多态性和内含子的变异外,未发现与疾病有关的外显子或邻近拼接位点的突变.②首次在中国人种中发现RP9基因的c.629A→G,(Lys210Arg)变异.结论 该文设计研究的突变基因未被检测到,表明此家系在11个RP相关致病基因的外显子和邻近拼接位点无基因突变.

  6. Comparative Analysis of Meat Quality and Storage Traits of Feeding Pigs and Hogwash Pigs%饲料猪和泔水猪肉质及贮藏特性比较研究

    Institute of Scientific and Technical Information of China (English)

    李建鲲; 李瑾瑜; 王子荣; 朱民望

    2012-01-01

    [目的]试验以同一来源的杜长大(DLY)为研究对象,对其分别进行常规饲料和泔水饲养研究其肉质和贮藏特性差异.[方法]采用单因素方差分析方法.[结果]饲料猪和泔水猪L*值、水分含量和肌苷酸含量存在显著差异(P<0.05);背膘厚、肌内脂肪、b*值和剪切力存在极显著差异(P<0.01);泔水猪肝脏、肾脏铜和铅含量较饲料猪高.泔水猪肉和饲料猪肉在2℃条件下贮藏,挥发性盐基氮(TVB -N)的含量和菌落总数随贮藏时间呈增长趋势;pH值随贮藏时间呈先下降后上升趋势,在24h时pH值降到最低;酸度氧化力系数随贮藏时间呈先上升后下降趋势,在第3d时酸度氧化力系数达到最高值.[结论]饲料猪与泔水猪相比,泔水猪在肉色、嫩度、风味物质含量方面具有一定的优势,但在食品安全方面存在风险.%[ Objective ] DLY that has the identical genetic traits was used as research object in this experiment. They were fed with feeding stuffs and hogwash respectively in order to study the difference in meat quality and storage traits. [Method] One-way ANOVA was used in this experiment. [Result] The L* value, total moisture and inosine acid content were significant (P <0.05) , back - fat thickness, intramuscular fat, b * value and shearing force were extremely significant (P < 0. 01) between feeding pigs and hogwash pigs. The content of copper and plumbum in liver and kidney of hogwash pigs is higher than that of feeding pigs. The trend of TVB - N and microbiology content of the meat stored at 2℃ was increasing with the storage time. When meat was stored to the twelfth day, it was considered as deteriorated meat. The trend of pH value was down first and then up with the storage time, and pH value reduced to the lowest after 24 hours. The trend of acidity versus oxidation force was up and then down with the storage time. Acidity versus oxidation force reached the highest value when the meat

  7. HCV genotyping and host genotyping: what role will they play in the antiviral treatment%丙型肝炎病毒基因型及其宿主基因型的检测及临床意义

    Institute of Scientific and Technical Information of China (English)

    王剑; 杨瑞锋; 魏来

    2012-01-01

    Persistent infection of hepatitis C virus (HCV) remains as a worldwide threat to public health,which involves a complex interaction between virus- and host related factors.HCV is classified as six genotypes and many subtypes according to the sequence heterogenecity.HCV genotype should be determined prior to treatment initiation since it plays a key role in selection of therapeutic regimen for chronic hepatitis C.Development of the antiviral treatment with protease inhibitor in combination with pegylated IFN-α and ribavirin requires the accurate determination of subtypes,e.g. 1a and 1b,as well.Genotyping methods based on sequence analysis, reverse hybridization or real-time PCR have been developed and evaluated.Some issues,however,should be settled to standardize the utility and result interpretation of these methods.More recently,host genotypes of IL28B have been found to be closely associated with HCV spontaneous clearance and the response to antiviral therapy.Moreover,polymorphisms in inosine triphosphate pyrophosphatase gene affect ribavirin-induced anemia.Therefore, host genotyping will be beneficial in predicting the outcome of chronic hepatitis C and monitoring the drug-induced adverse events.%丙型肝炎病毒(HCV)持续感染导致的慢性丙型肝炎(CHC)是一个全球性的公共卫生问题.HCV感染是病毒因素与宿主因素相互作用的复杂的过程,HCV基因型与宿主基因型均在其中发挥重要作用.HCV分为6个基因型和多个亚型,不同基因型对聚乙二醇干扰素α联合利巴韦林的标准化抗病毒治疗的应答不同,因此治疗前需要确定HCV基因型以制定相应的治疗方案.近几年随着HCV蛋白酶抑制剂等特异性靶向治疗药物的研究进展,要求基因型的检测要精确到“亚型”的水平.基于测序法、反相杂交或实时PCR的HCV分型方法已经被广泛应用,但必须解决几个问题以实现方法学的标准化.近两年来,宿主白细胞介素28B的基因型

  8. Analysis of the Correlation of PURH Genes Involved in De Novo Purine Biosynthesis on IMP Content in Suqin Silky%肌苷酸合成酶系PURH基因对苏禽乌骨鸡胸肌肌苷酸含量的关联分析

    Institute of Scientific and Technical Information of China (English)

    张学余; 束婧婷; 苏一军; 李国辉; 屠云洁

    2012-01-01

    This experiment was designed to'study the genetic effect of polymorpic loci in PURH on breast muscle inosine monophosphate acid (IMP) content in Suqin Silky. The polymorphism sites in exon 9 and exon 16 of PURH on Suqin Silky were identified by PCR-SSCP. Association analysis between the polymorphic sites and IMP contents in muscles of 90 days were tested by least square means. The results showed that TT genotype birds had significant higher IMP content than TC and CC genotype birds(P<0.01), The genotype of PURH gene exon 16(T17446C) had significant genetic effect on IMP content. The IMP content of the individuals with TT genotype was 0.757 mg/g higher than that of the individuals with CT genotype. The IMP content of the individuals with TT genotype was 1.083 mg/g higher than that of the individuals with CC genotype,TC genotype birds also had a significant higher IMP content than CC genotype birds(P<0.05). Therefore the conclusion could the putatively drawn that the genotype could be used as the molecular genetic marker to select the chicken for meat quality trait.%在分析氨基咪唑氨甲酰核苷酸转甲酰基酶/次黄嘌呤核苷酸环水解酶基因(fused IMP cyclohydrolase gene/phosphoribosylaminoimidazolecarboxamide formyltransferase PURH)对苏禽乌骨鸡鸡群体肌肉肌苷酸含量的遗传效应.采用PCR-SSCP方法检测苏禽乌骨鸡PURH基因外显子9和外显子16多态性,分析多态位点不同基因型与90日龄胸肌肌苷酸(IMP)含量的关联,各种基因型与胸肌IMP含量的方差分析结果表明:PURH 基因外显子16中多态位点T17446C与IMP含量显著相关,TT型个体胸肌IMP含量较CT型高出0.757 mg/g,TT 型个体胸肌IMP含量较CC型高出1.083 mg/g,均达到极显著水平(P<0.01),CT型个体IMP含量也显著地高于CC型(P<0.05).因此,可以利用该基因型对鸡的肉质风味性状进行标记辅助选择.

  9. 壳聚糖对肉仔鸡肉品质的影响%Effect of chitosan on meat quality in broilers/

    Institute of Scientific and Technical Information of China (English)

    刘梅

    2011-01-01

    An experiment was conducted to study the effects of different doses of chitosan on meat quality in broilers. A total of 120, 1 d old broilers were randomly allotted to five dietary treatments (6 birds per pen with 4 pens per treatment). The experiment period was 42 d. The control birds were fed the basal diet alone, the other treatments fed the basal diet supplemented with 50, 100, 150 mg· kg-1 chitosan (CHS), respectively, the fifth treatment was fed basal diet added 6 mg· kg-1 bambermycin. The results showed that supplementing chitosan significantly increased pH value (P<0.05), decreasing wear force and drip loss (P<0.05). Chitosan did not effect the contents of dry mater, crude protein and muscle fat (P>0.05), although the muscle fat decreased lineary and the meat flavor was negatively influenced. However, there was an increasing tendency in meat inosine monophsphate(IMP) by supplementing chitosan (P<0.05), so that meat flesh status was improved. In conclusion, chitosan in the diet of broilers increased meat tenderness and meat flavor, and promoted anti-oxidation function of muscle, thereby improving the quality of meat.%试验旨在探讨壳聚糖对鸡肉品质的影响.试验选用了体重相近的健康的1日龄AA肉仔鸡(公)120只,随机分成5组,每组设4个重复,每个重复6只鸡.在各组基础饲料中分别添加0、50、100、150mg·kg-1的壳聚糖,第5组在基础日粮中添加6mg·kg-1黄霉素,试验期42d.结果表明,肉仔鸡日粮中添加不同水平的壳聚糖,鸡肉pH呈上升趋势(P<0.05),肌纤维趋于变细(P>0.05),剪切力和滴水损失下降(P<0.05),MDA的含量在100、150mg·kg-1壳聚糖的处理组也呈显著下降趋势(P<0.05).添加壳聚糖对粗脂肪含量影响差异不显著(P>0.05),虽有所下降,但肌苷酸含量在150mg·kg-1壳聚糖的处理组与对照组比较,呈显著上升趋势且差异显著(P<0.05),改善肉的鲜味.以上结果显示,肉仔鸡日粮中添加壳聚糖

  10. 土霉素生产废水生物处理装置中微生物群落结构特征解析%Microbial community structures analysis in a biological plant treating oxytetracycline production wastewate

    Institute of Scientific and Technical Information of China (English)

    邓艳芹; 杨敏; 张昱; 李栋; 刘苗苗

    2012-01-01

    抗生素对细菌具有强抑制作用,从而会影响废水生物处理系统中的微生物群落结构。在用定量PCR方法对土霉素生产废水处理装置(进水中土霉素浓度为1 662.1±248.6μg/L)中的细菌16S rRNA基因和真菌18S rRNA基因的含量进行比较的基础上,利用16S rRNA基因克隆文库方法对污泥中的细菌群落结构进行了详细解析。定量PCR结果显示,土霉素废水活性污泥中真菌(18S rRNA)/细菌(16S rRNA)基因的拷贝数比例高达1.2,明显较高于非抗生素肌苷废水活性污泥中的比例1.52×10-6,表明真菌对于土霉素废水中有机物的去除可能发挥重要作用。细菌克隆文库分析结果显示,Alpha变形菌和Beta变形菌是主要的优势菌,比例分别为23.7%和22.0%,其次是酸杆菌(17.0%)和拟杆菌(11.9%)。%As antibiotics can cause strong inhibition of bacteria,their presence will influence microbial community structure in wastewater treatment plants.The abundances of bacterial and fungal populations in an oxytetracycline-production wastewater(influent oxytetracycline 1 662.1±248.6 μg/L) were measured by quantitative real-time PCR,and bacterial community structure was examined by 16S rDNA clone library construction.The ratio of fungi/bacteria valued 1.2 in oxytetracycline-production wastewater,much higher than that(1.52×10-6) in inosine-production wastewater with no antibiotic.The results indicate that fungi may play an important role in COD removal of oxytetracycline-production wastewater treatment.Bacterial community structure analysis shows that Alphaproteobacteria(23.7%) and Betaproteobacteria(22.0%) are predominant groups,and then Acidobacteria(17.0%) and Bacteroidetes(11.9%).

  11. Study on Thermal Sterilization Stability of Monosodium Glutamate and I + G%谷氨酸钠及I+G的热杀菌稳定性

    Institute of Scientific and Technical Information of China (English)

    王向阳; 金菲; 施青红; 李刚

    2011-01-01

    Monosodium glutamate ( MSG), disodium 5'-Inosinate (IMP) and guanosine 5'-monophosphate disodium salt(GMP) are main components that enhance flavour. I + G is composed of 50% IMP and 50% GMP. Study on processing stability of MSG and I + G has food production and processing significance. This experiment mainly used HPLC to measure the content change of monosodium glutamate and I + G which were treated with different sterilization temperature, sterilization time and acidity. The results showed that when sterilization temperature was 85℃ , 100℃ , 121℃ for 30 min,monosodium glutamate lost 3.16% , 13.21% ,30. 10% respectively, and I + G lost 8. 18% , 12.91% , 22.97% respectively. When sterilization time was 5min, 15min, 30minutes at 100℃, monosodium glutamate lost 1.43% ,7.25% ,8.63% respectively, and I + G lost 6.08% ,8.78% , 10.12% respectively. When acidity declined from pH 7 to pH 3 (pH 7,pH 6,pH 5,pH 4,pH 3) at 100℃ for 15min,monosodium glutamate lost 2.2% ,10.30% , 22.4% ,39.09% ,52.50% respectively, and I+ G lost 7.59% ,9.30% ,10.60% ,12.89% ,26.33% respectively.%谷氨酸钠(MSG,谷氨酸一钠)、5’-肌苷酸二钠(IMP)和5’-鸟苷酸二钠(GMP),是主要增味成分。I+G是IMP和GMP按照1:1混合的物质。研究谷氨酸钠及I+G的热处理稳定性,在食品烹调和食品加工过程中具有重要意义。实验主要采用高效液相色谱法测定杀菌温度、杀菌时间、pH对谷氨酸钠和I+G含量的影响。实验发现:杀菌时间为30min时,经85℃,100℃,121℃杀菌,谷氨酸钠浓度分别损失3.16%,13.21%和30.10%;I+G浓度分别损失8.18%,12.91%和22.97%。在1000C下,

  12. Studies on extractive components of Channa argus%乌鳢抽出物成分的研究

    Institute of Scientific and Technical Information of China (English)

    陈舜胜; 陆颖华; 山中英明

    2006-01-01

    分析了乌鳢(Channa argus)即杀后背肉、腹肉、尾部肉、肝脏、生殖腺中ATP关联化合物、游离氨基酸、多胺、糖元及糖酵解中间代谢物、有机酸等的含量.ATP关联化合物在肌肉中的总量为7.5~8.0 μmol·g-1.ATP在背肉含量为3.9 μmol·g-1,腹肉为4.1 μmol·g-1,尾部肉为4.7 μmol·g-1.运动激烈的尾部肉ATP占63%,含量相当高.肝脏中有少量的腺苷与肌苷酸一起被检出,据此可认为ATP的分解存在两个途径.游离氨基酸总量在背肉中为436.0 mg·(100g)-1,腹肉中为405.0 mg·(100g)-1,尾部肉中为356.3 mg·(100g)-1.牛磺酸和甘氨酸为主要氨基酸,占68%~73%.丙氨酸和谷氨酸也相当高的含量检出.肌肉中的多胺检出为精胺和亚精胺,肝脏和生殖腺中有较高浓度的腐胺及亚精胺和精胺检出.即杀后糖元的量约占肌肉的0.5%,还有相当多的葡萄糖和6-磷酸葡萄糖的糖酵解中间代谢物以及其最终产物乳酸的大量检出.%In this study, ATP and its related compounds, free amino acids, polyamines and monoamines, glycogen and glycolytic intermediates and organic acid were analyzed in the dorsal, ventral and caudal muscles of snakehead Channa argus together with the liver and gonad immediately after death. Total levels of ATP and its related compounds in the muscle ranged from 7.5 μmol·g-1 to 8.0 μmol·g-1. ATP concentrations were 3.9,4.1 and 4.7 μmol·g-1 in the dorsal, ventral and caudal muscles, respectively. ATP level was high (63%) in the caudal muscle which moves hard. Small amounts of adenosine was detected in the liver together with inosinic acid. It can be considered that there exist two metabolic pathways of ATP degradation in the liver of snakehead. The total amounts of free amino acids were 436.0,405.0 and 356.3 mg·(100 g)-1 in the dorsal, ventral and caudal muscles. Taurine and glycine were major free amino acids and accounted for 68% to 73%. Alanine and glutamic acids were detected in

  13. Tricarboxylic acid cycle intermediate pool size: functional importance for oxidative metabolism in exercising human skeletal muscle.

    Science.gov (United States)

    Bowtell, Joanna L; Marwood, Simon; Bruce, Mark; Constantin-Teodosiu, Dumitru; Greenhaff, Paul L

    2007-01-01

    expansion at the start of exercise (same absolute work intensity) in parallel with either equivalent or increased oxidative energy provision. Cycloserine inhibits alanine aminotransferase, which catalyses the predominant anaplerotic reaction in exercising human muscle. When infused into contracting rat hindlimb muscle, TCAi pool expansion was reduced by 25% with no significant change in oxidative energy provision or power output. Glutamine supplementation has been shown to enhance TCAi pool expansion at the start of exercise with no increase in oxidative energy provision. In summary, there is a consistent dissociation between the extent of TCAi pool expansion at the onset of exercise and oxidative energy provision. At the other end of the spectrum, the parallel loss of TCAi, glycogen and adenine nucleotides and accumulation of inosine monophosphate during prolonged exercise has led to the suggestion that there is a link between muscle glycogen depletion, reduced TCA cycle flux and the development of fatigue. However, analysis of serial biopsies during prolonged exercise demonstrated dissociation between muscle TCAi content and both muscle glycogen content and muscle oxygen uptake. In addition, the delay in fatigue development achieved through increased carbohydrate availability does not attenuate TCAi reduction during prolonged exercise. Therefore, TCAi concentration in whole muscle homogenate does not seem to be of functional importance. However, TCAi content can currently only be measured in whole muscle homogenate rather than the mitochondrial subfraction where TCA cycle reactions occur. In addition, anaplerotic flux rather than TCAi content per se is likely to be of greater importance in determining TCA cycle flux, since TCAi content is probably merely reflective of anaplerotic substrate concentration. Methodological advances are required to allow researchers to address the questions of whether oxidative phosphorylation is limited by mitochondrial TCAi content and

  14. 儿童口腔黏膜自伤性溃疡的临床研究%A Clinical Research on Children's Oral Mucosa Factitious Ulcer

    Institute of Scientific and Technical Information of China (English)

    邓雪莲; 潘昱; 劳均平; 陈巨峰

    2009-01-01

    目的 探讨儿童口腔黏膜自伤性溃疡的临床特点和治疗效果.方法 将36例口腔黏膜自伤性溃疡患儿分为三组,第1组:采用药物(肌苷片0.2 g,维生素B6片10 mg)早午晚服,局部用金因肽喷涂和心理辅导治疗,治疗1个月;第2组:采用正畸治疗纠正自伤性溃疡的习惯动作并结合心理辅导;第3组:第1组+第2组治疗方案,并随访追踪1个月,3个月,半年,1年,复诊了解患儿治疗情况和溃疡愈合情况.结果 在36例的研究对象中,多见于男性儿童,均有咬颊,咬舌,咬唇习惯.15例(41.67%)痊愈,9例(25.00%)显效,10例有效(27.57%),2例(2.78%)无效.第3组患儿的治疗效果明显优于第1第和第2组.结论 自伤性溃疡的习惯动作是本病重要的诊断依据.纠正自伤性习惯动作和心理治疗是本病重要的治疗方法 ,本病可能是儿童注意缺陷多动障碍的口腔表现.%Objective To explore the clinical manifestation and therapeutic effect of children's oral mucosa factitious ulcer.It aims to improve the diagnosis and therapy level of such ulcer.Method 36 cases with children's ulcer symptoms and mental attitudes were observed.Tothlcases and were divided into 3 groups.Group 1:Take medicine(Inosine Pill 0.2,Vitamin B6 Pill 10 mg)3 times a day;use EGF spray for some part,together with psyehotherapy for 1 month.Group 2:Adopt orthodontics to correct factitious habit and use psychotherapy.Group 3:Group 1+Group 2.Trackingrandomly for 1 month,3 months,half a year,1 year,check the therapeutic and ulcer healing-up progress by further consultation.Result According to 36 cases,boys are more vulnerable to such disease.They tend to bite cheek,tongue,lips.Ulcer used to happen on tongue brim mucosa,quantity 1~3,size 0.2~1.8 cm,irregular appearance,white keratosis surrounding,priority therapy is to correct factitious habit.15 cases(41.67%)healed,9 cases(25.00%)showed good result,10 cases(27.57%)were efficacious,2 cases(2.78%)ineffective.The thempeutic

  15. Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro%霉酚酸对人骨髓来源间充质干细胞的作用

    Institute of Scientific and Technical Information of China (English)

    曹伟杰; 刘丽珍; 来晓瑜; 王冲; 于晓虹; 黄河

    2011-01-01

    Objective: To investigate the effects of mycophenolic acid ( MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs were treated with MPA at the concentration of 1 μmol/L, 10 μmol/L, 50 μmol/L, and 100 μmol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5 '-monophosphate dehydrogenase (IMPDH ) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining,Ca2+ quantification and real-time PCR. Results; In the range of 1 μmol/L to 100 μmol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration- and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH Ⅰ and IMPDH Ⅱ . Von Kossa staining and Ca2 + quantification indicated that MPA inhibited osteogenic differentiation of MSCs,and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix. Conclusion; MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration- and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.%目的:研究霉酚酸( mycophenolic acid,MPA)对人骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)的作用及其机制.方法:在MSCs生长和分化过程中加入不同浓度的MPA,用CCK-8方法分析MSCs增殖的情况,用Annexin V/PI双染色法检测MPA各浓度组的细胞凋亡,RT-PCR方法分析MSCs次黄嘌呤核苷酸脱氢酶(IMPDH)的表达;应用von Kossa染色、钙定量和real-time PCR方法分析MPA对MSCs成骨分化的影响.结果:1~100 μmol/L的MPA呈时间浓度依赖性地抑制间充质干细胞的生长,添

  16. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2 in schizophrenic patients Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2 em pacientes esquizofrênicos

    Directory of Open Access Journals (Sweden)

    Gustavo Pimentel Dutra

    2010-09-01

    Full Text Available OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A (ADA1*2 exhibits 20-30% lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2 in schizophrenic patients and healthy controls. METHOD: The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. RESULTS: A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6% relative to controls (13/111 - 11.7%, p = 0.032, OR = 2.6. CONCLUSION: These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.OBJETIVO: A adenosina pode ter um papel importante na fisiopatologia da esquizofrenia, uma vez que modula a liberação de vários neurotransmissores, tais como glutamato, dopamina, serotonina e acetilcolina, diminui a atividade neuronal por hiperpolarização pós-sináptica e inibe a atividade dopaminérgica. A adenosina desaminase participa do metabolismo das purinas pela conversão de adenosina em inosina. O mais frequente polimorfismo funcional da adenosina desaminase (22G →A (ADA1*2 exibe uma diminuição de 20-30% da atividade funcional em indivíduos com genótipo G/A quando comparados com indivíduos com o genótipo G/G. O objetivo deste estudo foi avaliar o polimorfismo 22G→A (ADA1*2 em pacientes esquizofrênicos e em

  17. Wilson病铜过量负荷大鼠肝损伤的代谢组学研究%Research on metabolomics of liver injury rats with Wilson’ s disease due to copper overload

    Institute of Scientific and Technical Information of China (English)

    蒋怀周; 王键; 许晶晶; 董继扬

    2016-01-01

    Objective To explore the changes of small molecules in liver tissues of rats with copper overload and the influences of cop-per overload on metabolism of small molecules in liver tissues. Methods Sixteen Wistar rats were randomly divided into normal con-trol group and model group. Copper overload method was used to establish the model rats. 1 H-NMR was used to acquire the metabolic profile of rat liver tissues, and PLS-DA was used to analyze the changes of metabolites in rat liver tissues after copper poisoning. Re-sults Compared with normal control group, the contents of allantoin, taurine, myoinositol, lysine, nicotinamide, ethanolamine, ace-tate, glutamate, tyrosine, uridine, methionine, threonine, isoleucine, 3-hydroxybutyrate, valine, lactate, leucine, phenylalanine, N-acetylaspartate, fumarate, and adenosine/inosine were increased(P<0. 05), while the contents of creatine, asparagine, aspartate, phosphorylcholine and mannitol were decreased in model group(P<0. 05). Conclusion Copper damage in rat liver tissues may be involved in the metabolism of ornithine and Krebs cycles, lecithin, amino acid, energy, nucleotides and glucose.%目的:利用代谢组学技术研究铜负荷大鼠肝组织的小分子变化,探讨铜过量对肝脏小分子代谢的影响。方法16只Wistar大鼠随机分为正常组和模型组,以铜负荷法造模,通过核磁共振氢谱技术采集大鼠肝组织的代谢轮廓,以PLS-DA方法分析铜中毒后,大鼠肝组织代谢物的变化。结果与正常组对比,模型组大鼠肝组织中尿素囊、牛磺酸、肌醇、赖氨酸、尼克酰胺、乙醇胺、乙酸、谷氨酸、酪氨酸、尿苷、甲硫氨酸、苏氨酸、异亮氨酸、3-羟基丁酸、缬氨酸、乳酸、亮氨酸、苯丙氨酸、N-乙酰天冬氨酸、延胡索酸和腺苷/肌苷的含量升高(P<0.05),肌酸、天冬酰胺、天冬氨酸、磷酸胆碱和甘露醇的含量降低(P<0.05)。结论铜对大鼠肝组织的损伤可能涉

  18. Exploring metabolic pathway disruption in the subchronic phencyclidine model of schizophrenia with the Generalized Singular Value Decomposition

    Directory of Open Access Journals (Sweden)

    Morris Brian J

    2011-05-01

    Encyclopedia of Genes and Genomes (KEGG metabolite pathway database were altered in the PFC of PCP-treated rats. Several significant changes were discovered, notably: 1 neuroactive ligands active at glutamate and GABA receptors are disrupted in the PFC of PCP-treated animals, 2 glutamate dysfunction in these animals was not limited to compromised glutamatergic neurotransmission but also involves the disruption of metabolic pathways linked to glutamate; and 3 a specific series of purine reactions Xanthine ← Hypoxyanthine ↔ Inosine ← IMP → adenylosuccinate is also disrupted in the PFC of PCP-treated animals. Conclusions Network reordering via the GSVD provides a means to discover statistically validated differences in clustering between a pair of networks. In practice this analytical approach, when applied to metabolomic data, allows us to quantify the alterations in metabolic pathways between two experimental groups. With this new computational technique we identified metabolic pathway alterations that are consistent with known results. Furthermore, we discovered disruption in a novel series of purine reactions that may contribute to the PFC dysfunction and cognitive deficits seen in schizophrenia.

  19. Effects of Storage Conditions on Taste-active components in Mengshan Chicken%贮藏条件对蒙山草鸡肌肉鲜味成分的影响

    Institute of Scientific and Technical Information of China (English)

    李永洙; 陈常秀

    2011-01-01

    研究改善鸡肉风味的最佳成熟贮藏时期。利用高效液相色谱法对20周龄的蒙山草鸡不同部位、性别、贮藏条件下带骨骼的肌肉肌苷酸和氨基酸含量进行比较分析。结果表明:肌苷酸含量在室温条件下,0~8h间减少幅度较大,而肌苷酸分解物的次黄嘌呤含量在4~8h间显著增加(P〈0.05);在冷藏条件下,肌苷酸含量下降幅度比较缓慢,第5天后趋于平衡状态,次黄嘌呤含量在3~4d间显著增加(P〈0.05);在室温、冷藏条件下胸肉肌苷酸含量均高于腿肉,母鸡肌肉肌苷酸及相关核苷酸含量均显著高于公鸡(P〈0.05)。风味游离氨基酸占总氨基酸的比例,随贮藏时间延长呈显著下降趋势,腿肌在室温4~8h和冷藏4~5d极显著地降低(P〈0.01),而性别间无显著差异(P〉0.05)。结论:最佳成熟贮藏时期为室温条件下8h,冷藏条件下4d为宜。%In order to find out the optimal ripening time for improved taste of chicken during storage,the contents of inosinic acid(IMP) and amino acids in the skeletal muscles from different positions of 20-week-old female and male Menshan chickens were comparatively analyzed by high performance liquid chromatography(HPLC) during storage under different conditions.The results showed that IMP content was dramatically decreased during the first 8 hours of storage at room temperature with the result that the content of hypoxanthine,one of its decomposition products,was significantly increased(P〈0.05).During cold storage at 4 ℃,a slow decrease was observed in IMP content until day 5 followed by a stable trend and hypoxanthine content was significantly increased during day 3-4(P〈0.05).At both storage temperatures,IMP content was higher in the breast muscle compared with the thigh muscle and the contents of IMP and related nucleotides in the muscle of female chicken were significantly higher than those of male chicken(P〈0.05

  20. Effect of Zhenggan Tang decoction on the serum levels of leptin, adiponectin and insulin resistance on HBV-induced cirrhotic patients%中药正肝汤对乙型肝炎肝硬化患者血清瘦素、脂联素水平以及胰岛素抵抗影响的分析

    Institute of Scientific and Technical Information of China (English)

    徐建军; 潘锋; 徐虹

    2015-01-01

    Objective To evaluate the effect of Zhenggan Tang decoction on serum levels of leptin,adiponectin and insulin resistance on liver cirrhosis induced by chronic hepatitis B.Methods Sixty-six patients were recruited and randomly assigned either to a control group or to an intervention group,with 35 cases in the treatment and 31 in the control group respectively.Patients in the control group received inosine tablets and vitamin C treatment while patients in the treatment group were given Zhenggan Tang decoction additionally.After 3 months of treatment,the serum levels of leptin and adiponectin were detected and the index of insulin resistance calculated.Results There were no significant difference between the serum levels of leptin,adiponectin and the index of insulin resistance seen in the control group before and after the treatment.Serum levels of leptin and adiponectin and the index of insulin resistance in treatment group were reduced significantly after the treatment (P<0.05).There were significant difference in the serum levels of leptin and adiponectin between treat group and control group (P<0.05).Conclusion Zhenggan Tang decoction seemed to have reduced the serum levels of leptin,adiponectin and the index of insulin resistance among cirrhotic patients that induced by chronic hepatitis B.%目的 观察中药制剂正肝汤治疗乙型肝炎(乙肝)肝硬化患者对其血清瘦素(LEP)、脂联素(ADP)水平及胰岛素抵抗(IR)的影响.方法 将入选的66例乙肝肝硬化患者随机分为对照组(31例)和治疗组(35例),其中对照组选用肌苷片和维生素C口服治疗,治疗组在此基础上,应用正肝汤治疗,疗程为3个月,测定治疗前后患者血清LEP、ADP水平及IR指数.结果 对照组血清LEP、ADP水平和IR指数与治疗前比较,差异无统计学意义(P>0.05);治疗组血清LEP、ADP水平和IR指数较治疗前明显下降,差异有统计学意义(P<0.05),治疗组血清LEP和ADP水平

  1. Effects of Huanglian Jiedu Decoction on the Metabolic Features of Brown Adipose Tissue Extracts in Fructose-induced Insulin Resistance Model Based on NMR Metabonomics%基于核磁共振氢谱代谢组学研究黄连解毒汤对胰岛素抵抗大鼠棕色脂肪组织代谢组的影响

    Institute of Scientific and Technical Information of China (English)

    杨永霞; 王琳琳; 郑凌云; 王淑美; 黄榕波; 张磊; 黄耀庭

    2014-01-01

    With the application of 1H nuclear magnetic resonance(1 H NMR) spectroscopy, we studied the effect of Huanglian Jiedu decoction( HJD) on the metabonomics of brown adipose tissue extracts in high fruc-tose-induced insulin resistance model. 32 Wistar rats were divided into four groups, i. e. , controls, model group, positive drug group and HJD treated group, 8 in each group. The last three groups were given 100 g/L fructose water for 28 d to establish insulin resistance model, normal control group was given the equal volume of pure water at the same time. After 28 d, four groups of rats were given 100 g/L fructose water continuously. Rats in positive drug group were administered orally atorvastain at a dose of 10 mg/( kg·d) , and rats in HJD treated group were given by gavage with HJD water. The control and model groups were given by gavage with a certain volume of saline solution, and the experiment lasted 56 d. Brown adipose tissues were obtained and 1 H NMR spectra of each sample was performed and analyzed by principal component analysis( PCA) method. Compared with the control group, lactate, alanine, choline, phosphocholine/glycerol phosphocholine ( PC/GPC) , creatine/creatinine, taurine and inosine increased in the model and lipid decreased. In comparison with model group, the myo-inositol was increased in HJD group, and HJD had reversed the metabolites in the model group. HJD can regulate the body’ s energy metabolism and reduce the damaged cell membrances and the injury of liver and kidney. Therefore, this study elucidates the metabolic mechanism of HJD on insulin re-sistance( IR) .%采用基于核磁共振氢谱(1 H NMR)的代谢组学方法,研究了黄连解毒汤( HJD)对高果糖诱导胰岛素抵抗大鼠模型棕色脂肪代谢组的影响.选取Wistar大鼠32只,适应7 d后随机分为正常对照组、模型组、阳性药物对照组和黄连解毒汤组,每组8只.正常对照组给予纯净水喂养,其它3组给予100 g/L的果糖水喂饲.28

  2. Effects of Dietary Chitosan Oligosaccharide and Lactobacillus casei on Growth Performance, Meat Quality and Antioxidant Function of Broilers%饲粮添加壳寡糖和干酪乳杆菌对肉鸡生长性能、肌肉品质及抗氧化性能的影响

    Institute of Scientific and Technical Information of China (English)

    李阳; 常文环; 张姝; 郑爱娟; 刘国华; 蔡辉益; 刘伟

    2016-01-01

    casei, 120 mg/kg COS+2×106 CFU/g Lactobacillus casei, re⁃spectively. The trial lasted for 42 d. The results showed as follows:1) compared with the control group, dieta⁃ry supplementation with COS or COS and Lactobacillus casei significantly increased average daily gain, red⁃ness ( a∗) value of breast and thigh muscle, contents of intramuscular fat and inosinic acid, and contents of monounsaturated fatty acid ( MUFA) and polyunsaturated fatty acid ( PUFA) in breast muscle of broilers ( P<0.05), significantly decreased saturated fatty acid (SFA) content in breast muscle and yellowness (b∗) value of thigh muscle (P<0.05). 2) Supplementation with Lactobacillus casei in diet significantly increased intra⁃muscular fat content in thigh muscle and MUFA content in breast muscle while decreased SFA content in breast muscle ( P<0.05) . 3) Dietary supplementation with COS, Lactobacillus casei or two together significantly de⁃creased malondialdehyde (MDA) content in plasma, breast and thigh muscle (P<0.05), significantly in⁃creased the total superoxide dismutase ( T⁃SOD) activity and total antioxidant capacity ( T⁃AOC) in plasma, breast and thigh muscle (P<0.05), meanwhile decreased the creatine kinase (CK) activity in plasma (P<0.05) . The results indicate that dietary supplementation with COS, Lactobacillus casei or both together can in⁃crease growth performance and antioxidant function and promote meat quality of broilers, and 120 mg/kg COS supplementation gets the best effect.

  3. Cloning and expression analysis of aspartate aminotransferase cDNA in Fenneropenaeus chinensis following ambient ammonia stresses%中国明对虾天门冬氨酸转氨酶基因的克隆及氨氮胁迫对其时空表达的影响

    Institute of Scientific and Technical Information of China (English)

    李少飞; 何玉英; 李吉涛; 李健; 刘萍; 葛倩倩

    2014-01-01

    利用 RACE 技术克隆获得中国明对虾(Fenneropenaeus chinensis)天门冬氨酸转氨酶 GOT 基因(FcGOT)。FcGOT 基因cDNA全长为1910 bp,其中,开放阅读框1284 bp,编码427个氨基酸。同源性分析表明,中国明对虾天门冬氨酸转氨酶 GOT氨基酸序列与其他节肢动物高度保守,与克氏原螯虾(Procambarus clarkii)和桔粉蚧壳虫(Planococcus citri)的同源性分别为78%和73%。系统进化分析表明, FcGOT基因氨基酸序列与克氏原螯虾GOT聚为一支。组织表达分析发现FcGOT基因在肝胰腺、鳃、血细胞、肌肉、心脏、淋巴中均有表达,其中肝胰腺中表达量最高。氨氮胁迫后,荧光定量PCR分析结果表明, FcGOT基因在肝胰腺和鳃组织中的表达与对照组相比具有显著差异(P<0.05),表明 FcGOT 基因在氨氮代谢方面具有重要的作用,参与了中国明对虾机体的急性氨氮胁迫应答反应。%Fenneropenaeus chinensis is an important mariculture species in China. In aquaculture environments ammo-nia is a common toxic substance. In recent years, higher frequencies of ammonia nitrogen toxicity in shrimps have been reported. Therefore, it is necessary to investigate ammonia metabolism by F. chinensis. As an important member of the AAT-like family, the enzyme aspartate aminotransferase (GOT) is involved in many aspects of ammonia metabolism including participating in inosine monophosphate transdeamination, and the urea and citric acid cycles. Therefore, de-tailed understanding of the regulation of GOT is of great significance. In this study, we successfully cloned the aspartate aminotransferase cDNA of F. chinensis (FcGOT). The FcGOT cDNA, which was 1 910 bp in length, contained a 5′-untranslated region(UTR) of 83 bp, a 3′UTR of 543 bp, and an open reading frame (ORF) of 1 284 bp, encoded a 427 amino-acid polypeptide. FcGOT protein exhibited typical AAT-like family features, including a Lys catalytic residue and 10 pyridoxal-5

  4. 基于核磁共振氢谱代谢组学研究黄连解毒汤对胰岛素抵抗大鼠棕色脂肪组织代谢组的影响%Effects of Huanglian Jiedu Decoction on the Metabolic Features of Brown Adipose Tissue Extracts in Fructose-induced Insulin Resistance Model Based on NMR Metabonomics

    Institute of Scientific and Technical Information of China (English)

    杨永霞; 王琳琳; 郑凌云; 王淑美; 黄榕波; 张磊; 黄耀庭

    2014-01-01

    With the application of 1H nuclear magnetic resonance(1 H NMR) spectroscopy, we studied the effect of Huanglian Jiedu decoction( HJD) on the metabonomics of brown adipose tissue extracts in high fruc-tose-induced insulin resistance model. 32 Wistar rats were divided into four groups, i. e. , controls, model group, positive drug group and HJD treated group, 8 in each group. The last three groups were given 100 g/L fructose water for 28 d to establish insulin resistance model, normal control group was given the equal volume of pure water at the same time. After 28 d, four groups of rats were given 100 g/L fructose water continuously. Rats in positive drug group were administered orally atorvastain at a dose of 10 mg/( kg·d) , and rats in HJD treated group were given by gavage with HJD water. The control and model groups were given by gavage with a certain volume of saline solution, and the experiment lasted 56 d. Brown adipose tissues were obtained and 1 H NMR spectra of each sample was performed and analyzed by principal component analysis( PCA) method. Compared with the control group, lactate, alanine, choline, phosphocholine/glycerol phosphocholine ( PC/GPC) , creatine/creatinine, taurine and inosine increased in the model and lipid decreased. In comparison with model group, the myo-inositol was increased in HJD group, and HJD had reversed the metabolites in the model group. HJD can regulate the body’ s energy metabolism and reduce the damaged cell membrances and the injury of liver and kidney. Therefore, this study elucidates the metabolic mechanism of HJD on insulin re-sistance( IR) .%采用基于核磁共振氢谱(1 H NMR)的代谢组学方法,研究了黄连解毒汤( HJD)对高果糖诱导胰岛素抵抗大鼠模型棕色脂肪代谢组的影响.选取Wistar大鼠32只,适应7 d后随机分为正常对照组、模型组、阳性药物对照组和黄连解毒汤组,每组8只.正常对照组给予纯净水喂养,其它3组给予100 g/L的果糖水喂饲.28

  5. Nucleotide Mixture Supplementation Affects Non-specific Immune and Antioxidant Indices of Juvenile Litopenaeus vannamei%饲料中添加核苷酸混合物对凡纳滨对虾幼虾非特异性免疫和抗氧化指标的影响

    Institute of Scientific and Technical Information of China (English)

    许丹丹; 黄燕华; 曹俊明; 蓝汉冰; 王国霞; 张荣斌; 陈晓瑛; 严晶

    2011-01-01

    with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 g/kg the nucleotide mixture of adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP) disodium salt, inosine-5'monophosphate ( IMP ) disodium salt and guanosine-5'-monophosphate ( GMP ) disodium salt ( 1: 1: 1: 1:1,m/m), respectively. The results showed as follows: shrimp fed diets with 0.4 to 1.2 g/kg nucleotide mixture had a significantly higher total haemocyte count (THC) ( P < 0.05 or P < 0.01 ). Compared with the control group, serum superoxide dismutase (SOD) activity of 0.6 and 0.8 g/kg groups were significantly increased (P <0.05). The total antioxidation capacity (T-AOC) of 0.4 g/kg group was increased by 51.0% compared with the control group ( P > 0.05) and significantly higher than that of 1.2 g/kg group ( P < 0.05). With the increasing of dietary nucleotide mixture level, SOD activity and T-AOC in hepatopancreas of all groups increased at first and then decreased, and reached the highest value at 0.4 g/kg group. The activities of peroxidase (POD)and alkaline phosphatase (AKP) in serum and hepatopancreas were not significantly affected by nucleotide mixture supplementation ( P > 0.05). The anti-superoxide anion radical ( O2- · ) in 0.2, 0.4 and 0.6 g/kg groups was significantly higher than that of 1.0 and 1.2 g/kg groups ( P < 0.05), and there were no significant differences among the other groups ( P > 0.05). It is concluded that nucleotide mixture can improve the non-specific immunity and antioxidant capacity of juvenile Litopenaeus vannamei, and the optimal level of dietary nucleotide mixture for juvenile Litopenaeus vannarnei is 0.4 to 0.6 g/kg. [ Chinese Journal of Animal Nutrition, 2011, 23 ( 5 ): 828-835

  6. 饲料中添加核苷酸对凡纳滨对虾幼虾生长、肠道形态及抗氧化酶活力的影响%Effects of dietary nucleotides on growth performance, intestinal morphology and anti-oxidative activities of juvenile Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    许丹丹; 曹俊明; 黄燕华; 李雅琪; 蓝汉冰; 陈冰; 陈晓瑛; 严晶; 张荣斌

    2011-01-01

    There has been extensive research into the role of nucleotides and their related metabolic products in aquatic feeds. Nucleotides and metabolites play key roles in many biological processes and are considered conditionally essential nutrients. During periods of rapid growth or certain disease states, dietary nucleotides may spare the cost of de novo nucleotides synthesis and optimize the function of rapidly dividing tissues, such as those in the gastrointestinal and immune systems. Research on dietary nucleotides in aquatic animals has illustrated that they may improve diet palatability, enhance growth in early stages of development, and maintain intestinal and liver health, as well as increase immunity and disease resistance. Despite their apparent importance, little is known about the benefits of supplementary nucleotides in Litopenaeus vannamei. We evaluated the effects of dietary nucleotides on growth, body composition, midgut morphology, and anti-oxidant activity in the hepatopancreas and serum in juvenile L. Vannamei. We randomly assigned 960 shrimp (mean body weight: 1.01 g±0.02 g) into 8 triplicate groups. Group GO (control) was fed a base diet and the remaining seven groups (G0.1, G0.2, G0.4, G0.6, G0.8, G1.0, and G1.2) were fed the base diet supplemented with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 g/kg, respectively, of a nucleotide mixture containing adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP), uridine-5'- monophosphate disodium salt (UMP), inosine-S'-monophosphate disodium salt (IMP), and guanosine-5'-monophosphate disodium salt (GMP) (1:1:1:1:1 w/w, mix-NT). All groups were fed their respective diets three times a day (8:00, 15:00, and 20:00) at the same fixed rate, which ranged from 4% to 6% of body weight, for 7 weeks. Specific growth rate (SGR) and survival (SR) tended to increase as the concentration of the dietary mix-NT increased, peaking in the group supplemented with 0.6 g/kg, though the differences among the groups were

  7. 饲料中添加核苷酸对凡纳滨对虾幼虾生长、组织生化组成及非特异性免疫功能的影响%Effects of dietary nucleotides on growth performance, tissue biochemical composition and non-specific immunity of juvenile Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    曹俊明; 许丹丹; 黄燕华; 蓝汉冰; 陈冰; 赵红霞; 蒋卫亮; 陈晓瑛

    2011-01-01

    量、蛋白质沉积率、全虾粗脂肪和灰分含量,一定程度提高全虾粗蛋白和肝胰腺总蛋白含量,显著增加肝胰腺RNA、肠道总蛋白和RNA含量,提高对虾的非特异性免疫功能.%This study was undertaken to investigate the effects of dietary nucleotides on growth performance,body composition, tissue biochemical composition and non-specific immunity of juvenile shrimp (Litopenaeus vannamei).960 shrimp(0.43 ±0.01 ) g were randomly allocated into 8 groups.The control group was fed with the basal diet,while the other seven groups were fed with the basal diet added with 0.1,0.2,0.4,0.6,0.8, 1.0 and 1.2 g/kg mixture of adenosine-5'-monophosphate ( AMP ), cytidine-5'-monophosphate ( CMP), uridine-5'-mono- phosphate disodium salt ( UMP), inosine-5 '-monophosphate disodium salt (IMP) and gnanosine-5'-monophosphate disodium salt(GMP) ( 1: 1: 1: 1: 1 W/W, mix-NT) respectively.After 5 weeks feeding, the results showed that weight gain rate (WGR), specific growth rate (SGR)and feed intake (FI) in shrimp fed 0.4 g/kg mix-NT were significantly higher than those in the control group ( P < 0.05 ).Protein deposit rate (PDR) in 0.6 and 1.0 g/kg groups increased significantly compared with the control group.No significant difference was found among all the treatments in feed conversion rate ( FCR), survival rate ( SR ) and hepatosomafic index ( HSI ) ( P > 0.05 ).The crude lipid and ash content were significantly affected by the dietary mix-NT levels ( P < 0.05 ), while the dry matter and protein content showed no significant difference (P > 0.05).RNA content in hepatopancreas increased significantly (P < 0.05 ) with dietary mix~NT increasing, TP content was not significantly affected ( P > 0.05 ).TP and RNA content in intestine increased significantly with dietary nucleotides increasing( P <0.05).The uric acid( UA)content in serum decreased significantly in 0.6 g/kg group and glutamic-oxalacetic transaminease (GOT) activity increased